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TESIS DOCTORALESN.º 70

CATTLE NUTRITION AS A STRATEGY TO MITIGATEGASEOUS NITROGEN LOSSES FROM DAIRY FARMING

Haritz Arriaga Sasieta

UNIVERSITAT AUTÒNOMA DE BARCELONADEPARTAMENT DE CIÈNCIA ANIMAL I DELS ALIMENTS


First published: 1st july 2010

Edition: 50 copies

© Administration of the Autonomous Community of the Basque CountryEnvironment, Territorial Planning, Agriculture and Fishing Department

Internet: www .euskadi.net

Published: Eusko Jaurlaritzaren Argitalpen Zerbitzu NagusiaServicio Central de Publicaciones del Gobierno Vasco Donostia-San Sebastián, 1 - 01010 Vitoria-Gasteiz

Printed by: Grafo, S.A. Avda. Cervantes, 51 - 48970 Basauri

ISBN: 978-84-457-3082-9

Legal record: BI 2.091-2010

A catalogue record of this book is av ailable in the catalogue of the General Library of the Basque Governement: http://www.euskadi.net/ejgvbiblioteka


CATTLE NUTRITION AS A STRATEGY TO MITIGATE

GASEOUS NITROGEN LOSSES FROM DAIRY FARMING

Tesis doctoral presentada por

Haritz Arriaga Sasieta

bajo la dirección de

Dra. Pilar Merino Pereda

y

Dr. Sergio Calsamiglia Blancafort

Para acceder al grado de Doctor en el programa de Producción

Animal de la

UNIVERSITAT AUTÒNOMA DE BARCELONA

DEPARTMENT DE CIÈNCIA ANIMAL I DELS ALIMENTS

Bellaterra, Abril 2010


Pilar Merino Pereda, Investigadora del Departamento de Ecotecnologías de NEIKER-

Tecnalia

Certifico:

Que la memoria titulada “Cattle Nutrition as a Strategy to Mitigate Gaseous

Nitrogen Losses from Dairy Farming” presentada por Haritz Arriaga Sasieta para

optar al grado de Doctor por la Universitat Autònoma de Barcelona, ha sido realizada

bajo mi dirección, y considerándola concluída, autorizo su presentación para que sea

juzgada por la comisión correspondiente.

Y para que así conste, firmo el presente en Derio, Abril de 2010.

Dr. Pilar Merino Pereda


Sergio Calsamiglia Blancafort, Catedrático del Departament de Ciència Animal i dels

Aliments de la Facultat de Veterinaria de la Universitat Autònoma de Barcelona

Certifico:

Que la memoria titulada “Cattle Nutrition as a Strategy to Mitigate Gaseous

Nitrogen Losses from Dairy Farming” presentada por Haritz Arriaga Sasieta para

optar al grado de Doctor por la Universitat Autònoma de Barcelona, ha sido realizada

bajo mi dirección, y considerándola concluída, autorizo su presentación para que sea

juzgada por la comisión correspondiente.

Y para que así conste, firmo el presente en Bellaterra, Abril de 2010.

Dr. Sergio Calsamiglia Blancafort


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CATTLE NUTRITION AS A STRATEGY TO MITIGATE GASEOUS NITROGEN LOSSES FROM DAIRY FARMING

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ESKERRONAK / AGRADECIMIENTOS

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RESUMEN

La optimización de la nutrición del ganado vacuno lechero es una herramienta clave en

la mitigación de la problemática ambiental derivada de la acumulación de N en las explotaciones lecheras. Los problemas derivados del exceso de N en la producción de

leche se han centrado históricamente en el estudio de la contaminación por nitratos. Sin embargo, en los últimos años los estudios se han focalizado en las emisiones gaseosas

de N a la atmósfera. En este contexto, la presente tesis doctoral se planteó con dos objetivos principales: a) estudiar la viabilidad de la manipulación de las raciones en la

reducción de la acumulación de N en las explotaciones comerciales de la Comunidad Autónoma del País Vasco (CAPV); b) estudiar el efecto de la manipulación de las dietas

(adecuado aporte de energía y N a nivel ruminal o ajuste de N de la ración a las necesidades animales) en el balance de N a nivel animal, en las características del purín resultante y la consiguiente emisión de NH3, N2O y NO en el establo y/o las praderas

fertilizadas con purín.

En el primer ensayo llevado a cabo en 64 explotaciones comerciales de la CAPV, los resultados mostraron que la sobrealimentación proteica es una práctica común para la

cabaña en lactación. El 69,7% de las raciones muestreadas excedieron en la ingestión de N, estimándose en un 7,4% el exceso de proteina metabolizable ingerida. Dado que la

ingestión de N fue la mejor variable predictora de la excreción de N (R2 = 0,7), el ajuste de la proteína bruta (PB) de la ración a los requerimientos del ganado podría ser una

estrategia viable en la reducción de la excreción de N en las granjas comerciales. Otras estrategias nutricionales como la manipulación de la calidad proteica de la ración, la

reformulación de las dietas, la separación de los animales en lotes de alimentación o el empleo de diferentes sistemas de alimentación no mejoraron significativamente el uso

de N en el animal. Considerando la variabilidad existente en la excreción de N animal por litro de leche producido, la reducción de N excretado por la cabaña en lactación

podría alcanzar un 35,5% al producir el total de la cuota láctea. Sin embargo, si la excreción de N es referida a la disponibilidad de tierras (grado de intensificación), los

resultados mostraron que el efecto de la manipulación de la ración puede hallarse seriamente limitada en explotaciones altamente intensificadas (la manipulación de la PB


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de la ración explicó únicamente el 11,2% de la varianza de la excreción de N por parte de la cabaña en lactación y por hectárea de suelo disponible).

En el segundo experimento realizado, se estudió el efecto del aporte de energía a dietas

isonitrogenadas en el aprovechamiento de N a nivel animal, la excreción de N y la concentración de N en los purines resultantes. Los purines resultantes de las diferentes

dietas fueron posteriormente aplicados en pradera para la evaluación de la volatilización de los gases de N (NH3, N2O y NO). La modificación de la ración de una dieta con bajo

contenido en forraje (45:55) (dietas energéticas empleadas en granjas tecnificadas) a una dieta con alto contenido en forraje (75:25) (dietas menos energéticas y consideradas

más sostenibles ambiental y alimentariamente) mostró que el aumento de consumo de forraje puede limitar el consumo voluntario de materia seca (especialmente si se

emplean forrajes con alto contenido en FND). Como consequencia de esta limitación, se reducirá la ingestión de N, la consiguiente excreción de N y la acumulación de N-NH4

+

en el purín resultante. Sin embargo, la eficiencia de uso de N en leche (NUE) o la excreción de N por litro de leche producido puede no ser mejorada debido a la

disminución en la producción de leche. La reducción del contenido de N-NH4+ del purín

puede afectar al manejo de los purines aplicados sobre pradera en función de si las

aplicaciones se realizan en base a los requerimientos de N-NH4+ de las especies

vegetales o si se realizan en base a la aplicación de materia fresca de purín para

favorecer el vaciado de fosas de almacenamiento. El tipo de manejo seleccionado tendrá posteriormente implicaciones ambientales. El patrón de emisión de NH3, N2O y NO

será similar tras la aplicación de los purines obtenidos a partir de dietas con alto o bajo contenido en forraje si la tasa de aplicación de N-NH4

+ en campo es la misma (120 kg N-NH4

+ ha-1 en el presente estudio). El factor de emisión de gases de N en las dietas con

alto contenido en forraje alcanzó el 15,6% (17,8 kg N ha-1) mientras que para dietas bajas en contenido forrajero fue del 9,6% (11,5 kg N ha-1). La volatilización de NH3

representó el 60% de las pérdidas gaseosas de N tras 2 meses de medidas en campo. El patrón de emisión y la emisión acumulada de NH3, N2O y NO estarán supeditados al

manejo de los purines en pradera.


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RESUMEN

En el tercer experimento llevado a cabo, se estudió el efecto del aporte nitrogenado a dietas isoenergéticas en el aprovechamiento de N en vacas de mitad o final de lactación,

la excreción fecal y urinaria de N y la acumulación de gases de N (NH3 y N2O) en el suelo del establo (estabulación permanente). Los resultados mostraron que la reducción

de la concentración de PB de la ración (dietas de 14,0% PB 16,0 PB y 17,0% PB), disminuyó la excreción de N. Sin embargo, la disminución de la concentración de PB de

la ración también contribuyó a la reducción del rendimiento lechero. De este modo el ajuste de la PB de la ración no contribuyó significativamente a mejorar el uso de N en

leche, aunque la excreción de N por litro de leche producido tendió a disminuir. Como consecuencia de la mayor excreción de N en las dietas con mayor contenido proteico, la

concentración de NH3 procedente del suelo del establo aumentó con dichas raciones. Las concentraciones de NH3 aumentaron desde 7,1 mg NH3 m-3 en dietas de bajo

contenido proteico a 10,8 mg NH3 m-3 en dietas de alto valor proteico (concentración atmosférica, 0,4 mg NH3 m-3). Esta reducción supuso que por cada unidad de PB

reducida, la concentración de NH3 disminuía un 13%. La menor concentración de NH3

en bajas concentraciones de PB podría ser explicada por la disminución de la

concentración de la urea urinaria en dichas dietas. Al contrario que el NH3, la concentración de N2O en el suelo del establo no respondió al efecto de la manipulación

de la ración. El tratamiento de bajo contenido proteico fue de 1,21 mg N2O m-3, similar al valor medio obtenido para las mayores concentraciones de PB con 1,10 mg N2O m-3.

A pesar de la falta de respuesta a la manipulación de las raciones, los resultados mostraron que la concentración de N2O de los establos puede diferir de la concentración

atmosférica (0,55 mg N2O m-3).

Debido al estrecho rango de temperaturas medidas durante el ensayo (12.6ºC-18.0ºC),

unido a la variabilidad existente en la concentración de N de las muestras fecales y urinarias, no se observó ningún efecto de la temperatura en las concentraciones de NH3

y N2O del establo. Sin embargo, los resultados obtenidos a partir de incubaciones de muestras de heces y orina (ratio 2:1) realizadas a diferentes temperaturas (4ºC, 19ºC and

29ºC) mostraron que la temperatura afecta significativamente en la concentración de NH3 para todas las concentraciones de PB. Además, la concentración de NH3 puede

diferir entre dietas con diferente contenido en PB a altas temperaturas. Por el contrario, el aumento de temperatura no se relaciona con un aumento de la concentración N2O.


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ABSTRACT

Optimisation of dairy cow nutrition has been described as a key factor to reduce N pollution from dairy farms. Nitrogen pollution derived from dairy farming has been

historically focused on nitrate losses. However, the study of gaseous N losses has become the main objective in the recent times. Therefore, this PhD-thesis was planned

with two main objectives: a) the evaluation of the feasibility of dietary strategies to reduce N pollution in commercial dairy farms from the Basque Country; b) the study of

the effect of dietary manipulation (correct protein and energy balance in the rumen or fitting N intake to animal requirements) on cow N balance, slurry characteristics and the

subsequent NH3, N2O and NO volatilisation from dairy barn floors and/or slurry amended grasslands.

The first trial conducted on 64 commercial farms from the Basque Country showed that

protein overfeeding is a common practice for lactating herds. In fact, 69.7% of sampled rations exceeded in N intake, in which exceeding metabolizable protein was estimated

by 7.4%. As N intake was the best predictor of N excretion (R2 = 0.7), fitting dietary crude protein (CP) content to cow requirements may be a feasible strategy to reduce N

excretion in commercial farms. Other strategies such as the manipulation of the quality of protein fed, the periodical ration reformulation, the use of different feeding groups in

lactating herds and the selection of a feeding system did not improve cow N use efficiency. Considering the variability of N excreted per milk kilogram in sampled

herds, we estimated that N reduction might reach up to 35.5% in lactating herds when whole milk quota is produced. However, when cow N excretion was referred to farmland availability (intensification characteristics) results showed that the effect of

dietary manipulation may be limited in highly intensified farms (dietary N manipulation explained 11.2% of variance on herd N excretion per hectare).

In the second experiment, we studied the effect of energy supplementation to

isonitrogenous rations on cow N use in order to reduce animal N excretion and the subsequent N accumulation in diet-derived slurries. Slurries were afterwards applied on

grassland and gaseous N losses were measured (NH3, N2O and NO). Ration


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modification from low forage content diets (45:55) (high energy content diets usually used in specialised farms) to high forage content diets (75:25) (lower energy content

diets which are usually considered feed and environmentally sustainable) showed that increasing forage content of diets might limit voluntary dry matter intake (especially for

high NDF content forages). As a consequence, N intake will be reduced in lactating cows, minimizing therefore N excretion and slurry NH4

+-N content. However, milk N

use efficiency (NUE) or N excreted per milk kilogram may not be improved due to the lower response of milk yield. Reducing slurry NH4

+-N content may involve

management and environmental implications when slurry is applied on grassland whether slurry is applied on field to fit plant N requirements or is applied on fresh

matter basis to empty slurry storages. The emission pattern of NH3, N2O and NO gases will be similar from high or low forage content diets derived slurries whether equal

amount of NH4+-N is applied (120 kg NH4

+-N in the current study). The emission factor of high forage treatment was 15.6% (17.8 kg N ha-1), while averaged 9.6% (11.5 kg N

ha-1) in low forage diet. Ammonia volatilisation represented 60% of total N gas losses after 2 months of measurements. Ammonia, N2O and NO emission pattern and

cumulative emission will vary depending on slurry management on grassland.

In the third experiment, we studied the effect of varying dietary CP in isoenergetic diets on mid-late lactating cow N use, and fecal and urinary N excretion. In addition, NH3

and N2O accumulation was measured in barn floors (tie-stall). Results showed that reducing dietary CP (diets contained 14.0% CP, 16.0% CP and 17.0% CP) N excretion

decreased. However, milk yield also decreased with lower N intake. Although milk N use efficiency was not improved significantly, N excreted per milk yield tended to decrease through lowering dietary CP content. Increasing CP intake enhanced NH3

concentration from barn floors, whose values ranged from 7.1 mg NH3 m-3 in low protein diets to 10.8 mg NH3 m-3 in high protein diets (atmospheric concentration, 0.4

mg NH3 m-3). This result meant that NH3 concentration is reduced 13% per each unit of dietary CP reduced. The lower NH3 concentration in low CP diets might be explained

because urinary urea N also tended to decrease. In contrast to NH3 concentration, on-farm N2O concentrations did not respond to the different dietary CP levels. Treatments

averaged 1.21 mg N2O m-3 in low protein diets while high protein rations showed concentrations around 1.10 mg N2O m-3. Nevertheless, these data support that N2O


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ABSTRACT

concentration in dairy barns may be significantly different from atmospheric concentration (0.55 mg NH3 m-3).

Temperature monitored during the experiment ranged between 12.6ºC and 18.0ºC. The

narrow range of temperatures together with the variability detected in N concentration of urinary and fecal samples contributed to mask the effect of temperature on NH3 and

N2O accumulation. However, results recorded from fecal and urinary incubations (ratio 2:1) at 3 temperatures (4ºC, 19ºC and 29ºC) demonstrated that temperature affects NH3

concentration in different CP contets. In addition, NH3 concentration may differ between different CP contents at high temperatures. In contrast to NH3, increasing

temperature did not enhance N2O concentration.


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TABLE OF CONTENTS

1. INTRODUCCIÓN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291.1. Modelo Productivo en Vacuno de Leche: Intensificación . . . . . . . . . . . . . . . . . . . . . . 311.2. El Sector Vacuno Lechero en la Comunidad Autónoma del País Vasco . . . . . . . . . . . 34

1.2.1. Evolución de las Explotaciones de Vacuno de Leche . . . . . . . . . . . . . . . . . . . 341.2.2. El Sector de Vacuno de Leche de la CAPV y el Medio Ambiente . . . . . . . . . 36

1.3. El Ciclo del Nitrógeno en las Explotaciones de Vacuno de Leche . . . . . . . . . . . . . . . 381.4. Emisiones Gaseosas de Nitrógeno en Explotaciones de Vacuno de Leche . . . . . . . . . 42

1.4.1. Emisiones de NH3 en Explotaciones de Vacuno de Leche . . . . . . . . . . . . . . . 431.4.1.1. Formación de NH3 en las Explotaciones de Vacuno de Leche . . . 431.4.1.2. Contribución del Sector Vacuno Lechero a las Emisiones de NH3. . 441.4.1.3. Emisiones de NH3 en Establos y el Sistema de Almacenamiento

de Purines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461.4.1.4. Emisiones de NH3 Procedentes de la Aplicación del Purín en Pra-

deras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471.4.2. Emisiones de Óxidos de Nitrógeno en Explotaciones de Vacuno de Leche . . . 49

1.4.2.1. Formación de N2O y NO en las Explotaciones de Vacuno de Leche 491.4.2.2. Contribución del Sector Vacuno Lechero a las Emisiones de Óxi-

dos de N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511.4.2.3. Emisiones de Óxidos de N en Establos y el Sistema de Almacena-

miento de Purines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531.4.2.4. Emisiones de Óxidos de N Procedentes de la Aplicación del Purín

en Praderas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541.5. Visión General de las Estrategias de Minimización de las Emisiones de N en Explo-

taciones de Vacuno Lechero . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561.6. Nutrición del Ganado Vacuno Lechero como Estrategia de Minimización de las Emi-

siones de los Gases de N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581.6.1. Ajuste de la Proteína de la Ración en el Ganado Vacuno de Leche . . . . . . . . 60

1.6.1.1. Requerimientos Proteicos en Vacuno de Leche. Proteína Metaboli-zable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

1.6.1.2. Proteína de la Ración, Eficiencia NUE y Excreción de N . . . . . . . 611.6.1.3. Degradación de la Proteína, Síntesis de NH3 y Excreción de Urea 63

1.6.2. Ajuste de la Calidad de la Proteína en Ganado Vacuno Lechero . . . . . . . . . . 651.6.2.1. Síntesis de la Proteína Microbiana del Rumen . . . . . . . . . . . . . . . . 661.6.2.2. Suplementación con PNDR e Infusión Intestinal de Aminoácidos . . 671.6.2.3. Otras Estrategias Nutricionales . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

2. OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713. NUTRITIONAL AND MANAGEMENT STRATEGIES ON NITROGEN AND PHOS-

PHORUS USE EFFICIENCY OF LACTATING DAIRY CATTLE ON COMMER-CIAL FARMS: AN ENVIRONMENTAL PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . 753.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

3.2.1. Dairy Farm Survey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793.2.2. Cornell Net Carbohydrate and Protein System (CNCPS 5.0) . . . . . . . . . . . . . 803.2.3. On-site Sample Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803.2.4. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

3.3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.3.1. Description of Farms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.3.2. Description of Lactating Herd Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843.3.3. Lactating Cow Nutrient Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

3.3.3.1. Lactating Cow N Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873.3.3.2. Lactating Cow P balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

3.3.4. Management Practices to Improve N and P Utilization Efficiency . . . . . . . . 933.3.5. Effect of Intensification on N and P Excretion . . . . . . . . . . . . . . . . . . . . . . . . 94

3.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96


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4. DIETARY MODIFICATION IN DAIRY CATTLE: FIELD MEASUREMENTS TO ASSESS THE EFFECT ON AMMONIA EMISSIONS IN THE BASQUE COUNTRY 974.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

4.2.1. Animals, Diets and Manure Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014.2.2. Application of Slurries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034.2.3. Measurement of NH3 Emissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044.2.4. Cumulative Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054.2.5. Soil Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054.2.6. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

4.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064.3.1. Nitrogen Excretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064.3.2. Ammonia Emissions from Slurry Applied to Soil . . . . . . . . . . . . . . . . . . . . . 1084.3.3. Soil Mineral N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

4.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1094.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

5. EFFECT OF DIET MANIPULATION IN DAIRY COWN BALANCE AND NITRO-GEN OXIDES EMISSIONS FROM GRASSLANDS IN NORTHERN SPAIN . . . . . . 1135.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

5.2.1. Animals, Diets and Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.2.2. Field Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205.2.3. N2O Emission Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205.2.4. NO Emission Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215.2.5. N2O and NO Cumulative Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215.2.6. Soil Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215.2.7. Yield Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225.2.8. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

5.3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1235.3.1. Composition of Diets and Ruminal Processes . . . . . . . . . . . . . . . . . . . . . . . . 1235.3.2. Milk Yield, N Use Efficiency and N Excretion . . . . . . . . . . . . . . . . . . . . . . . 1265.3.3. Slurry Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285.3.4. N2O Fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1295.3.5. NO Fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1335.3.6. Grass Yield and N Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

5.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356. DIETARY CRUDE PROTEIN MODIFICATION ON AMMONIA AND NITROUS

OXIDE CONCENTRATION ON A TIE-STALL DAIRY BARN FLOOR . . . . . . . . . . 1376.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

6.2.1. Animals, Diets and Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416.2.2. On-Farm Trace Gas Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446.2.3. Laboratory Incubations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446.2.4. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1466.3.1. Feeding Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1466.3.2. Milk Yield and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1466.3.3. Cow N Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1486.3.4. On-Farm NH3 and N2O Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

6.3.4.1. Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1496.3.4.2. Nitrous Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

6.3.5. Effect of Temperature on NH3 and N2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1516.3.5.1. Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1516.3.5.2. Nitrous Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

6.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537. GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155


25

ÍNDICE

7.1. Is it feasible to reduce farm N surplus through dietary strategies in commercial dairyfarms from the Basque Country? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

7.2. Dietary strategies to improve milk NUE and reduce N excretion in lactating cows . . 1617.3. Dietary strategies to alter slurry composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667.4. Dairy cow nutrition and ammonia emissions from barn floors and grasslands . . . . . . 1677.5. Dairy cow nutrition and N oxides emissions from stalls and grasslands . . . . . . . . . . 1697.6. Gaseous N losses in the future and other comments . . . . . . . . . . . . . . . . . . . . . . . . . . 173

8. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1759. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179


26


INDEX OF TABLES

Tabla 1.1. Evolución del consumo mundial de los alimentos (1961-2005) . . . . . . . . . . . . . . 31Tabla 1.2. Explotaciones de vacuno lechero de la CAPV por tamaño de rebaño . . . . . . . . . . 35Tabla 1.3. Distribución de la cuota láctea de la CAPV por tamaño de explotación . . . . . . . . 36Tabla 1.4. Censo de vacuno de leche y producción láctea de la CAPV por provincias . . . . . 37Tabla 1.5. Entradas de N por concentrados y fertilizantes en explotaciones de vacuno de le-

che intensivos de la UE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Tabla 1.6. Emisiones de NH3 en establos y depósitos de almacenamiento de purines . . . . . 46Tabla 1.7. Fuentes y factores de emisión de N2O en explotaciones de vacuno lechero . . . . . 52Tabla 1.8. Factores de emisión de N2O en diferentes sistemas de estabulación . . . . . . . . . . . 53Tabla 1.9. Estrategias de minimización de NH3 en explotaciones de vacuno de leche . . . . . 57Tabla 1.10. Estrategias de minimización de N2O en explotaciones de vacuno de leche . . . . . 58Tabla 1.11. Principales ingredientes en vacuno de leche: PB y degradabilidad ruminal . . . . . 64Table 3.1. Herd size, milk yield and land use in farms from the Basque Country . . . . . . . . . 84Table 3.2. Ration and ingredients, management and milk yield by feeding groups . . . . . . . . 85Table 3.3. N balance for lactating cows on farms from the Basque Country . . . . . . . . . . . . . 88Table 3.4. Prediction equations for manure, fecal and urinary N output1 . . . . . . . . . . . . . . . . 89Table 3.5. N balance for lactating cows on farms from the Basque Country . . . . . . . . . . . . . 91Table 3.6. Prediction equations for manure, fecal and urinary P output1 . . . . . . . . . . . . . . . . 92Table 3.7. Effect of feeding groups, feeding systems, reformulation and degree of intensifica-

tion on N and P utilization in milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Table 3.8. Daily and annual N and P excretion by herds concerning land area . . . . . . . . . . . 95Table 4.1. Ration composition of HF and LF diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Table 4.2. Characteristics of the slurries applied to soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Table 4.3. N use efficiency in milk and N excretion in urine and feces . . . . . . . . . . . . . . . . . 107Table 4.4. Daily N intake and excretion in lactating cows fed HF and LF diets . . . . . . . . . . 107Table 5.1. Mean (SD) chemical composition of ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . 123Table 5.2. Ingredients and chemical composition of HF and LF diets . . . . . . . . . . . . . . . . . . 124Table 5.3. Effect of dietary manipulation on N metabolism from HF and LF diets . . . . . . . . 128Table 5.4. Chemical composition of HF and LF diet derived slurries . . . . . . . . . . . . . . . . . . 129Table 5.5. Cumulative N2O and NO emissions from HF and LF treatments . . . . . . . . . . . . . 132Table 5.6. Grass yield, botanical composition and N uptake from HF and LF slurries . . . . . 135Table 6.1. Composition of LP, MP and HP diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142Table 6.2. Effect of dietary CP content on N metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Table 6.3. Effect of temperature on NH3 concentration (mg m-3) in incubated jars . . . . . . . 151Table 6.4. Effect of temperature on N2O concentration (mg m-3) in incubated jars . . . . . . . 153


27

ÍNDICE

INDEX OF FIGURES

Figura 1.1. Carga ganadera por vacuno de leche en municipios de la CAPV . . . . . . . . . . . . . 37Figura 1.2. Principales flujos de N en el sistema integrado suelo-planta-animal de una explo-

tación de vacuno de leche (Fuente: Rotz. 2004) . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Figura 1.3. Distribución de las emisiones de NH3 en Europa (Fuente: Erisman et al., 2003) . . 45Figura 1.4. Contenidos de PB y producción de leche según la fase de lactación (Fuente: Wu y

Satter, 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Figura 1.5. Relación entre la ingestión de PB y la eficiencia NUE (Fuente: Kalscheur et al.,

2006. 6,8% PDR; ❍ 8,2% PDR; 9,6% PDR; × 1 1,0% PDR) . . . . . . . . . . . 62Figura 1.6. Relación entre la ingestión de N y excreción de N en ganado vacuno de leche

(Fuente: Yan et al., 2006) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Figura 1.7. Esquema de la degradación proteica en el rumen del vacuno de leche . . . . . . . . . 65Figure 3.1. Herd mean milk yield and nitrogen use efficiency (NUE) . . . . . . . . . . . . . . . . . . . 88Figure 3.2. Relationship between total N intake and fecal, urinary and milk N output . . . . . . 90Figure 3.3. Relationship between nitrogen use efficiency (NUE) and phosphorus use efficien-

cy (PUE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Figure 4.1. Pattern of ammonia emissions from HF and LF diet derived slurry . . . . . . . . . . . 108Figure 4.2. Evolution of soil ammonium content from HF and LF diet derived slurry . . . . . . 109Figure 5.1. Nitrous oxide emission pattern from HF and LF treatments . . . . . . . . . . . . . . . . . 130Figure 5.2. Soil ammonium content in the grassland soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Figure 5.3. Soil nitrate content in the grassland soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Figure 5.4. Nitric oxide emission pattern from HF and LF treatments . . . . . . . . . . . . . . . . . . . 134


1 INTRODUCCIÓN


31

1.1. Modelo Productivo en Vacuno de Leche: Intensificación

El informe titulado “El Estado Mundial de la Agricultura y la Alimentación” emitido por la Organización de las Naciones Unidas para la Agricultura y la Alimentación

(FAO, 2007) ha remarcado el incremento del consumo de alimentos a escala mundial (en países desarrollados y en países en vías de desarrollo) en las últimas 4 décadas

(1961-2005) (Tabla 1.1). Destaca especialmente el aumento en el consumo de cultivos oleaginosos (4,0%), huevos (3,4%), hortalizas (3,2%) y productos cárnicos (3,0%).

Según la FAO, la evolución del consumo de leche a escala mundial también ha mostrado una tendencia al alza, aunque su incremento es algo inferior a los productos

anteriormente citados con un crecimiento anual de 1,4%. Sin embargo, este aumento en el consumo de leche ha sido desigual en las diferentes regiones mundiales, mostrándose

superior en los países en vía de desarrollo (3,2%).

Tabla 1.1 Evolución del consumo mundial de los alimentos (1961-2005). 1961-76 1977-91 1992-05 1961-2005

Cambio porcentual anual (%) Promedio (%)

Mundo 3,5 1,8 1,3 2,2 Cereales

Países en Desarrollo 3,9 2,8 1,5 2,8

Mundo 2,9 4,8 4,2 4,0 Cultivos

Oleaginosos Países en Desarrollo 3,1 5,0 4,9 4,4

Mundo 3,4 2,3 0,8 2,2 Azúcar


Mundo 0,8 1,5 0,9 1,1 Legumbres


Mundo 1,3 0,5 1,5 1,1 Raíces

Tubérculos Países en Desarrollo 3,0 1,6 2,2 2,3

Mundo 1,8 3,2 4,7 3,2 Hortalizas


Mundo 3,0 3,4 3,6 3,4 Huevos


Mundo 3,5 3,0 2,6 3,0 Carne


Mundo 1,6 1,4 1,2 1,4 Leche



32


Los estudios poblacionales pronostican que la población mundial podría alcanzar los 9,3 billones de habitantes en 2030 (Nakicenovic et al., 2000), debido principalmente al

aumento poblacional de los países en vía de desarrollo. Por lo tanto, la evolución del consumo de alimentos y los pronósticos de aumento de población a escala mundial

permiten predecir una mayor demanda de leche en el futuro.

Ante tal hipótesis, la pregunta que surge a continuación es la siguiente: ¿Cómo se va a satisfacer tal demanda de leche? o ¿Qué modelo productivo se empleará para garantizar

la producción de leche requerida? En virtud de la tendencia observada en vacuno de leche durante los últimos años, todo apunta a que será el modelo de producción

intensivo el sistema de producción empleado para la producción de leche (Bouwman et

al., 2005). Este sistema de producción se caracteriza por el aumento del tamaño medio

de la cabaña, el aumento del nivel productivo de la misma, la mejora genética de la cabaña mediante el uso generalizado de la raza lechera Frisona/Holstein y un manejo de

la cabaña en condiciones de estabulación permanente. Sin embargo, esta especialización de las explotaciones lecheras conlleva en muchas ocasiones fenómenos como la

excesiva carga ganadera, lo cual ha incidido directamente en la casi desaparición del pastoreo como sistema de producción en muchas zonas (CEAS, 2002). A este respecto,

cabe destacar el incremento de la producción mundial de leche y carne procedente del ganado rumiante (+40%) en relación al aumento de la superficie de pasto (+4%) durante

el periodo comprendido entre 1970 y 1995 (FAO, 2001). La rentabilidad económica del modelo intensivo de producción en vacuno de leche (van Arendock y Liinamo, 2003) y

el aumento de la demanda mundial de alimentos conforman la base del avance de la intensificación tanto en sociedades occidentales como en sociedades en transición (CEAS, 2002).

En la Unión Europea (UE), el modelo intensivo de producción de leche aglutinaba hace

una década el 85% de la cuota láctea europea y el 83% del censo de ganado vacuno de leche (EUROSTAT, 1995). El amplio desarrollo de este modelo productivo está

relacionado con la política de ayudas económicas establecida por la política agraria común europea (PAC), el cual a fin de garantizarse la propia autosuficiencia alimentaria

y rebajar la dependencia de las fluctuaciones del mercado lácteo mundial (Suzuki y Kaiser, 2005), impulsó durante años la producción de leche mediante ayudas

económicas directas a la producción (Beck et al., 1991). El mantenimiento de las primas


33

INTRODUCCIÓN

económicas a la producción láctea hasta la Reforma de la PAC de 2003 (aplicado en 2006), junto con el estancamiento del precio de la leche en origen y el continuo aumento

de los gastos de producción, han contribuido a la necesidad de aumentar la producción al menor coste posible por medio de la intensificación (van Arendonck y Liinamo

2003). Sin embargo, la sostenibilidad de este modelo productivo ha sido ampliamente cuestionada, dado que es un modelo productivo ligado a problemas medioambientales y

sociales (Hadjigeorgiou et al., 2005). Además, y a pesar de su reconocida rentabilidad económica, la actual coyuntura del sector de vacuno de leche de la UE (fuerte caída del

precio de la leche en origen e incremento de los precios de concentrados y fertilizantes) hace plantearse en la actualidad la rentabilidad económica del modelo de producción en

intensivo a largo plazo.

Desde la publicación del Informe Brundtland (Anonymous, 1987), el concepto de desarrollo o producción sostenible es un término a aplicar a cualquier actividad

económica, incluida la producción de leche. Esta sostenibilidad del sistema productivo debe integrar los aspectos económicos, medioambientales y sociales derivados de la

propia actividad (Hadjigeorgiou et al., 2005). En este sentido, la actual PAC aboga por el desarrollo de un sector agroganadero sostenible, un sector que aporte productos de

calidad mediante sistemas de producción seguros y respetuosos con el medio ambiente (van Passel et al., 2007). Para ello la PAC se dotó de la Reforma de la Agenda 2000, el

cual impulsó la implantación de ayudas económicas basadas en consideraciones medioambientales en contraposición con las tradicionales ayudas económicas ligadas a

la producción. La política agraria de la UE se halla encaminada por tanto a aumentar la sostenibilidad de los ecosistemas agro-ganaderos, integrándolos en la política de mercado y de renta mediante conceptos como la ecocondicionalidad o la modulación de

medidas agroambientales.

El efecto de la intensificación de la actividad ganadera sobre el medio ambiente debe enfocarse desde la perspectiva de la creación de unas áreas sobreexplotadas y otras

áreas abandonadas (CEAS, 2002). En las áreas sobreexplotadas, la reducción de la pérdida de nutrientes se ha convertido en la mayor preocupación medioambiental

(Kuipers y Mandersloot, 1999; Gerber et al., 2005). La intensificación del sector vacuno lechero en áreas sobreexplotadas es considerada responsable de procesos de

eutrofización de las aguas superficiales o contaminación de las aguas subterráneas por la


34


excesiva acumulación de nitratos (NO3-) y fósforo (P), de procesos de contaminación

atmosférica por gases de efecto invernadero como el dióxido de carbono (CO2), el

metano (CH4) y el óxido nitroso (N2O), de fenómenos de lluvia ácida por las emisiones de amoníaco (NH3) o de la destrucción de la capa de ozono por las emisiones de óxido

nítrico (NO). Además, la sobreexplotación también se halla ligada a otros problemas ambientales como los procesos de alteración y erosión de los suelos por el excesivo uso

de fertilizantes orgánicos y/o minerales o una excesiva carga ganadera, la acumulación en el suelo de aditivos alimenticios, pesticidas o metales pesados, problemas por los

malos olores procedentes de las deyecciones ganaderas o la pérdida de biodiversidad y hábitat (disminución de pastos, simplificación de especies vegetales, pérdida de razas

animales) (CEAS, 2002). Por tanto, desde el punto de vista medioambiental, la percepción actual permite asegurar que la intensificación de la producción agro-

ganadera supone numerosos efectos negativos sobre el medio ambiente (Oenema, 2004, Flamant et al., 1999).

1.2. El Sector Vacuno Lechero en la Comunidad Autónoma del País Vasco

1.2.1. Evolución de las Explotaciones de Vacuno de Leche

Durante varias décadas el sector vacuno lechero ha liderado las cuentas de la economía

agraria de la Comunidad Autónoma del País Vasco (CAPV), aportando entorno al 25% del total de la producción final agraria. Dicho sector ha desempeñado durante años un

papel central y de liderazgo en el proceso de profesionalización y desarrollo de la ganadería de la CAPV, marcando las pautas para el desarrollo de sectores como el ovino

lechero o el vacuno de carne. Sin embargo, la importancia del sector vacuno lechero ha cambiado notablemente desde la adopción de la PAC en 1986. Como dato representativo, si en el año 1986 la producción de leche (bovino y ovino) suponía el

52% de la producción final ganadera y el 23,5% del total de la producción final agraria (87 millones €, 98,8% por vacuno de leche), en 2000 su aportación había descendido al

43% de la producción final ganadera y al 11,7% de la producción final agraria (68 millones €). Desde la implantación de las cuotas lácteas, el sector vacuno lechero de la

CAPV tendió a la intensificación de los sistemas de producción, con una mayor concentración de la cabaña ganadera en explotaciones altamente profesionalizadas, la

desaparición de muchas explotaciones familiares y la pérdida de cuota láctea a nivel del


35

INTRODUCCIÓN

territorio (Ruiz et al., 2006). Datos publicados por los censos agrarios de 1989 y 1999 (EUSTAT, 2005) muestran el impacto de la entrada en la UE sobre el sector vacuno

lechero de la CAPV. Así, mientras que en 1989 existían 69.549 cabezas repartidas en 11.274 explotaciones, 10 años más tarde el tamaño de la cabaña ganadera se había

reducido a 40.649 animales, distribuidos en 3.157 explotaciones. Esta tendencia ha continuado en el tiempo, tal y como han demostrado los datos publicados en el Plan

Sectorial de Vacuno de la CAPV entre los años 2004 y 2005, el cual cifraba en 2.411 el total de explotaciones de vacuno lechero para una cabaña de 38.621 vacas lecheras (la

producción total de leche en la CAPV alcanzaba los 243 millones de litros anuales). Según los últimos datos publicados por el Departamento de Medio Ambiente,

Planificación Territorial, Agricultura y Pesca del Gobierno Vasco (DMAPTAP) correspondientes a 2008, se censaron 1.262 explotaciones de vacuno de leche, con una

producción global de 204 millones de litros de leche (DMAPTAP, 2009). Actualmente, la producción de leche procedente del sector vacuno de la CAPV representa el 4% de la

cuota nacional (FEGA, 2009).

Otros datos que corroboran la profesionalización y tecnificación de las explotaciones de vacuno de leche se comprueban en la evolución de parámetros como el incremento del

tamaño medio de las cabañas ganaderas, el aumento de la producción media por vaca o el aumento de la cuota media de las explotaciones. La Tabla 1.2 muestra la evolución de

las explotaciones por el tamaño de cabaña ganadera entre los años 1995 y 2008 (EUSTAT, 2009).

Tabla 1.2. Explotaciones de vacuno lechero de la CAPV por tamaño de rebaño.

1995 2008

Nº vacas n % n %

1 a 4 2.603 46,8 654 51,8 5 a 9 1.294 23,2 119 9,4

10 a 19 876 15,7 121 9,6 20 a 49 641 11,5 180 14,3

> 50 154 2,8 188 14,9 Total 5.568 1.262 Fuente: EUSTAT (2009)


36


Destaca la fuerte caída de las explotaciones de menor tamaño, desapareciendo más de 3.000 explotaciones de tamaño inferior a 10 vacas, y el aumento porcentual de las

explotaciones con un número mayor a 50 vacas, pasando de representar un 2,8% en 1995 a un 14,9%, en 2008. El aumento del rendimiento lácteo de las cabañas de la

CAPV se debe al avance en la mejora genética de las cabañas, la optimización de la nutrición o la mejora de las instalaciones. De este modo, y según datos recogidos por el

Instituto Vasco de Estadística-EUSTAT, el aumento de la producción media por animal pasó desde los 4.934 kg en 1996 hasta los 7.185 kg anuales en 2003 (EUSTAT, 2005).

Acorde con el incremento del tamaño medio de las cabañas ganaderas y la producción láctea de las mismas, las cuotas lácteas de las explotaciones de la CAPV también han

aumentado durante estos últimos años. De este modo, mientras que los datos recogidos en 2004 situaban la cuota media en 185.000 litros por explotación, ésta alcanzaba los

320.000 litros en 2006 (Ruiz et al., 2006). En la siguiente tabla se muestra la distribución de la cuota láctea de la CAPV en 2006 por tamaño de explotación (Tabla

1.3).

Tabla 1.3 Distribución de la cuota láctea de la CAPV por tamaño de explotación.

Rango cuota, kg < 160.000 160.000-320.000 > 320.000

Explotaciones, nº 348 135 232 Explotaciones, % 48,7 18,9 32,5

Cuota, % 10,3 12,4 76,3 Fuente: Informe PROBEHI (Ruiz et al., 2006)

1.2.2. El Sector de Vacuno de Leche de la CAPV y el Medio Ambiente

En el actual contexto bio-geográfico, las explotaciones de vacuno de leche de la CAPV

se distribuyen mayoritariamente a lo largo de la vertiente Atlántica del territorio, con tres principales zonas de producción: el valle de Karrantza en Bizkaia, el norte de la

provincia de Araba (valle de Aiara y estribaciones del Gorbea) y la zona Asteasu-Aia en Gipuzkoa (Figura 1.1). Según datos correspondientes al año 2007, entorno al 80% del

ganado y la producción láctea de la CAPV era localizado en los territorios históricos de Bizkaia y Gipuzkoa (Tabla 1.4) (EUSTAT, 2009). Sin embargo, la vertiente atlántica de

la CAPV se caracteriza por su orografía montañosa, una elevada parcelación del terreno


37

INTRODUCCIÓN

y una fuerte presión demográfica e industrial sobre el entorno rural, lo cual supone un importante obstáculo para el uso del suelo para fines ganaderos.

Figura 1.1. Carga ganadera por vacuno de leche en municipios de la CAPV.

Tabla 1.4. Censo de vacuno de leche y producción láctea de la CAPV por provincias.

Territorio Histórico Censo % Leche (T año-1) %

Bizkaia 9.831 35,7 71.886 34,8

Araba 5.830 21,1 51.817 25,0

Gipuzkoa 11.885 43,2 82.964 40,2 Fuente: EUSTAT (2009)

La intensificación del sector lechero ha contribuido a la aparición de problemas

ambientales en las zonas con mayor tradición lechera de la CAPV. Del inventario de residuos orgánicos de Lekuona et al. (2002) se observó que las tres principales áreas de

producción lechera presentaban los mayores niveles de excedentes de purín, alcanzando volúmenes excedentarios de 32.864, 42.416 y 32.311 m3 año-1 en el valle de Karrantza,

las estribaciones del Gorbea y el área de Asteasu-Aia, respectivamente. Un proyecto conjunto de NEIKER-Tecnalia y la Diputación Foral de Gipuzkoa ha constatado que el

excedente de purines de vacuno es una realidad en el territorio histórico de Gipuzkoa desde hace 12 años. El 70% de los pastos analizados para el mencionado estudio (2.092

ha) mostraron la falta de necesidad de abonado mediante purines de vacuno de leche,

Karrantza Asteasu-Aia

Valle de Aiara y Estribaciones del

Gorbea


38


dado que los niveles de N y P en el suelo se hallaban en concentraciones próximas a originar la contaminación de las aguas (Merino y Berano, 2006). Ante tales evidencias,

se están impulsando desde la Administración Pública medidas encaminadas a la minimización de los problemas ambientales derivados de la actividad lechera. Así, el

Gobierno Vasco publicó en 2007 la Guía de Buenas Prácticas para Explotaciones de Vacuno de Leche, vigente hasta la actualidad, en la que se recogía la Ley General de

Protección del Medio Ambiente del País Vasco (Ley 3/1998 de 27 de febrero de 1998) y el Real Decreto sobre protección de aguas contra la contaminación producida por los

NO3- procedentes de fuentes agrarias (RD 261/1996 de 16 de febrero de 1996). Además,

existe en la CAPV la Norma Técnica de Producción Integrada de Vacuno de Leche

(BOPV, nº 119 de 24 de junio de 2005) en el que se consideran aspectos de protección medioambiental, impulsando una actividad ganadera que respete el entorno (suelo,

agua, aire) y la biodiversidad de flora y fauna propia del territorio. En relación a N y P, esta Norma Técnica prohíbe el empleo de raciones con concentraciones superiores a

17,5% de proteína bruta (PB) y 0,40% de fósforo (P). También las instancias provinciales como la Diputación Foral de Bizkaia están impulsando en la actualidad

ayudas económicas para la aplicación de medidas agroambientales tales como el Plan de Gestión de Estiércoles y Purines y de Fertilización en Explotaciones con Base

Territorial, ayudas para la conservación de prados de siega de interés o las ayudas para una producción integrada. En colaboración con la Administración Pública, desde el

sector privado también se están impulsando estrategias que mejoren la sostenibilidad ambiental de las explotaciones. Este es el caso del valle de Karrantza (Bizkaia), donde

se sitúan más del 80% de las explotaciones del territorio histórico, y donde el exceso de purines del valle ha posibilitado el planteamiento de un proyecto de implantación de una planta de tratamiento de purines y cogeneración de energía.

1.3. El Ciclo del Nitrógeno en las Explotaciones de Vacuno de Leche

Debido a su importancia desde el punto de vista productivo y medioambiental, el ciclo

del N de las explotaciones de vacuno de leche ha sido uno de los ciclos más estudiados durante los últimos años (Rotz et al., 1999; Spears et al., 2003b; Rotz, 2004; Nevens et

al., 2006; Fangueiro et al., 2008; Sonneveld et al., 2008). Desde el punto de vista productivo, el N resulta un nutriente esencial tanto para el crecimiento de las especies

forrajeras empleadas en la nutrición ganadera como para el crecimiento, desarrollo y


39

INTRODUCCIÓN

producción láctea del ganado vacuno. Desde el prisma medioambiental, la intensificación del sector lechero ha aumentado la preocupación por el conocimiento del

ciclo de N de las explotaciones, dado que la necesidad de incorporar N externo al sistema a fin de garantizar una mayor producción lechera y forrajera, provoca

importantes pérdidas de N al medio ambiente. A pesar de la variabilidad existente entre las explotaciones de vacuno de leche en cuanto al sistema de producción

intensivo/extensivo, el tipo de cultivo forrajero o el empleo del pastoreo como sistema de producción, la Figura 1.2 muestra un ciclo típico de N en las explotaciones de

vacuno de leche, el cual integra aspectos ligados con la producción animal, el suelo y la producción forrajera (Rotz, 2004).

Figura 1.2. Principales flujos de N en el sistema integrado suelo-planta-animal de una

explotación de vacuno de leche (Fuente: Rotz. 2004).

El ciclo del N de las explotaciones de leche contempla una serie de entradas y salidas de N en el sistema. Las principales entradas (inputs) de N corresponden a la compra de

alimentos y fertilizantes minerales, seguido de otras incorporaciones como la compra de

Animal

Cosecha

Suelo

Praderas/Cultivos

Pastoreo

Purín/Estiércol

Granja

Venta de leche/animal

Compra alimentos, camas, etc

Purín/Estiércol exportado Volatilización gaseosa

Compra fertilizantes Deposición atmosférica

Lixiviación y Escorrentía Volatilización gaseosa

Fijación de leguminosas Venta forraje Volatilización gaseosa


40


materiales para el encamado (paja, etc), la compra de ganado externo, la importación de estiércoles o purines, o la fijación de N por parte de los cultivos de leguminosas. Las

principales salidas de N incluyen la venta de leche, animales y/o forrajes producidos en la explotación, así como la exportación de estiércoles o purines a otras explotaciones

(Dou et al., 1996). Los alimentos (forrajes y concentrados) y los fertilizantes minerales constituyen las principales entradas de N en el sistema, especialmente en los modelos

intensificados, debido a que el aumento del tamaño de las cabañas y el mayor rendimiento lácteo de las mismas crean tanto la necesidad de suplementar las raciones

de base forrajera propia con alimentación externa como la necesidad de aumentar la producción forrajera propia mediante el empleo de fertilizantes (CEAS, 2002). De este

modo, el modelo intensivo de producción lechera se convierte en un modelo basado en la constante entrada de N externo, obviando en muchos casos estrategias de

optimización de los recursos propios o los recursos disponibles en el entorno más próximo a la explotación (van Bruchem et al., 1999). La Tabla 1.5 muestra la entrada

porcentual de N por medio de alimentos y fertilizantes en explotaciones intensivas de diferentes países de la UE, representando la suma de ambas fuentes más del 75% del

total de N importado en muchos de los casos.

Tabla 1.5. Entradas de N por concentrados y fertilizantes en explotaciones de vacuno de leche intensivos de la UE.

Territorio Concentrados Fertilizantes Fuente

Holanda 63% 35% Ondersteijn et al. (2002)

Francia 27% 59% Bos et al. (2005)

Alemania 31% 56% Bos et al. (2005) Italia 67% 25% Bos et al. (2005)

Dinamarca 40% 33% Nielsen y Kristensen (2005)

CAPV 59% 9% Del Hierro et al. (2006)

Flandes 25% 42% Nevens et al. (2006)

Portugal 70% 24% Fangueiro et al. (2008)

En este balance neto de entradas y salidas de N en forma de producto (leche, carne,

forraje y/o purín), las entradas de N superan en la mayoría de los casos, y en especial en las explotaciones intensificadas, las cantidades de N exportado, dando lugar al

denominado surplus o exceso de N de las explotaciones. Aunque las pérdidas de N


41

INTRODUCCIÓN

ligadas a la producción lechera son generalizadas en todos los modelos productivos, Steinshamn et al. (2004) resumieron a partir de trabajos desarrollados en diferentes

explotaciones de la UE que el modelo de producción intensivo se relacionaba con el mayor exceso o surplus de N. El grado de surplus de N de una explotación ganadera es

considerado como un indicador válido para la evaluación de la potencialidad de riesgos ambientales derivados de la actividad ganadera (Schröder et al., 2003), expresando la

potencial pérdida de N en términos de volatilización, desnitrificación y lixiviación (Børsting et al., 2003).

Las pérdidas de N se deben a las ineficiencias en el uso del N dentro de los diferentes

sistemas integrados en una explotación lechera (suelo, planta y animal) (Rotz, 2004). Comenzando el ciclo de N en la alimentación de la cabaña ganadera, ésta se caracteriza

por presentar una limitación fisiológica en el aprovechamiento de la proteína de la ración en leche (Tamminga, 1992; Jonker et al., 2002; Powell et al., 2006), lo cual

implica que la mayor parte del N consumido por el ganado sea excretado al medio (el establo en condiciones de estabulación permanente o el pasto en sistemas de pastoreo).

Además, y a pesar de los avances realizados en la nutrición del vacuno de leche, el N en forma de proteína es habitualmente sobreutilizado en relación a los requerimientos

animales (Børsting et al., 2003; Arriaga et al., 2009). En explotaciones con un sistema de estabulación permanente y altamente intensificadas, la acumulación de N se ve

acrecentada por la alta densidad ganadera de las mismas, lo cual provoca una mayor acumulación de residuos tanto en establos como en fosas de almacenamiento. A pesar

del avance tecnológico existente en relación a las estrategias para la disminución de las pérdidas de N durante la fase de estabulación o almacenamiento de purines, una elevada proporción del N excretado por el ganado es perdido por volatilización gaseosa antes de

su aplicación en campo (Rotz, 2004). En sistemas de manejo en pastoreo, las excretas animales se depositan directamente en el pasto, dando lugar igualmente a pérdidas de N

a la atmósfera por la volatilización de gases de N o a pérdidas de N por lixiviación o escorrentías de NO3

-. Cuando las enmiendas acumuladas en fosas o estercoleros son

aplicadas en campo (suplementadas en ocasiones con fertilizantes minerales) para favorecer la producción forrajera, también se producen pérdidas de N por volatilización,

lixiviación o escorrentía debido a la ineficiencia en la absorción de N por parte de las plantas. Con el empleo de los forrajes producidos en la explotación para la nutrición de

la cabaña ganadera se cierra el ciclo de N de las explotaciones lecheras. Considerando


42


estas premisas, Rotz (2004) estableció que desde un punto de vista integral de las explotaciones de vacuno de leche, el correcto manejo del N debería encaminarse

principalmente a aumentar la eficiencia de uso de N por parte del ganado (animal), la minimización de las pérdidas de N durante el almacenamiento de las deyecciones y su

posterior aplicación en campo (suelo) y la correcta aplicación de las enmiendas orgánicas y fertilizantes minerales para el óptimo crecimiento de los cultivos forrajeros

(planta).

1.4. Emisiones Gaseosas de Nitrógeno en Explotaciones de Vacuno de Leche

La lixiviación de NO3- a las aguas subterráneas ha sido considerada como el problema

medioambiental más preocupante derivado de la actividad ganadera en el pasado

reciente. Sin embargo, esta máxima preocupación por la contaminación de las aguas ha sido relevada paulatinamente por un creciente interés en las emisiones de gases de N a

la atmósfera (Rotz, 2004). Las salidas de N de las explotaciones ganaderas en términos de volatilización gaseosa ocurren principalmente en forma de amoníaco (NH3), óxido

nitroso (N2O) y óxido nítrico (NO). Las emisiones de NH3 contribuyen a problemas ambientales tales como la fertilización, acidificación y eutrofización de diversos

ecosistemas, las emisiones de N2O se consideran responsables del fenómeno del calentamiento global de la tierra mientras que el NO regula el balance oxidativo de la

troposfera y supone un riesgo ambiental dada su relación con la eliminación de precursores para la síntesis del ozono troposférico (NRC, 2003).

Las emisiones de NH3 comienzan con la deposición urinaria en los establos y ocurren posteriormente durante las fases de manejo de purines en el establo, el almacenamiento

de los purines en las fosas de purines y la aplicación de los purines en campo (Misselbrook et al., 2005). Las emisiones de N2O se hallan ligadas con los procesos de

nitrificación y desnitrificación que ocurren tanto en las fosas de almacenamiento de purines como tras la aplicación de los purines en campo. Las emisiones de NO ligadas a

las explotaciones de vacuno de leche proceden fundamentalmente de las aplicaciones de los purines en campo.


43

INTRODUCCIÓN

1.4.1. Emisiones de NH3 en Explotaciones de Vacuno de Leche

Los purines producidos en las explotaciones ganaderas constituyen la mayor fuente de contaminación por emisión de NH3 tanto en Europa como en Estados Unidos (Bussink y

Oenema, 1999; Webb et al., 2005), emisiones de NH3 que se relacionan con problemas de acidificación y eutrofización de ecosistemas edáficos y acuáticos (Amann et al.,

2007). Junto a los problemas ambientales, las emisiones de NH3 pueden también afectar a la productividad de la explotación al disminuir las condiciones de bienestar animal y

puede afectar negativamente también sobre la salud humana (Rumburg et al., 2008a). Las principales fuentes de emisión de NH3 en las explotaciones lecheras son la propia

granja (zona de establos y almacenamiento de purines), la aplicación de los purines en campo y las deyecciones urinarias del ganado manejado en régimen de pastoreo (Smits

et al., 2003), aunque en general se consideran mayoritarias las pérdidas de NH3 durante la fase de estabulación y la aplicación de los purines en campo (van Duinkerken et al.,

2005).

1.4.1.1. Formación de NH3 en las Explotaciones de Vacuno de Leche

La volatilización de NH3 se describe como un proceso que implica la conversión

química del ión NH4+ presente en el purín a NH3 disuelto, y el posterior transporte físico

del NH3, compuesto extremadamente volátil, a la atmósfera. La principal causa de las

pérdidas de NH3 en las explotaciones de vacuno de leche parte de la limitación fisiológica del ganado bovino en el aprovechamiento de la proteína de la ración,

principal causa de la acumulación de N-NH4+ en el purín. La habitual eficiencia de uso

del N por parte del ganado lechero en lactación en explotaciones de vacuno de leche comerciales se sitúa entorno al 20% o 30% (Tamminga, 1992; Jonker et al., 2002;

Powell et al., 2006; Arriaga et al., 2009). De modo que el N que no ha sido extraído en forma de leche (o carne), es excretado al medio (establo o pasto) tanto por vía urinaria

como por vía fecal (Castillo et al., 2000; Kebreab et al., 2001; Broderick et al., 2003; Yan et al., 2006). El factor determinante en la minimización de las emisiones NH3

consiste en el control sobre la excreción de N urinario, más que la reducción del N fecal, dado que en la orina se encuentra la urea (CO(NH2)2), molécula precursora de la síntesis

de NH3, y compuesto nitrogenado que puede representar hasta el 90% del N presente en


44


la orina (Bussink y Oenema, 1998). La urea urinaria es un producto químico derivado de la liberación de NH3/NH4

+ en el rumen, originado por la degradación de la proteína a

nivel ruminal. La acumulación de NH3/NH4+ ruminal debe ser eliminado del organismo

animal al ser tóxico en concentraciones altas, proceso que se realiza mediante la

destoxificación de ambas moléculas a la molécula de urea a nivel hepático. La urea urinaria excretada por el ganado será convertido a ión NH4

+ al entrar en contacto con las

enzimas de actividad ureasa presentes en las heces (emisiones en granja) o en el suelo (emisiones procedentes de las deposiciones urinarias del ganado en pastoreo). A

continuación se presenta la serie de reacciones químicas para la síntesis del ión NH4+ a

partir de la urea presente en la orina:

CO(NH2)2 + H2O (NH4)2CO3 2NH4+ + CO3

2-

La magnitud de la emisión de NH3 a partir del N-NH4+ acumulado variará en función de

factores físico-químicos como la temperatura atmosférica, la concentración de NH4+ en

la solución o el pH de la solución y del suelo, de factores ligados a la ventilación de los

establos (natural o forzada, orientación del establo) o aspectos meteorológicos como la velocidad del viento o las precipitaciones en forma de lluvia, y de factores relacionados

con el manejo del ganado (estabulación o pastoreo, el tiempo de estabulación, etc), el tipo de encamado empleado (paja de cereal, residuos de serrería, camas comerciales) o

el manejo de los purines (sistema de extracción de deyecciones, sistemas de limpieza de establos, tipos de fosa de almacenamiento, modo de aplicación de los purines) (de Boer

et al., 2002; Frank et al., 2002; Misselbrook et al., 2005; Smith et al., 2007; Powell et

al., 2008; Rumburg et al., 2008a,b).

1.4.1.2. Contribución del Sector Vacuno Lechero a las Emisiones de NH3

Las emisiones de NH3 a nivel mundial alcanzan unos niveles de emisión de entorno a

45-54 Tg N anuales (Bouwman et al., 1997; van Aardenne et al., 2001), en el que el sector agroganadero representa el 79% de las emisiones globales (van Aardenne et al.,

2001). Dentro del sector agroganadero destaca la contribución de la producción animal, al representar el 50% de las emisiones globales de NH3 y el 80-90% de las emisiones

comunitarias (ECETOC, 1994; Hutchings et al., 2001). En este sentido, y tal y como se


45

INTRODUCCIÓN

observa en el mapa de emisiones de NH3 del año 1998 (Figura 1.3), los focos de mayor concentración de NH3 coincidían con las áreas de mayor concentración ganadera

(Erisman et al., 2003). Los inventarios realizados en diferentes estados de la UE en relación a la emisión de NH3 avalan la idea de la elevada contribución de la producción

animal a las emisiones globales de NH3. Así, se conoce que el sector ganadero de Suecia era responsable del 90% de las emisiones nacionales de NH3 (50.200 t N-NH3

anuales) (Frank y Swensson, 2002), que la producción animal en Dinamarca alcanzaba recientemente niveles de emisión de NH3 similares a los de Suecia (51.000 t N-NH3

anuales) (Kai et al., 2008) o que las actividades agroganaderas ligadas al empleo de deyecciones representaban el 95% de las emisiones de NH3 en Holanda (Braam et al.,

1997b). A pesar de que diversos trabajos han demostrado la contribución de los sectores porcino, ovino o avícola a las emisiones de NH3, destaca sobremanera el sector vacuno

lechero como la principal fuente de emisión de NH3 a la atmósfera (Bussink y Oenema, 1998; Hyde et al., 2003). En estados con una gran tradición lechera como Holanda o

Suiza, la contribución del sector vacuno lechero alcanzó en el pasado niveles de emisión de NH3 de entorno al 50% de las emisiones globales del territorio (van Duinkerken et

al., 2005; Reidy et al., 2008).

Figura 1.3. Distribución de las emisiones de NH3 en Europa (Fuente: Erisman et al., 2003).


46


1.4.1.3. Emisiones de NH3 en Establos y el Sistema de Almacenamiento de Purines

La creciente atención suscitada por la problemática ambiental originada dentro de las granjas de vacuno lechero, la consideración positiva de la mejora del bienestar animal y

la mayor preocupación por la salud humana ha provocado que diversos grupos científicos hayan trabajado desde la década de los 90 en la reducción de la volatilización

de NH3 en las granjas de vacuno de leche. Estas granjas, las cuales incluyen el conjunto de establos y fosas de almacenamiento de purines o estercoleros, constituyen una fuente

importante de emisión de NH3 en la explotación lechera. Monteny y Erisman (1998) establecieron que las emisiones de NH3 procedentes de los establos representaban el

28% del total de emisiones de NH3 vinculados al sector agro-ganadero holandés. A pesar de la variabilidad existente en las emisiones de NH3 procedentes de las granjas de

vacuno lechero, se estima que las pérdidas de NH3 pueden alcanzar entre un 50% y un 75% del N total excretado por el conjunto de la cabaña ganadera (van Horn et al., 1994;

Rotz, 2004). La variabilidad existente en la emisión de NH3 procedente de las granjas lecheras también se observa al revisar referencias bibliográficas en relación a la tasa

anual de emisión por animal (Tabla 1.6).

Tabla 1.6 Emisiones de NH3 en establos y depósitos de almacenamiento de purines. Emisión N-NH3, kg vaca-1 año-1 Referencia

Tipo de Establo y Fosa Establo Fosa

Estabulación libre 60 _ Moreira y Satter (2006)

Estabulación libre 40 _ Rumburg et al. (2008)a Estabulación libre 8,1 _ Sonneveld et al. (2008) Estabulación libre 20 _ Flesch et al. (2009)

Estabulación fija 9,8 _ Monteny y Erisman (1998)Estabulación fija 27,5 _ Powell et al. (2008)

Estabulación fija 3,1 _ Sonneveld et al. (2008) Fosa anaeróbica _ 55 Rumburg et al. (2008)b

Estiércol apilado _ 8,9 Sonneveld et al. (2008)

La variabilidad existente en las emisiones de NH3 de las granjas lecheras se halla ligada

a los múltiples factores que interactúan en dicha emisión (Ndegwa et al., 2008). Factores como la nutrición proteica del ganado (James et al., 1999; de Boer et al., 2002;


47

INTRODUCCIÓN

Frank y Swensson, 2002; Smits et al., 2003; Swensson, 2003; Misselbrook et al., 2005; Powell et al., 2008), el sistema de estabulación en libre o fijo (Swensson y Gustafsson.,

2002; Rotz, 2004), el diseño de los patios (formas de V, rampas para separación de la fracción sólida y líquida) (Braam et al., 1997a,b), el tipo de encamado (Powell et al.,

2008), el tiempo de estancia de la cabaña en el establo (Gilhespy et al., 2006), el sistema de acumulación (emparrillado o fosa externa) y extracción de los purines (arrobaderas

mecánicas, limpieza por agua a presión, frecuencia de extracción) (Kroodsma et al., 1993; Hartung y Phillips, 1994; Monteny y Erisman, 1998), el empleo de inhibidores de

la actividad ureasa, sustancias acidificantes y biofiltros (Ogink y Kroodsma, 1996; Monteny y Erisman; 1998; Martinec et al., 2001; van der Stelt et al., 2007; Kai et al.,

2008), el encostramiento natural de los purines (Misselbrook et al., 2005), la tasa de ventilación de las granjas (Snell et al., 2003) o la estacionalidad de la toma de medidas

(Powell et al., 2008; Harper et al., 2009) contribuyen a la variabilidad observada en las tomas de medida in situ.

Dada la complejidad de la medida de la emisión de NH3 se han desarrollado en los

últimos años numerosos modelos mecanísticos para la estimación de la emisión en granja. Los modelos mecanísticos permiten predecir la emisión de NH3 según los

procesos de transporte en la fuente de emisión de NH3 y los mecanismos de transferencia a la atmósfera (Teye y Hautala, 2008). Así, Ni (1999) elaboró una revisión

sobre 30 modelos ya existentes en la época y paulatinamente fueron desarrollándose otros modelos predictivos considerando diferentes parámetros implicados en la emisión

de NH3 (de Boer et al., 2002; Monteny et al., 2002; Smits et al., 2003; Pinder et al., 2004; Rumburg et al., 2008a; Teye y Hautala, 2008).

1.4.1.4. Emisiones de NH3 Procedentes de la Aplicación del Purín en Praderas

Las emisiones de NH3 ligadas a la aplicación de purines de vacuno lechero en praderas han sido estudiadas por numerosos equipos de investigación durante las últimas

décadas. El amplio estudio de tales emisiones en la UE podría estar justificado con que el 10% de las emisiones totales de NH3 proceden de la aplicación de las deyecciones

ganaderas en campo (ECETOC, 1994). De acuerdo con esta preocupación ambiental, diversos inventarios nacionales han destacado la importancia de la aplicación de los


48


purines en praderas como la fuente principal de emisión de NH3 en el sector lechero. Misselbrook et al. (2000) estimaron para el Reino Unido que la emisión total de NH3

procedente de las aplicaciones de purines de vacuno de leche en pradera alcanzaban 45 kt N-NH3 año-1 en 1997, englobando el 38,2% de las emisiones de NH3 del sector a

nivel nacional. Hutchings et al. (2001) estimaron para el caso danés que las emisiones procedentes de la aplicación de purines de vacuno alcanzaban 11,8 kt N-NH3 año-1

(42,1% de las emisiones del sector). En Irlanda, Hyde et al. (2003) estimaron que la emisión de NH3 procedente de las aplicaciones de los purines de vacuno de leche en

praderas alcanzaba 27,0 kt N-NH3 año-1 en 1990 (38,7% de las emisiones del sector), prediciendo unas emisiones de 26,2 kt N-NH3 año-1 para 2010 (29,1% de las emisiones

del sector). Más recientemente Reidy et al. (2008) han publicado para el sector vacuno lechero suizo que las emisiones de NH3 ligadas a la aplicación de purines alcanzaban

27,6 kg N-NH3 año-1 vaca-1, representando éste el 69,0% de las emisiones del sector. Havlikova et al. (2008) determinaron en la República Checa que las emisiones de NH3

debido a la aplicación de purines de vacuno de leche en campo alcanzaban entorno a 6,0 kg N-NH3 año-1 vaca-1 (20,7% de las emisiones del sector). En concordancia con estos

inventarios nacionales, varios modelos desarrollados en diversos países de la UE con el propósito de estimar las emisiones de NH3 procedentes del sector de vacuno de leche y

proponer medidas para su mitigación (DYNAMO en Suiza, NARSES en el Reino Unido, GAS-EM en Alemania, MAM y FARMMIN en Holanda), estimaron que los

factores de emisión procedentes de la aplicación de purines en campo pueden representar entre el 43% (NARSES) y el 68% (MAM) de la emisión global ligada las

explotaciones de vacuno de leche (Reidy et al., 2008).

Las emisiones de NH3 procedentes de la aplicación de purines en campo (praderas o

cultivos) son variables, de modo que dichas emisiones pueden oscilar entre 0 y 60% del N-NH4

+ aplicado (Sommer et al., 2003). Esta variación en el factor de emisión puede

oscilar en función de las características físico-químicas tanto del purín aplicado como del suelo receptor, las condiciones meteorológicas en la zona de aplicación y finalmente

del área y tiempo de aplicación de los purines (Sommer y Hutchings, 2001).


49

INTRODUCCIÓN

1.4.2. Emisiones de Óxidos de Nitrógeno en Explotaciones de Vacuno de Leche

La contaminación ambiental por la emisión de los óxidos de N procedentes de la actividad lechera hace referencia a la emisión de N2O y NO. Las emisiones de N2O se

relacionan con los procesos de calentamiento global (su poder de calentamiento es 320 veces superior a la capacidad calorífica del CO2) y la destrucción de la capa de ozono

(Crutzen, 1970). La presencia de NO contribuye a la formación fotoquímica del ozono troposférico (Crutzen, 1979), un gas altamente contaminante para la salud humana

(Staffelbach et al., 1997) y en presencia del radical OH- o el O3, la presencia de NO puede contribuir a la producción de ácido nítrico (HNO3), dando lugar a deposiciones

ácidas (Logan, 1983).

El estudio bibliográfico de las emisiones de N2O y NO ligadas a la producción lechera muestra que el N2O ha sido estudiado en mayor profundidad que el NO, del que se

desconocen todavía muchos aspectos y del que apenas se tienen referencias bibliográficas. A este respecto, históricamente ha existido la duda sobre si el NO es un

verdadero intermediario o un subproducto de los procesos de nitrificación y denitrificación (Amundson y Davidson, 1990), aunque actualmente se cree que es un

intermediario obligatorio. En relación a las emisiones de N2O procedentes de la actividad lechera, Velthof et al. (1998) identificaron 11 fuentes directas de emisión de

N2O. La contribución de cada una de las fuentes a la emisión de N2O varia en función del modelo productivo desarrollado en la explotación, aunque la interacción entre el

suelo (pasto)-fertilizante (orgánico o inorgánico) y la acumulación de purines en las fosas son considerados como las principales fuentes de emisión a la atmósfera (Velthof et al., 1998; Chadwick et al., 1999).

1.4.2.1. Formación de N2O y NO en las Explotaciones de Vacuno de Leche

El proceso de formación de los óxidos de N (N2O y NO) es regulado por una serie de

reacciones bioquímicas denominadas nitrificación y desnitrificación (Firestone y Davidson, 1989). El proceso de la nitrificación consiste en la transformación bioquímica

del ión NH4+ en ión NO3

- en condiciones aeróbicas. Por el contrario, la desnitrificación consiste en un proceso anaerobio por el que el ión NO3

- es reducido hasta una molécula


50


final de N2. La descomposición de la urea urinaria debido a las enzimas de actividad ureasa es el origen del NH4

+ necesario para los posteriores procesos de nitrificación y

desnitrificación, responsables de las emisiones de N2O y NO a la atmósfera. Junto con los principales procesos de nitrificación y desnitrificación, la desnitrificación de los

nitrificantes ha sido también propuesta como una tercera fuente de síntesis de N2O y NO (Poth y Focht, 1985; Wrage et al., 2001).

La nitrificación es un proceso aeróbico desarrollado en 2 pasos y llevado a cabo por

microorganismos tanto autótrofos como heterótrofos (Haynes, 1986). Los nitrificantes primarios (géneros Nitrosomonas, Nitrosospira, Nitrosolobus, Nitrosovibrios y

Nitrosococcus), que se hallan tanto en las fosas de almacenamiento de purines como en los suelos de praderas o cultivos, completan el primer paso de la nitrificación con la

oxidación del ión NH4+ al ión intermediario nitrito (NO2

-). El segundo paso de la nitrificación, regulado por los nitrificantes secundarios (género Nitrobacter), completa

la oxidación del ión NO2- al ión NO3

-. El proceso de la nitrificación es un proceso relativamente constante en los diferentes ecosistemas, y se halla regulado por la

disponibilidad del ión NH4+ y la molécula de O2 (Firestone y Davidson, 1989).

NO NO NO

NH4+ + O2 + 2H+ NH2OH HNO NO2

- NO3-

N2O

Los microorganismos desnitrificantes son fundamentalmente heterótrofos y anaerobios

facultativos que utilizan el ión NO3- o el ión NO2

- como aceptores finales de electrones en condiciones de anaerobiosis (condiciones que se cumplen tanto en fosas como en

praderas). La función de intermediario del N2O en la cadena de reacciones del proceso de desnitrificación fue contrastada por Firestone et al. (1979), siendo la consideración

del NO como intermediario necesario motivo de discusión durante años. A este respecto, un trabajo de investigación de Amundson y Davidson. (1990) en la década de

los años 90 comprobó la necesidad de NO en la reducción del ión NO3- a la molécula de

N2. La producción de los óxidos de N durante los procesos de desnitrificación se halla

regulada por el contenido hídrico de los suelos, la presión parcial de O2, la


51

INTRODUCCIÓN

disponibilidad de carbono orgánico, la disponibilidad del ión NO3- y la temperatura

atmosférica (Bouwman et al., 1993).

NO3- NO2

- NO N2O N2

1.4.2.2. Contribución del Sector Vacuno Lechero a las Emisiones de Óxidos de N.

La concentración de los óxidos de N (N2O y NO) en la atmósfera muestra una evolución al alza, iniciada con el impulso de la industrialización del siglo XVIII y continuada por

las actividades económicas humanas hasta el presente siglo XXI. Según datos emitidos por el IPCC (2001), la concentración de N2O en la troposfera aumentó desde los 270

ppb en el año 1750 hasta los 314 ppb del año 2001. De la misma manera, la acumulación de NO en la atmósfera ha aumentado (Lee et al., 1997). Las actividades

antropogénicas son las principales fuentes de las emisiones de N2O y NO a la atmósfera (Isermann, 1994; Lee et al., 1997), siendo el sector agro-ganadero, junto con la

producción industrial y el sector energético los principales sectores vinculados a las emisiones de N2O y NO a la atmósfera. Según datos emitidos por la Agencia Europea

de Medio Ambiente para el N2O, la contribución del sector primario de la UE-15 alcanzaba en su conjunto aproximadamente entre un 45% y un 65% de las emisiones

globales de N2O (emisiones directas e indirectas) en 2004. Entre las diferentes fuentes clasificadas destacaron las emisiones procedentes de los suelos (31%), de la producción

animal (8%) y de los sistemas de almacenamiento de deyecciones (6%). En relación a la emisión de NO, la bibliografía existente sitúa a los combustibles fósiles (carbón,

petróleo) como la principal fuente de la emisión de NOx (NO + NO2) a la atmósfera con 20 millones de toneladas anuales, mientras la aportación del sector agro-ganadero alcanzaba las 11 millones de toneladas anuales (Isermann, 1994).

La producción animal es considerada como una importante fuente de óxidos de N a la

atmósfera, en especial las emisiones de N2O. Dentro de los diferentes sectores ligados a la producción animal, la producción del ganado rumiante es considerada como la

principal fuente de emisión de N2O. Freibauer (2003) estimó que la producción animal rumiante era la responsable del 55% de las emisiones de N2O procedentes del sector

primario europeo. Dentro de la producción de rumiantes, el ganado vacuno es el principal contribuyente a las emisiones de N2O, siendo el sector vacuno lechero la


52


principal fuente de emisión de N2O (Velthof et al., 1998). A pesar de que existen pocos inventarios nacionales en relación a la contribución de la producción lechera a las

emisiones de N2O, Velthof y Oenema (1997) estimaron que el 35% de las emisiones nacionales de Holanda procedían del sector vacuno lechero (13,7±5,1 Gg N anuales), lo

cual suponía entre un 3,2 y un 4,6% del surplus de N de las explotaciones lecheras. Velthof et al. (1998) identificaron 11 fuentes directas y 3 fuentes indirectas de emisión

de N2O ligadas a la producción del vacuno lechero en régimen intensivo (Tabla 1.7). Cabe reseñar la importante contribución de las emisiones de N2O procedente de los

suelos, en especial de las aplicaciones de purines sobre praderas.

Tabla 1.7. Fuentes y factores de emisión de N2O en explotaciones de vacuno lechero.

Fuente de emisión de N2O Media (Desv.Est)

Directa

Emisión del suelo (background) (g N-N2O ha-1 año-1) 900 ± 300 Fertilizante N en suelo (g N-N2O kg-1 N fert) 10 ± 5

Purín aplicado en suelo (g N-N2O kg-1N purín)

Aplicación superficial 3 ± 3

Inyección de purín 5 ± 5

Emisión del suelo pastado (g N-N2O kg-1N excretado) 25 ± 15

Fijación biológica de N (g N-N2O kg-1N fijado) 5 ± 5

Fosa de purines (g N-N2O kg-1N purín) 0,05 ± 0,05

Ganado rumiante (g N-N2O kg-1N ingerido) 0,05 ± 0,05

Ensilados (g N-N2O kg-1N-NO3-) 15 ± 10

Uso de energía (g N-N2O GJ-1) 1 ± 1

Lixiviación de N (g N-N2O kg-1N lixiviado) 25 ± 25

Volatilización de NH3 (g N-N2O kg-1N-NH3) 5 ± 5

Indirecta Fertilizante (N) comprado (g N-N2O kg-1 N fert) 5 ± 5

Forrajes comprados (g N-N2O kg-1 N forraje) 20 ± 10

Concentrados comprados (g N-N2O kg-1 N conc) 10 ± 5


53

INTRODUCCIÓN

Por el contrario, escasas referencias bibliográficas recogen la contribución de las explotaciones lecheras a las emisiones de NO, hallándose unas pocas referencias sobre

la contribución de los purines a las emisiones de NO cuando son aplicados en praderas (Davidson, 1991; Yamulki et al., 1997; Pinto et al., 2004; del Prado et al., 2006;

Menéndez et al., 2008; Sánchez-Martín et al., 2008; Arriaga et al., 2010).

1.4.2.3. Emisiones de Óxidos de N en Establos y el Sistema de Almacenamiento de

Purines

La contribución de las granjas (establos y sistemas de almacenamiento de purines) a la

emisión de N2O ha sido estudiada por diferentes equipos científicos desde finales de la década de los 90 (Velthof y Oenema, 1997; Velthof et al., 1998; Chadwick et al., 1999;

Amon et al., 2001; Jungbluth et al., 2001; Külling et al., 2001; Külling et al., 2003; Amon et al., 2006; Monteny et al., 2006; Weiske et al., 2006; Gac et al, 2007;

Havlikova et al., 2008). Por el contrario, las referencias a la emisión de NO en granjas lecheras es nula. Al contrario de las emisiones de NH3, donde tanto el establo como las

fosas de purines son importantes fuentes de emisión, la emisión de N2O procede básicamente de las fosas de purines. La menor bibliografía existente entorno a las

emisiones de N2O en la zona de estabulación se debe a la dificultad de determinar el flujo de ventilación natural y la reducida acumulación de N2O en los establos, siendo en

ocasiones la concentración de N2O atmosférico superior a la concentración en el establo (Jungbluth et al., 2001). Una revisión bibliográfica de las emisiones de N2O medidas en

los establos de vacuno lechero aporta los siguientes factores de emisión (Tabla 1.8):

Tabla 1.8. Factores de emisión de N2O en diferentes sistemas de estabulación

Estabulación Factor de Emisión N2O

(g vaca-1 día-1)

Notas Referencia

Estabulación Fija 0,14-1,19 Mínimo-máximo Amon et al., 2001

Estabulación fija 0,61 Media anual Amon et al., 2001

Cama caliente 2,01 Datos verano Amon et al., 2001

Estabulación libre 0,8 Salida a pasto Sneath et al., 1997

Estabulación libre 1.6 Media anual Jungbluth et al., 2001


54


Las fosas de almacenamiento de purines se consideran como la segunda fuente de emisión de N2O en las explotaciones de vacuno lechero tras las emisiones procedentes

de la aplicación fertilizantes en campo (Chadwick et al., 1999, Monteny et al., 2006). Las fosas de purines son depósitos estrictamente anaerobios, condiciones que inhiben

los procesos de nitrificación o desnitrificación responsables de la síntesis de N2O. Sin embargo, son habitualmente sometidas a una aireación forzada o una dilución con agua

con el objetivo de optimizar el manejo y la extracción de los purines. Dichas maniobras contribuyen de manera indirecta a la creación de una atmósfera apta para los

microorganismos desnitrificantes (anaerobios facultativos), los cuales dan lugar a los procesos de desnitrificación que emiten el N2O a la atmósfera (Monteny et al., 2006).

La contribución de las fosas a las emisiones N2O presenta una gran variabilidad (Sommer et al., 2000; Monteny et al., 2001; Külling et al., 2003; Amon et al., 2006),

motivo por el cual la determinación de las emisiones de N2O en las fosas de purines requiere un método que sea continuado en el tiempo y que disminuya la heterogeneidad

de medida. La capacidad de emisión de N2O durante la fase de almacenamiento puede variar en función de parámetros como el contenido en N y C del purín, el periodo de

duración del almacenamiento y el tipo de tratamiento sometido (Amon et al., 2006).

1.4.2.4. Emisiones de Óxidos de N Procedentes de la Aplicación del Purín en Praderas

La aplicación de los purines en praderas es considerada como la principal fuente de

emisión de óxidos de N (principalmente de N2O) a la atmósfera, motivo por el que muchos equipos investigadores han procedido a su estudio desde los años 90 (Estavillo

et al., 1994; Velthof et al., 1996; McTaggart et al., 1997; Velthof et al., 1998; Merino et

al., 2001a,b; Machefert et al., 2002; Merino et al., 2002; Sozanska et al., 2002; Merino et al., 2005; Menéndez et al., 2006; Cárdenas et al., 2007; Flechard et al., 2007; Jones et

al., 2007; Soussana et al., 2007; Sánchez-Martín et al., 2008; Arriaga et al., 2009). Las emisiones de N2O procedentes de las praderas oscilan entre 100 y 1.000 kg C-CO2

equiv ha-1 anuales (Machefert et al., 2002; Sozanska et al., 2002), con una emisión media anual de 2,0 kg N-N2O ha-1 (Freibauer, 2003). De este modo, se estiman unas

emisiones de 160 kt N2O anuales procedentes de las praderas de la UE, según los 80 millones de hectáreas de praderas censadas en la UE-25 (EEA, 2005).


55

INTRODUCCIÓN

Los mecanismos responsables para la emisión de N2O y NO en praderas son de orden microbiológico y químico (Bremner et al., 1980). Mientras que la síntesis de N2O se

debe principalmente a procesos estrictamente microbiológicos, la síntesis de NO puede llevarse a cabo también mediante reacciones químicas (van Cleemput y Baert, 1984).

Entre los procesos microbiológicos, la nitrificación aeróbica autótrofa (oxidación del NH4

+ a NO3-) y la desnitrificación anaeróbica heterótrofa (reducción anaeróbica del

NO3- a N gaseoso) son consideradas como las responsables de la síntesis de N2O y NO

(Davidson, 1991). Ambos procesos son controlados por la disponibilidad de N mineral,

la temperatura y humedad del suelo, la textura del suelo y el C orgánico acumulado en las praderas (Granli y Bøckman, 1994; Jones et al., 2007). El tipo de N aplicado

mediante los purines o los fertilizantes inorgánicos (NH4+, NO3

- o N orgánico) sobre las praderas favorecerán la nitrificación o la denitrificación en cada caso, donde suelos con

baja humedad y textura gruesa favorecerán la nitrificación y suelos con elevada humedad y textura fina contribuirán a procesos desnitrificantes. La presencia de C

orgánico rápidamente oxidable procedente de la aplicación de purines favorecerá la síntesis de N2O mediante desnitrificación. De manera general se considera que las

praderas ligadas a las explotaciones de vacuno de leche presentan un alto contenido en C por la frecuente aplicación de purines así como que se localizan en áreas de elevada

humedad de modo que en principio se considera que son condiciones ideales para la desnitrificación (Granli and Bøckman, 1994). De cualquier manera, dado que pueden

coexistir microespacios aeróbicos como anaeróbicos en los suelos, ambos procesos pueden ocurrir simultáneamente (Kuenen y Robertson, 1994). El patrón de emisión de

N2O difiere según se apliquen fertilizantes orgánicos o inorgánicos sobre praderas (Jones et al., 2007). Mientras que la aplicación de fertilizantes inorgánicos produce emisiones de N2O de menor duración en el tiempo, la aplicación de purines produce

mayores emisiones, y de mayor duración, debido a que en muchos casos se observan efectos residuales de aplicaciones anteriores. Además, la adición de fuentes de C

aumenta las pérdidas por desnitrificación y también se produce la mineralización del N orgánico (Jones et al., 2007). Los factores de emisión de N2O para los fertilizantes

inorgánicos se sitúan entre 0,1% y 1,4 % (Slemr y Seiler, 1984) mientras que pueden alcanzar valores superiores a 4,3% tras la aplicación de purines (Jones et al., 2007).


56


1.5. Visión General de las Estrategias de Minimización de las Emisiones de N en Explotaciones de Vacuno Lechero

La adopción del Protocolo de Gotemburgo (UNECE, 1999) para luchar contra la

acidificación, la eutrofización y el ozono troposférico y el Protocolo de Kyoto (UNFCCC, 1997) para disminuir las emisiones de gases de efecto invernadero han

obligado a los estados firmantes a reducir las emisiones de NH3, N2O y NO. La firma del Protocolo de Gotemburgo obligaba a los 29 estados europeos firmantes a la

reducción del 12% de las emisiones de NH3 entre 1990 y 2010 (Erisman et al., 2003). Con respecto a la adopción del Protocolo de Kyoto, en él se acordó reducir las

emisiones de gases invernadero a nivel comunitario en un 8% para el período 2008-2012, tomando de base las emisiones de 1990. Con el objetivo de cumplir a nivel estatal

y regional los Protocolos de Gotemburgo y Kyoto, así como para incorporar los aspectos medioambientales de la PAC a nivel de explotación (ecocondicionalidad y

medidas agroambientales), se han propuesto en los últimos años diversas estrategias de minimización de NH3, N2O y NO ligadas al sector vacuno lechero. Estas estrategias

incluyen medidas de optimización de la eficiencia de uso de N tanto a nivel de animal como de planta, la gestión de los animales estabulados o en pastoreo y los purines

acumulados en fosas, el diseño de naves y fosas que permitan reducir las emisiones o la aplicación de diversas tecnologías desarrolladas para tal efecto. En la Tabla 1.9 y la

Tabla 1.10 se recogen las principales estrategias descritas para la minimización de las emisiones de NH3 y los óxidos de N, respectivamente, en las explotaciones de vacuno

lechero.

La aplicación de ciertas estrategias (la reducción de la proteína de la ración, el ajuste de

la tasa de aplicación de N en campo, etc) puede provocar interacciones positivas entre los diferentes gases, de modo que se minimicen tanto las emisiones de NH3 como de los

óxidos de N. En este sentido, Brink et al. (2001) observaron que la minimización de las emisiones de NH3 implicaba la reducción de las emisiones de N2O en un 15% tras

aplicar estrategias de minimización de la emisión de NH3 en granja y en la aplicación de purines en campo. Sin embargo, otras estrategias provocan efectos antagónicos entre los

gases. Por ejemplo, la mezcla de paja de cereal con el purín para disminuir la emisión de NH3 aumenta las emisiones de N2O (Chambers et al., 2002). Por tanto es importante

considerar la reducción de la emisión desde un punto de vista integral.


57

INTRODUCCIÓN

Tabla 1.9. Estrategias de minimización de NH3 en explotaciones de vacuno de leche.

Estrategia Ámbito de la explotación Referencias

Reducción de la excreción de N animal por ajuste de la cantidad y calidad de CP de la ración

Estabulación

Nennich et al., 2005 Yan et al.. 2006 Merino et al., 2008

Manejo del ganado (tipo de estabulación, horas de estabulación, etc) Estabulación

Swensson y Gustafsson, 2002 Gilhespy et al., 2006

Diseño de patios (tipo de suelo, emparrillado, pendientes, etc)

Estabulación Braam et al., 1997a,b Monteny y Erisman, 1998

Manejo de las deyecciones (arrobaderas, manguera de agua, tiempo de acumulación, etc)

Estabulación Kroodsma et al., 1993 Lachance et al., 2005

Uso de aditivos en deyecciones (enzimas, ácidos)

Estabulación Monteny y Erisman, 1998 Martinec et al., 2001

Lavadores de gases Estabulación Melse y Ogink, 2005

Impermeabilización y encostramiento natural Fosa de purín Misselbrook et al., 2005

Webb et al., 2005 Uso de aditivos en fosa (enzimas, ácidos, etc) Fosa de purín Kai et al., 2008

van der Stelt et al., 2007

Incremento de C/N en purín Fosa de purín Misselbrook et al., 2005

Precipitación química Fosa de purín Arogo et al., 2006

Nitrificación biológica Fosa de purín Arogo et al., 2006

Preparación del suelo Purín en campo Sommer y Hutchings, 2001

Menor viscosidad purín Purín en campo Sommer y Hutchings, 2001

Tasa de aplicación de purín Purín en campo Sommer y Hutchings, 2001

Inyección de purín Purín en campo Sommer y Hutchings, 2001

Uso de aditivos (ácidos, inhibidores ureasa,) Purín en campo Jarvis y Pain, 1990

Merino et al., 2002


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Tabla 1.10. Estrategias de minimización de N2O en explotaciones de vacuno de leche.

Estrategia Ámbito de la explotación Referencias

Reducción de la excreción de N animal por ajuste de la cantidad de CP de la ración

Estabulación Külling et al., 2001 Cárdenas et al., 2007 Arriaga et al., 2009

Impermeabilización y encostramiento natural Fosa de purín Misselbrook et al., 2005

Webb et al., 2005

Digestión anaeróbica de purines Fosa de purín Amon et al., 2006 Weiske et al., 2006

Purines líquidos Fosa de purín Groenestein y van Faassen, 1996

Laboreo del suelo Purín en campo Pinto et al., 2004

Uso fertilización amoniacal Purín en campo Dobbie y Smith, 2003

Dosis de plicación de purín Purín en campo Velthof et al., 1998 Weiske et al., 2006

Uso de inhibidores nitrificación Purín en campo Merino et al., 2001a,b Merino et al., 2005 Menéndez et al., 2006

A pesar de las numerosas estrategias existentes para la reducción de las emisiones de gases de N en las explotaciones de vacuno de leche, se considera que la reducción de la

excreción de N de origen animal mediante la manipulación de las raciones es la estrategia más rentable tanto ambiental como económicamente para disminuir las

emisiones de N (Rotz, 2004). La reducción de la excreción de N conlleva por una parte la extracción de una mayor cantidad de N en forma de producto (leche, carne) y por otro parte conlleva la reducción de la acumulación de N en los establos y las fosas de

purines, permitiendo una gestión más fácil de los purines acumulados.

1.6. Nutrición del Ganado Vacuno Lechero como Estrategia de Minimización de las Emisiones de los Gases de N.

La eficacia de la manipulación de las raciones en la reducción de la emisión de gases de

N en vacuno lechero, principalmente para el NH3, ha sido descrita por diferentes grupos de investigación. El efecto de la manipulación proteica sobre las emisiones de NH3 ha


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sido estudiada a nivel de granja (Frank et al., 2002; Frank y Swensson, 2002; Smits et

al., 2003; van Duinkerken et al., 2005; Powell et al., 2008), en las fosas de

almacenamiento de purín (Külling et al., 2001; Külling et al., 2003), tras la aplicación de los purines en campo (Reijs et al., 2007; Merino et al., 2008) mediante incubaciones

en el laboratorio (Paul et al., 1998; James et al., 1999; Misselbrook et al., 2005; van der Stelt et al., 2007) o a través de la elaboración de modelos (de Boer et al., 2002). Las

publicaciones que relacionan la nutrición del ganado lechero con las emisiones de N2O a la atmósfera son menos numerosas (Külling et al., 2001; Külling et al., 2003;

Cárdenas et al., 2007).

La reducción de las emisiones de N a la atmósfera (NH3, N2O o NO), implica el control de la excreción de N animal, la reducción de la pérdida de N durante el almacenamiento

de los purines y el control de las pérdidas de N durante las aplicaciones de los purines y de los fertilizantes minerales en campo (van Horn et al., 1996). A pesar de las diferentes

estrategias existentes (detalladas anteriormente), se considera que la minimización de las pérdidas de N en todos aquellos procesos englobados en la producción láctea se debe

iniciar con el control de la excreción de N en la cabaña ganadera (especialmente en vacas de producción) mediante la optimización de la eficiencia de uso del N en leche

(NUE) (Rotz, 2004).

NUE = (Producción de leche, kg d-1 x Proteína Lactea, %) x 100 N ingerido, g d-1

La NUE puede ser optimizada mediante el ajuste del contenido proteico de las raciones y el ajuste de su calidad proteica a los requerimientos animales, así como mediante el

aumento del rendimiento productivo de la cabaña (Jonker et al., 2002; Rotz, 2004; Huhtanen y Hristov, 2009). No obstante, se considera que el control sobre la excreción

de N resulta ser más eficiente mediante el ajuste de la proteína de la ración y la mejora de la calidad de la misma en comparación con la estrategia de mejorar el rendimiento

lácteo de la cabaña (Rotz, 2004; Huhtanen y Hristov, 2009). Junto con la manipulación de la ración, otras estrategias nutricionales y de manejo animal como la agrupación de la

cabaña en diferentes lotes de alimentación en función de la edad o la fase de lactación (St-Pierre y Thraen, 1999), o la reformulación de las raciones pueden contribuir también


60


a la minimización de la excreción de N mediante la optimización de su uso (Jonker et

al., 2002).

1.6.1. Ajuste de la Proteína de la Ración en el Ganado Vacuno de Leche

1.6.1.1. Requerimientos Proteicos en Vacuno de Leche. Proteína Metabolizable

Numerosos trabajos han demostrado que una nutrición proteica precisa del ganado vacuno lechero implica una mejora en la eficiencia de uso de N sin que ello repercuta en

una merma de de la producción de leche (Huhtanen y Hristov, 2009). Históricamente, tanto la falta de incentivos económicos que permitieran incrementar la NUE como la

sobrealimentación proteica que evitara la pérdida de la capacidad productiva del ganado, han provocado que las explotaciones de vacuno de leche hayan alimentado su

cabaña ganadera por encima de sus necesidades (vandeHaar y St-Pierre, 2006). Sin embargo, en los últimos 30 años se ha producido un importante avance en el desarrollo

de modelos predictivos de los requerimientos proteicos de las diferentes cabañas ganaderas. Los diferentes modelos desarrollados (AFRC en Reino Unido, INRA en

Francia, GER en Alemania, NRC en Estados Unidos, CNCPS en Estados Unidos) han establecido los requerimientos proteicos del ganado lechero a partir del concepto de

proteína metabolizable. La proteína metabolizable se define como la proteína verdadera que es digerida tras su paso por el rumen, y cuyos aminoácidos son absorbidos en el

intestino delgado para garantizar las funciones básicas animales de mantenimiento, crecimiento, reproducción y lactación.

Debido a la fisiología digestiva de los rumiantes, el cumplimiento de los requerimientos proteicos en vacuno de leche implica la necesidad de satisfacer los requerimientos de N

tanto en el rumen (síntesis de proteína microbiana) como en el intestino delgado (absorción de aminoácidos). El incumplimiento de estos requerimientos proteicos

implica problemas en el desarrollo y producción animal y por tanto una reducción en la productividad de la explotación. El déficit de proteína en rumen se relaciona con efectos

como la minimización de la síntesis de proteína microbiana, la reducción en la ingestión de alimentos, una disminución en la ganancia de peso vivo en el ganado y la pérdida de

producción lechera. Una baja absorción intestinal de aminoácidos puede ocasionar la


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reducción del aumento de peso en el ganado, la disminución de la producción lechera y la falta de eficiencia reproductiva. También la sobrealimentación proteica del ganado

lechero provoca trastornos en la fertilidad de la cabaña, pérdidas de peso vivo y producción de leche o la disminución de la calidad de la leche. Por tanto, el uso

eficiente de N es uno de los pilares básicos para el desarrollo de sistemas productivos agro-ganaderos sostenibles, puesto que un uso ineficiente de ellos no solo supone un

riesgo potencial para el medio ambiente sino que puede afectar también al desarrollo económico del sistema productivo (Oenema y Pietrzak, 2002; Nevens et al., 2006).

1.6.1.2. Proteína de la Ración, Eficiencia NUE y Excreción de N

La histórica sobrealimentación proteica ha provocado que los ganaderos hayan

alimentado su cabaña ganadera en lactación con no menos de un 18% de PB (vandeHaar y St-Pierre, 2006). Sin embargo, los avances realizados en la nutrición del vacuno

lechero han permitido rebajar el consumo medio de PB en las raciones. La importancia del ajuste del PB de la ración se debe a que la ingestión de N ha sido descrita como la

variable que mejor predice la pérdida de N al medio en el ganado vacuno lechero (Yan et al., 2006). El aumento de la PB de la ración se relaciona con el aumento de la

producción lechera (Wu y Satter, 2000) (Figura 1.4) aunque su relación no es lineal a partir de un umbral de ingestión proteica.

Figura 1.4. Contenidos de PB y producción de leche según la fase de lactación (Fuente:

Wu y Satter, 2000).


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Así, Wu y Satter (2000) demostraron que raciones con concentraciones de PB entre 16,0% y 17,5% podían garantizar altas producciones de leche (11.000 kg año-1 vaca-1).

Broderick (2003) constató que la producción láctea puede no verse comprometida en vacas de alta producción (34,7 kg día-1) con niveles de PB en la ración de 16,7% (593 g

N vaca-1 día-1). Olmos-Colmenero et al. (2006) confirmaron que raciones de 605 g N vaca-1 día-1 (16,5% PB) alcanzaban la máxima producción láctea (38,3 kg vaca-1 día-1)

en relación a las raciones con una concentración proteica superior e Ipharraguerre et al. (2005) concluyeron que la ingestión de N diario podría reducirse a 600-650 g N vaca-1

día-1 (16,8% PB) siempre que la ración aporte la suficiente energía y se halle balanceada en cuanto a la calidad proteica. Sin embargo, cuando el consumo de PB no se traduce en

una mayor producción de leche, se produce una pérdida en la eficiencia de uso del N en leche (NUE) (Kalscheur et al., 2006) (Figura 1.5), aumentando de esta manera la

excreción de N al medio.

Figura 1.5. Relación entre la ingestión de PB y la eficiencia NUE (Fuente: Kalscheur et al.,

2006. 6,8% PDR; 8,2% PDR; 9,6% PDR; x11,0% PDR).

Tamminga (1992) describió valores de NUE de entorno a 20% para el ganado vacuno

frisón holandés. Posteriormente se han descrito eficiencias de uso superiores (25%-28%) en explotaciones de ganado vacuno de leche de raza Holstein (Castillo et al.,

2001a,b; Jonker et al., 2002; Yan et al., 2006; Arriaga et al., 2009). De acuerdo con


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estos datos, Yan et al. (2006) describieron la relación existente entre la ingestión y la excreción diaria de N con un coeficiente de correlación (r2) de 0,90, estimando que el

72,2% del N ingerido es excretado por el ganado vacuno lechero en lactación (Figura 1.6). Este porcentaje de excreción de N coincide además con trabajos publicados por

otros autores (Castillo et al., 2000; Nennich et al., 2005).

Figura 1.6. Relación entre la ingestión de N y excreción de N en ganado vacuno de leche (Fuente: Yan et al., 2006).

La causa de la baja eficiencia en el uso de la proteína de la ración y la elevada excreción

de N al medio es el bajo rendimiento del ciclo de N en el interior del rumen (Tamminga, 1992), donde hasta el 30% del N ingerido es perdido debido a la ineficiencia metabólica

del rumen.

1.6.1.3. Degradación de la Proteína, Síntesis de NH3 y Excreción de Urea

La eficiencia de uso del NH3 ruminal procedente de la degradación de la proteína resulta el factor principal en la determinación del impacto ambiental ligada a la producción de

leche (Hristov y Pfeffer, 2005). Los ingredientes empleados en la nutrición del ganado vacuno lechero contienen 2 formas nitrogenadas: la proteína verdadera o el N proteico

(albuminas, globulinas, glutelinas, prolaminas, colágeno, queratina, elastina, etc) y el N no proteico (N amoniacal, ácidos nucleicos, aminoácidos libres, péptidos de cadena

corta, amidas y aminas). La proteína verdadera se clasifica a su vez en 2 grupos en


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función de su comportamiento en el rumen: la proteína degradable (PDR) y la proteína no degradable en rumen (PNDR). En función del contenido de PB de la ración, la

concentración de proteína degradable (PDR), la tasa de degradación de la misma y la disponibilidad de energía de los microorganismos ruminales, variará la acumulación de

NH3 en el rumen. La Tabla 1.11 recoge el contenido en PB y su degradabilidad de los principales ingredientes empleados en la nutrición del ganado vacuno lechero.

Tabla 1.11 Principales ingredientes en vacuno de leche: PB y degradabilidad ruminal.

Forrajes Concentrados

Ingrediente PB,% Degr, % Ingrediente PB,% Degr, %

Alfalfa Deshidr 16,7 40 Cebada 11,3 75

Alfalfa Heno 13,4-20,8 65-80 Maíz 7,7 45 Paja Cereales 3,5 30 Trigo 11,2 78 Ensilado de Maíz 7,6-9,4 68 DDGS maíz 24,5 55

Heno de Hierba 9,4-20,5 60-80 Harina de Soja 44,0 63 Ensilado Hierba 10,7-18,2 65-80 Gluten Maíz 19,0 75

Ensilado Triticale 9,9-11,9 75 Gluten Maíz 60 60,0 30 Hierba Fresca 8,0-19,7 65-80 Tercerillas Trigo 14,9 78 Fuente: FEDNA, 2003.

El NH3 ruminal procede de la degradación de las proteínas y la posterior desaminación

de los aminoácidos en el rumen (Figura 1.7). Durante muchos años se asumió que la desaminación de los aminoácidos era llevado a cabo por algunas de las especies

bacterianas más importantes numéricamente en el rumen (Butyrivibrio fibrisolvens,

Megasphaera elsdenii, Prevotella ruminicola, Selenomonas ruminantium y

Streptococcus bovis) (Cotta y Russell, 1982). Sin embargo, se ha observado que estas bacterias poseen una actividad de desaminación relativamente baja, existiendo otro

grupo bacteriano numéricamente poco importante pero con una elevada actividad desaminadora como son las bacterias hiperproductoras de amoníaco (HAP) (Chen y

Russell, 1989). El NH3 acumulado en el rumen es parcialmente empleado por la flora microbiana ruminal para la síntesis de la proteína microbiana (proteína de alta calidad),

pero el NH3 excedentario resulta ser un compuesto químico tóxico para el organismo animal, el cual debe ser excretado al exterior. La mayor parte del NH3 excedentario es

transformado en una molécula no tóxica (urea) en el hígado y expulsado fuera del


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organismo por medio de la excreción urinaria. Bristow et al. (1992) confirmó que entre el 60 y 90% del N urinario era N en forma de urea. Por este motivo, se considera que la

manipulación del contenido en PB de la ración es una estrategia más eficaz en el control de la excreción de N urinario que en la reducción de la excreción fecal. El N fecal

procede del N alimentario no digerido, parte de la proteína microbiana no absorbida en el intestino y el N endógeno procedente de la actividad metabólica del animal (saliva,

células epiteliales, etc), factores menos manipulables desde la ración. Kebreab et al. (2001) observaron que a partir de un consumo diario de 400 g N vaca-1, la excreción

urinaria de N aumentaba exponencialmente en tanto que el crecimiento del N fecal seguía una tendencia lineal.

AGV NH3 + AGV NH3

Aminoácidos

Aminoácidos

Proteína Proteína Péptidos Ración Soluble

Proteína Microbiana

Extracelular Intracelular

Figura 1.7. Esquema de la degradación proteica en el rumen del vacuno de leche.

1.6.2. Ajuste de la Calidad de la Proteína en Ganado Vacuno Lechero

El ganado vacuno requiere de la absorción de aminoácidos para la producción de leche y la síntesis de proteína láctea, aminoácidos que son suministrados principalmente por

parte de la proteína microbiana sintetizada en el rumen y el paso de la PNDR. Las estrategias de minimización de la excreción de N desde la manipulación de la calidad

proteica de la ración incluyen la reducción de la ingestión de proteína PDR hasta los valores mínimos que permita mediante la sincronización de la energía la optimización


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de la síntesis de la proteína microbiana y la correcta suplementación con proteína PND o aminoácidos libres (lisina y metionina) (Børsting et al., 2003).

1.6.2.1. Síntesis de la Proteína Microbiana del Rumen

La proteína microbiana es la fuente principal de suministro de proteínas de alta calidad

al intestino delgado para su posterior incorporación a la leche y la proteína láctea, por lo que resulta fundamental su optimización desde la manipulación de las raciones con el

fin de mejorar la NUE en leche y reducir la excreción de N al medio. La eficiencia de la síntesis de proteína microbiana depende principalmente de dos factores nutricionales: la

disposición de N y carbohidratos en el rumen, y la correcta sincronización en la degradación de ambas (NRC, 2001). La manipulación de la ración y la relación entre la

cantidad de proteína ruminal y disposición de carbohidratos como estrategia de optimizar la síntesis de proteína microbiana han sido estudiados por numerosos grupos

de investigación (Herrera-Saldaña et al., 1990; Hoover y Stokes, 1991). En este sentido, algunos estudios han demostrado que la sincronización para una rápida fermentación de

almidón y proteína de rápida degradabilidad estimula la síntesis de proteína microbiana (Herrera-Saldaña et al., 1990) mientras que la falta de sincronización entre la

degradación de carbohidratos y proteína reduce la síntesis de proteína microbiana (Sinclair et al., 1993).

Además del aspecto productivo de la síntesis de la proteína microbiana (producción de

leche y contenido en proteína láctea), la optimización de la síntesis es fundamental desde el punto de vista medioambiental. El aumento de la síntesis de proteína conlleva la reducción de la excesiva acumulación de NH3 en el rumen. Diversos equipos

científicos han tratado de determinar la concentración de NH3 necesaria para garantizar una síntesis de proteína microbiana adecuada, y las evidencias sitúan entre 5 y 11 N-

NH3 mmol L-1 la concentración necesaria para maximizar los flujos de N microbiano desde el rumen (Hume et al., 1970). Sin embargo, los resultados también muestran que

las pérdidas de N a nivel ruminal comienzan a aumentar a partir del umbral situado en 5 N-NH3 mmol L-1 (Ahvenjärvi et al., 2002; Korhonen et al., 2002). De cualquier manera,

a pesar de los numerosos estudios llevados a cabo en relación a la sincronización de alimentos proteicos y energéticos, no han podido establecerse aún las combinaciones


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más precisas que garanticen una máxima producción de proteína microbiana. Los intentos para definir los requerimientos de N para alcanzar un óptimo crecimiento de la

población microbiana del rumen ha sido un continuo tema de debate y estudio. Ello se debe principalmente a la complejidad del metabolismo de N a nivel ruminal, las

diferencias metabólicas de N en las distintas especies de microorganismos que lo habitan, la constante incertidumbre de desconocer las especies microbianas presentes en

el rumen en función de la alimentación y el desconocimiento de las interacciones existentes entre las especies de microorganismos. Además, a pesar de que variando los

ingredientes de la ración se optimice y mejore la sincronización de la degradación de la proteína y la energía, no es posible llegar a discernir si el aumento de la síntesis de

proteína microbiana se debe a la propia sincronización o al hecho de variar los ingredientes y la relación que se establece entre ellos (Dewhurst et al., 2000). La

bibliografía resume que cuando la fermentación ruminal es normal, existe muy poca capacidad de mejora en la síntesis de proteína microbiana alterando las tasas de

degradabilidad de proteína y energía (NRC, 2001).

1.6.2.2. Suplementación con PNDR e Infusión Intestinal de Aminoácidos

A fin de garantizar la presencia de aminoácidos esenciales en la absorción intestinal, puede ser habitual el empleo de diversas fuentes de aminoácidos by-pass en la nutrición

del ganado vacuno lechero. Las estrategias más habituales son el uso de alimentos con alta concentración en PNDR (gluten meal, granos de destilería, DDGS de maíz o la

cebadilla) o la infusión de aminoácidos sintéticos, principalmente lisina y metionina (Schwab et al., 1992), en el intestino delgado (Johnson-vanWieringen et al., 2007). Aunque el empleo de proteínas by-pass ha sido relacionado con el aumento de la

producción lechera y la síntesis de la proteína láctea (Noftsger y St-Pierre, 2003), no todos los estudios avalan esta hipótesis. Santos et al. (1998) observaron a partir de

trabajos publicados a lo largo de 12 años que únicamente el 17% de los resultados mostraron una relación positiva entre la suplementación con PNDR y la producción de

leche. De igual manera, Bach y Stern (1999) mostraron la inexistencia de una relación significativa entre el nivel de PND de la ración, la producción de leche y el porcentaje

de proteína en leche. La falta de respuesta en la producción de leche se atribuye a la reducción de la síntesis de proteína microbiana, la falta de aminoácidos esenciales en la


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PNDR suministrada y la baja digestibilidad intestinal de los ingredientes ricos en PNDR (Santos et al., 1998). En relación a la falta de respuesta en el contenido proteico de la

leche, Bach y Calsamiglia (2002) atribuyeron tal déficit a un error de cálculo en el contenido de PNDR asignado a los ingredientes, al hecho de que la PDR de la ración

puede ser suficiente para cubrir las necesidades proteicas del animal, a una baja digestión intestinal de la PNDR y a que los aminoácidos aportados en la PNDR no

satisfagan los requerimientos animales. Como consecuencia de tal variabilidad en los resultados, la suplementación con PNDR tampoco ha mostrado una respuesta uniforme

como estrategia de mejora de la NUE. A pesar de que Børsting et al. (2003) señalaran la manipulación de la calidad proteica (incluidos el uso de PNDR y/o de lisina y metionina

sintéticas) como estrategias de minimización de la excreción de N, otro trabajos no mostraron una mejora de la NUE en leche debido al mayor uso de PNDR en las

raciones (Castillo et al., 2001b; Leonardi et al., 2003). Por motivo de la amplia casuística hallada en la bibliografía, los resultados obtenidos sugieren que el aumento de

PNDR no mejora de manera general la productividad del ganado lechero (Santos et al., 1998; Ipharraguerre y Clark, 2005).

1.6.2.3. Otras Estrategias Nutricionales

Como alternativa al tradicional manejo nutricional de la ración, existe el recurso de la

utilización de aditivos alimentarios para mejorar la eficiencia en la producción lechera y reducir las pérdidas de nutrientes, estimulando o inhibiendo el metabolismo energético

y/o nitrogenado. Hasta su prohibición en 2006 (1 de enero del 2006, Reglamento 1831/2003/CE), antibióticos como la monensina se han utilizado de manera generalizada en la nutrición del vacuno lechero como promotores del crecimiento.

Como resultado de la prohibición del uso de estos aditivos puede aumentar la incidencia de algunas patologías como la acidosis o el timpanismo, aumentar los costes de

producción y/o en la emisión de sustancias contaminantes al medio. A este último respecto, se ha estimado que la supresión del uso de estos promotores de crecimiento en

la alimentación del ganado porcino, vacuno y avícola en Alemania, Francia y el Reino Unido aumentará la emisión de N y P en 78.000 Tn anuales.


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Por tanto la producción ganadera se halla en la necesidad de buscar productos alternativos a los tradicionales promotores del crecimiento. Actualmente destacan como

sustancias alternativas los probióticos y prebióticos, los ácidos orgánicos, las enzimas, y los extractos vegetales. En los últimos años, se han prodigado mucho los estudios de los

extractos de plantas y los aceites esenciales como una de las alternativas más eficaces como alternativa a los antibióticos (Wallace et al., 2002; Cardozo et al., 2004; Busquet

et al., 2006; Calsamiglia et al., 2007). Los extractos de plantas (principalmente compuestos fenólicos y terpenoides) son compuestos naturales a los que se les atribuyen

propiedades antisépticas, antifúngicas, antioxidantes y antitumorales (Aureli et al., 1992; Youdim y Deans, 1999). Se considera que estos aditivos podrían mejorar la

eficiencia de utilización de los alimentos modificando la estructura de la población microbiana y, en consecuencia, el perfil de fermentación ruminal. En este sentido,

algunos estudios han mostrado su eficiencia en la reducción del NH3 ruminal (Busquet et al., 2006). De similar manera, se ha demostrado la efectividad de los aceites

esenciales como agentes antimicrobianos o antisépticos frente a bacterias patógenas. En rumiantes, su capacidad antimicrobiana parece actuar inhibiendo selectivamente a una

parte de la población microbiana ruminal resultando en una reducción de las pérdidas energéticas y/o proteicas a nivel ruminal (Calsamiglia et al., 2007).


2 OBJECTIVES


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The current PhD-thesis was planned based on the environmental pollution detected in high density dairy production areas from the Basque Country (northern Spain),

worsened by the increasing intensification of dairy farms, and all previous knowledge cumulated in NEIKER-Tecnalia on N gas emissions from dairy slurry applications on

grassland. As optimisation of animal feeding has been described as a key factor for the reduction of N pollution, this thesis was proposed with two main objectives:

1. To evaluate the feasibility of dietary strategies to reduce N accumulation in

commercial dairy farms from the Basque Country.

2. To study the effect of dietary manipulation on slurry characteristics and the subsequent NH3, N2O and NO gas accumulation in dairy barn floors and after

diet-derived slurry application on grassland.

The fulfillment of the first objective will add new and valuable information at regional level because nutrition had never been studied as a N reduction strategy on commercial

dairy farms. As N overfeeding was a common practice among farmers, the adoption of such strategy might let farmers, advisors and technicians improve N balance at farm

level. In addition to the environmental benefits, dietary manipulation might also contribute to improve the profitability of dairy farms.

In relation to the second objective, we consider that it brings a whole perspective of

what dairy farming is in terms of animal production (nutrition, ruminal processes, milk yield, milk N use) and environment (N excretion, slurry composition, NH3, N2O and NO emission). This study will join two scientific areas with a large literature

background each one (animal nutrition and environment), but in which literature references are fewer on the interaction of both areas.

The above mentioned two objectives were studied through the following experimental

objectives:

1. To describe N intake and N excretion level of lactating herds in commercial farms from the Basque Country, and to detect therefore nutritional strategies to

reduce N excretion. In addition, the relative weight of nutrition on herd N


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excretion per hectare was studied due to the increasing intensification of dairy farming in the region.

2. To study the effect of dietary manipulation (change of forage:concentrate ratio)

on lactating cow N balance, on chemical characteristics of diet-derived slurry and on NH3, N2O and NO emission after applying slurry on grassland.

3. To study the effect of dietary protein content modification on lactating cow N

balance and NH3 and N2O concentration from barn floors.


3 NUTRITIONAL AND MANAGEMENT STRATEGIES ON NITROGEN AND PHOSPHORUS USE EFFICIENCY OF LACTATING DAIRY CATTLE ON COMMERCIAL FARMS: AN ENVIRONMENTAL PERSPECTIVE


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Nutritional and Management Strategies on Nitrogen and Phosphorus Use Efficiency of Lactating Dairy Cattle on Commercial Farms: An Environmental Perspective

Abstract

Dairy farm activities contribute to environmental pollution through the surplus nitrogen (N) and phosphorus (P) that they produce. Optimization of animal feeding and

management has been described as a key strategy for reducing N and P excretion in manure. Sixty four commercial dairy farms were studied in order to assess the

efficiency of N and P use in lactating herds and to identify dietary and management factors that may contribute to improving the efficiency of nutrient use for milk

production, and reduce N and P excretion. The average ration was formulated to 50:50 forage:concentrate ratio with grass silage and corn silage as the main forage sources.

Mean N and P intakes were 562 g d-1 (16.4 CP%) and 84.8 g d-1 (0.40 P%), respectively. Milk yield averaged 29.7 kg d-1 and contributed to 25.8% (SD = 2.9) of N

utilization efficiency (NUE) and 31.9% (SD = 4.5) of P utilization efficiency (PUE). Dietary N manipulation through fitting the intake of crude protein (CP) to animal

requirements showed a better response in terms of reducing N excretion (R2 = 0.70) than that estimated for P nutrition and excretion (R2 = 0.30). Improvement in NUE

helped increase PUE, despite the widespread use of feedstuffs with a high P content. Management strategies for lactating herds, such as the use of different feeding groups,

periodical ration reformulation and selection of feeding system did not show any consistent response in terms of improved NUE and PUE. The optimization of NUE and PUE contributed to reducing the N and P excretion per unit of milk produced, and

therefore reductions in N and P excretion of between 17 and 35%, respectively, were estimated. Nevertheless, nutritional and herd management strategies were limited when

N and P excretion were considered in relation to the whole lactating herd and farmland availability. Dietary CP manipulation was estimated to reduce herd N excretion by 11%

per hectare, whereas dietary P manipulation would be reduced by no more than 17%. We conclude that the correct match between the ingested and required N and P, together

with an increase in milk productivity may be feasible strategies for reducing N and P excretion by lactating herds on commercial farms.


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Key Words: nitrogen, phosphorus, dairy farm Abbreviation key: CF = Component feeding, CNCPS 5.0 = Cornell Net Carbohydrate

and Protein System model, CP = Crude protein, LU = Livestock unit, MUN = Milk urea nitrogen, N = Nitrogen, NUE = Nitrogen use efficiency, P = Phosphorus, PCF =

Purchased complete feed, PUE = Phosphorus use efficiency, RFV = Relative feed value, TMR = Total mixed ration.

3.1. Introduction

One of the major objectives of the European Common Agricultural Policy (CAP) is to

develop a sustainable farming system with environmental-friendly production management (Van Passel et al., 2007). Efficient use of nutrients is one of the major

assets of sustainable agricultural production systems because inefficient nutrient use not only results in excessive and potentially harmful losses to the environment, but also

affects economic performance (Oenema and Pietrzak, 2002).

Dairy farm activities have been described as contributors to N and P environmental pollution (Spears et al., 2003a,b). Nitrogen pollution from dairy farms affects water, by

nitrate leaching, which contributes to eutrophication, and also air, through the emissions of gaseous N compounds such as ammonia (NH3) and nitrogen oxides (N2O and NO).

Accumulated P from dairy farms can leach into groundwater or cause eutrophication of surface waters due to run-off from agricultural land (Sims et al., 1998).

The application of extensive farming methods in dairy production, the reduction of external nutrient inputs and the efficient use of nutrients at farm or regional level have

been described as advisable strategies for environmentally sustainable farming activity (Tamminga, 2003). Extensive farming and strategies to reduce nutrient inputs are

difficult to develop in the Basque Country (northern Spain). Rural land prices have increased dramatically in this mountainous region in recent years because of the

historical division of rural land for inheritance purposes and high industrial pressure. Moreover, the current trend is for intensified milk production on commercial farms in

the Basque Country. Data from 1996 to 2003 showed that although the dairy cattle population was reduced by 31.6%, the milk quota in the territory remained constant

(250,000 t) due to the increased mean milk yield of the herds from 4,934 to 7,241 kg


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NUTRITIONAL AND MANAGEMENT STRATEGIES ON NITROGEN AND PHOSPHORUS USE EFFICIENCY OF LACTATING DAIRY CATTLE ON COMMERCIAL ...

cow-1 year-1. Intensified dairy farms (high input:output) are characterized by the widespread use of purchased concentrates and mineral fertilizers (CEAS, 2002), which

make the minimization of external inputs in the dairy system difficult to achieve. Optimization of N and P use may therefore be the most realistic strategy for reducing

environmental N and P pollution in the Basque Country.

Optimization of animal feeding and management has been described as a key strategy for the reduction of N and P excretion in manure (CAST, 2002; Cerosaletti et al., 2004;

Ipharraguerre and Clark, 2005). The correct match between the quantity and quality of protein required by the animal, together with an increase in animal productivity helps

improve the efficiency of N use for milk production and the reduction in N excretion (Rotz, 2004). Similarly, the reduction in P overfeeding, the use of feedstuffs with high

amounts of available P and the optimization of production may lead to reduced P excretion in manure (Maguire et al., 2005). Furthermore, herd management practices

such as animal grouping (St Pierre and Thraen, 2001), the increased frequency of ration balancing and the feeding system used (Jonker et al., 2002) may also contribute to

reducing nutrient excretion in manure.

Some options have been considered in the territory to mitigate the impact of dairy farming on environmental pollution (Merino et al., 2002; Merino et al., 2005; Menéndez

et al., 2006). However, there is no evidence that improved nutritional strategies would be useful for minimizing the excretion of N and P at the farm level. The first objective

of the present study was to determine the current N and P utilization efficiency and excretion levels in lactating dairy herds in the Basque region. The second objective was to identify dietary and management factors that may help to improve the efficiency of N

and P use. The third objective was to relate nutrient use improvement to N and P excretion regarding the farm intensification level.

3.2. Materials and Methods

3.2.1. Dairy Farm Survey


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A confidential survey was conducted between September 2003 and April 2004. A total of 76 commercial dairy farms were selected by four Milk Producers Advisory Centers

in order to represent different ranges of milk yield and feeding systems. These farms represented 7.0% of all dairy farms in the Basque Country in 2004. Farmers were

contacted by telephone and interviewed in person. The survey included information on adult dairy herd characteristics (breed, number and distribution by parity of milking

animals and number of dry cows in the herd). Farmers were also questioned about milk production level and quality, and all data were checked by the Advisory Centers. Ration

data for milking herds were also requested (types and amounts of forage and concentrate, costs of daily rations) together with nutritional management strategies

(feeding groups for lactating herds, frequency of ration balancing, feeding system, mineral supplementation or grazing activity). Feeding systems were classified as total

mixed rations (TMR), purchased complete feed (PCF) or component feeding (CF). Information on concentrate composition was obtained from the corresponding

commercial feed companies. Data regarding the land in relation to dairy activity (grassland or cropland), use of land (for homegrown forage production or slurry

fertilization) and ownership (owned or rented) were also obtained from the survey.

3.2.2. Cornell Net Carbohydrate and Protein System (CNCPS 5.0)

The CNCPS 5.0 model (Fox et al., 2004) was used in order to simulate an average lactating cow on each farm. Cow-related data were fitted to the average features of

Basque commercial dairy farms: age (47 months), body weight (650 kg), pregnant days (40 days), days from calving (150 days), lactation number (2 lactations), breed (Holstein/Friesian), calving interval (13 months), age at first calving (29 months) and

condition score (3.0). Environmental data (temperature, wind speed and humidity) for the sampling day were obtained from the Basque Meteorological Service (Euskalmet).

3.2.3. On-site Sample Collection

On-site sample collection included feed, fecal, urinary and milk samples from lactating

herds. Feed samples were placed inside portable freezers and transported to the


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laboratory. The feed samples were then immediately dried in a forced air oven (60ºC, 72 h) for dry matter (DM) determination. The samples were ground to pass a 1.5 mm

screen (0.2 mm screen for P determination). Each feed sample was analyzed for CP (Kjeldahl N method), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid

detergent insoluble protein (ADIP), acid detergent lignin (ADL) (Van Soest et al., 1991), ash, ether extract (920.39/90 method in AOAC, 1990) and P (ICP spectrometry

UV-Spectrophotometry VARIAN CARY 100). Daily N and P intakes were estimated on the basis of the concentrations of N and P in individual feeds (or imported blends)

and information about the ration formulation was supplied by the farmers. Milking cows were divided in different feeding groups (high/low production) and when only an

average herd milk production was known, the N and P intakes were averaged for the herd in proportion to the number of cows in each feeding group. Finally, the percentage

of purchased N and P in the ration was calculated by taking into account the origin of the feedstuffs (home-grown forage or purchased feedstuff).

Freshly deposited feces were sampled from the barn floor just after being excreted by

lactating cows. The fecal samples were collected from 5% to 15% of the lactating cows included in the herd, depending on lactating herd size. Each subsample was mixed to

make a composite fecal sample (nearly 1 kg), which represented the mean sample for the herd. Samples were transported in portable freezers to the laboratory and analyzed

for total N (N-Kjeldahl method), C/N ratio and total P (ICP spectrometry UV-Spectrophotometry VARIAN CARY 100). Daily fecal volume excretion was estimated

by the CNCPS 5.0 model and this amount was multiplied by fecal N and P concentration in order to estimate the mean N and P excretion per day.

Urine samples were collected by a non-invasive method, by use of buckets, and collected samples represented between 5% and 15% of the total urine excreted by the

herd. These samples were mixed to make a composite urine sample (nearly 1 L) with 10% H2SO4 solution in order to avoid losses of ammonia. Samples were divided into

different subsamples in the laboratory before being frozen at -20ºC. Urine was analyzed for total N (N-Kjeldahl method) and urinary urea-N (UUN) (diacetyl monoxime method

by Douglas and Bremner, 1970). Urinary P was not analyzed as it is generally assumed that it is excreted in low quantities. Daily urinary volume was estimated by CNCPS 5.0

and daily urinary N excretion was estimated by multiplying the excretion volume by the


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analyzed N content. After determination of total N and P excreted, purchased N and P excretion was estimated by applying the same percentage of purchased N and P intake

to the total N and P excretion, according to Powell et al. (2002).

Milk samples were collected from the bulk tank (100 mL) and analyzed for fat, CP, lactose and milk urea nitrogen (MUN) by the Milk Institute of Lekunberri (Instituto

Lactológico de Lekunberri, Navarra, Spain). Milk fat and CP were analyzed according to AOAC (1990) procedures, and MUN concentrations were determined by the diacetyl

monoxime method (Douglas and Bremner, 1970). A milk P concentration of 0.09% was assumed (NRC, 2001). Daily milk N and P outputs were estimated considering the

mean milk yield and the analyzed N content and estimated P concentration, respectively. The NUE and PUE values were obtained considering the N and P content

of the milk compared with the amount of N and P ingested.

3.2.4. Statistical Analysis

Statistical analyses were carried out with Statview software (SAS Inst., Inc., Cary, NC) and each farm was considered as an experimental unit. When farmers did not answer all

the questions or some answers were considered misleading, the corresponding herds were excluded from the statistical analysis. Thus, 64 out of 76 sampled commercial

farms were finally considered for statistical analysis. Descriptive statistics were analyzed for the complete dataset. Treatment means were tested by regression analysis,

for continuous variables, or ANOVA, for discrete variables. A multivariate regression was developed with N/P excretion by the lactating herd per day and per hectare as the dependant variable and herd size, land area, N/P intake, ration CP and P concentration,

N/P fecal or urinary excretion and milk yield of the herd as independent variables. Normality test and equality of variances F-test were performed before ANOVA. The

Tukey-Kramer test was used for post-hoc analysis, at a significance level of P < 0.05. For application of the analysis to annual basis data (e.g., annual herd N/P excretion per

hectare) only those farms in which the farmers agreed not to change the ration were selected.


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3.3. Results and Discussion

3.3.1. Description of Farms

The selected farms represented 5.9% of total commercial dairy farms in the Basque

Country for the period 2003/2004. These farms included 5,618 adult Holstein/Friesian cows representing on average 16.4% of the whole dairy cow population in the territory,

and 19.2% of total quota allocated to the Basque Country. Large ranges of herd size and milk production were observed in the survey (Table 3.1). Cow herds ranged from 19 to

596 cows and annual milk yield ranged from 5,713 to 12,165 kg cow-1 year-1. Mean milk yield (9,057 kg cow-1 year-1 or 29.7 kg d-1) and mean herd size (87 cows) were

higher than mean values described for the region in 2003/2004, which reached 7,401 kg cow-1 year-1 and 32 cows, respectively (EUSTAT, 2004; FEPLAC, 2004). The selected

farms were biased to more intensive farms, representing the current trend of dairy farming in the Basque Country. The level of intensification was much higher than that

reported for commercial dairy farms in other countries in the European Union (EU) (Table 3.1) (Bos et al., 2004). According to the ranking defined by Berentsen et al.

(2003) (low < 12,000 kg ha-1; 12,000 kg ha-1 < medium < 15,000 kg ha-1 ; high > 15,000 kg ha-1), 46.9% of surveyed farms were classified as highly intensive farms, 15.6% were

classified as having a medium level of intensification and 37.5% as having a low level of intensification. The average cow stocking rate was 2.1 cows ha-1, just above the

suggested stocking rate for the EU (Tamminga, 2003). Univariate and multivariate regression analysis of the present data confirmed land availability as the main factor that

affected the observed overall high stocking rates (R2 = 0.17, P < 0.05; negative slope).

The average farmland area for sampled dairy farms was 47.9 ha and was used for grass

and/or corn production and for slurry spreading (Table 3.1). On average, 89.1% of surveyed farms included farmland rented from neighbouring areas and therefore only

48.5% (23.2 ha) of farmland belonged to dairy producers. Grassland production was the main use of farmland area and represented on average 89.6% of the total area (Table

3.1). Grassland is usually managed as permanent or rotational grassland, with ryegrass (Lolium perenne, Lolium multiflorum) and/or festuca (Festuca arundinacea, Festuca

rubra) as the main grass species (I.Albizu pers. comm). Most grass forage was used to


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produce silage (90.6%) and only 26.6% of the herds grazed on pasture in the spring/summer period (when the current study was carried out all the herds were

confined to stalls).

Table 3.1. Herd size, milk yield and land use in farms from the Basque Country.

Parameter Mean SD 10th percentile 90th percentile

Cows

Total 87 76 33 156 Milking 76 68 29 136

Dry 11 9 3 23 Production

Annual yield, kg cow-1 9,057 1,395 7,213 10,522 Milk yield, kg cow-1 d-1 29.1 4.83 22.6 34.6 Farmland availability

Grassland, ha 42.9 30.2 14.9 80.0 Cropland, ha 5.0 11.7 0.0 16.7

Slurry spreading area, ha 50.5 36.8 16.9 91.0 Farmland property, % 48.5 32.3 3.2 100.0

Stocking rate, cows ha-1 2.1 0.15 1.0 3.6 Milk yield per area, kg ha-1 16,767 9,522 8,248 27,186

3.3.2. Description of Lactating Herd Ration

The ration parameters, the mean use of the forage and concentrate ingredients, management strategies and milk production data for the different feeding systems are

shown in Table 3.2. The choice of each feeding system basically depended on farmland availability and herd size (P < 0.05). Farms with large herds and low land availability

imported PCF blends daily, as home-grown forage production was limited (P < 0.05). On the other hand, large farms chose TMR or CF rations, allowing farmers to use more

home-grown forage (P < 0.05). Grass silage was the main forage used in TMR or CF rations rather than corn silage, mainly used in PCF (P < 0.05). The average

forage:concentrate ratio was around 50:50 for each feeding system. Soybean meal and corn grain were main ingredients in the concentrates.


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Table 3.2. Ration and ingredients, management and milk yield by feeding groups.

TMR PCF CF Mean

Ration data

DMI, kg d-1 21.9a 21.5a 20.5a 21.3 Ration cost, € cow-1 d-1 3.9a 4.2a 3.5b 3.8

Forage:concentrate 49:51a 49:51a 54:46a 50:50 CP, % 16.5ab 17.3a 16.0b 16.4

P, g kg DM-1 4.00a 4.16a 3.87a 3.98 Home-grown forage, % of ration DM 27.8a 7.7b 34.9a 27.2

Ingredients, % of the diet Grass silage, % 21.0a 6.7b 24.5a 19.9

Corn silage, % 15.0b 25.2a 6.1c 13.2 Alfalfa hay, % 9.2a 14.4a 10.4a 10.6 Grass hay, % 1.2b 1.9ab 8.5a 4.3

Soybean meal, % 18.6a 7.5b 16.0a 15.6 Corn grain, % 16.5a 10.9b 12.4b 13.9

Gluten feed, % 8.3a 11.0a 10.8a 9.8 Cottonseed, % 6.1a 4.3a 5.9a 5.7

Beet pulp, % 2.6ab 0.4b 4.7a 3.1 Barley grain, % 4.8ab 3.1b 6.3a 5.1

Wet brewers’s grains, % 0.8b 28.6a 0.0b 5.3 Management and milk parameters

Herd size, number of cows 114a 93a 58b _ Farmland area, ha 64.4a 32.2b 39.2b _

Stocking rate, cows ha-1 2.0b 3.1a 1.8b _ Milk yield, kg cow-1 day-1 30.6a 31.1a 26.6b _ a,b,cDifferent superscripts within the same row indicate significant differences (P < 0.05).

On selected farms, mean DM intake of milking cows was 21.3 kg cow-1 day-1 and it was

similar among different feeding systems. Dietary CP concentrations ranged from 12.7 to 18.4% with a mean content of 16.4%, and differed between feeding groups (P < 0.05).

The concentration of CP was higher for PCF (17.3%) than TMR (16.5%) and CF (16.0%). Recent researches have shown that a CP concentration of between 16.5% and

17% may be sufficient to produce milk yields >30 kg d-1 (Broderick, 2003;


86


Ipharraguerre and Clark, 2005; Olmos Colmenero and Broderick, 2006). On average, only one third of surveyed rations were formulated with > 17% CP concentration and

the highest milk yield (39.9 kg d-1) was achieved with a grass and corn silage based TMR ration (40:60 forage concentrate ratio) with a mean CP content of 16.9%. The

wide range of CP content observed in the diets was due to the variability in concentration of CP in grass silage (7.5% to 17.7%) and commercial concentrates

(17.5% to 24.2%). Mean ruminal degradable protein (RDP) was estimated from CNCPS 5.0 as 62.9% of total CP and the use of grass silage was related to a higher proportion of

RDP (P < 0.05). On the contrary, the greater use of concentrates helped increase the proportion of ruminal undegradable protein (RUP) in the ration (P < 0.05) and the

higher RUP content was related to higher mean milk yields (R2 = 0.10, P < 0.05; positive slope).

The range of dietary P content was just above NRC (2001) recommendations (0.32% -

0.38%) and ranged from 0.28 to 0.54% (mean, 0.40%) and there were no differences between feeding systems. The variability in the surveyed data in this trial was lower

than that reported by Dou et al. (2003) (range from 0.36% to 0.70%; mean 0.44%), and Powell et al. (2002) (range from 0.23% to 0.85%; mean 0.40). According to previous

studies, which reported grass silage as a large contributor to dietary P intake (Valk et al., 2000; Kebreab et al., 2005), the present survey confirmed grass silage as the main

source of forage P. The average P content of grass silage was 0.33% and ranged from 0.12 to 0.93%; this variability suggested an opportunity to reduce the mean P content of

silage through correct grassland P fertilization (Valk et al., 2000). Nevertheless, the use of forage with a high P content has been related to difficulties in minimizing the P content of rations (Cerosaletti et al., 2004). As expected, concentrates contained more P

than grass silage (range between 0.38 and 0.69%; mean 0.53%). The substitution of grass silage by corn silage and the greater use of low P content by-products (beet pulps

or citrus pulp) rather than other by-products such as wheat middlings or soybean meal may contribute to reducing the P content of the ration (Dou et al., 2003; Cerosaletti et

al., 2004). Nevertheless, the heterogeneity of the ingredients used in the surveyed rations did not allow identification of specific ingredients that could be used to reduce

the ration P content. Some authors also consider that minimizing mineral P supplementation may be a key strategy to minimize ration P content (Dou et al., 2003).


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In this sense, P mineral supplementation was detected in 67% of surveyed herds, although the mineral P in concentrates represented only 0.2% of concentrate feedstuffs.

3.3.3. Lactating Cow Nutrient Balance

3.3.3.1. Lactating Cow N Balance

The mean, standard deviation and percentile distribution of N intake, utilization and

excretion for lactating cows across all herds are given in Table 3.3. Mean N intake was 562 g d-1 and the estimated metabolizable protein supply was 7.4% higher than

estimated requirements. The highest milk yield (39.9 kg d-1) corresponded to a grass and corn silage-based TMR ration (40:60 forage concentrate ratio) with a mean daily N

intake of 608 g cow-1. According to recommendations on protein intake made by Ipharraguerre and Clark (2005), the intake of N by high-producing dairy cows could be

decreased to about 600 to 650 g per day. Mean N intake from home-grown forage was 21.0% (SD = 1.8), but depended on the level of farm intensification (P < 0.05).

Mean NUE (%) was 25.8% (SD = 2.9) and ranged from 19.2 to 32.3% (Table 3.3).

Average NUE was lower than reported by other authors for grass silage based rations, which reached about 28% (Castillo et al., 2001b; Yan et al., 2006). Maximum NUE

value corresponded to the above mentioned grass and corn silage based TMR ration (40:60). Milk yield was positively correlated to NUE (%) values (R2 = 0.21, P < 0.05)

(Figure 3.1) although a high variability was detected between herds. This variability might be attributed to the different N intake levels as milk CP content was not related to

milk yield (P > 0.05). Thus, protein nutrition could be still improved on dairy farms in the Basque Country. The MUN has been also proposed as an indicator of the protein nutrition and efficiency of N utilization in dairy cows. The mean MUN value was 10.4

mg dL-1 (SD = 2.7) (Table 3.3) and ranged from 3.8 to 15.5 mg dl-1. Although a univariate regression model confirmed that MUN was not an accurate estimator of NUE

in this study (P > 0.05), mean MUN value was lower than reported by Nousiainen et al. (2004) of 16.0 CP% and 507 g day-1 N intake rations (MUN = 13.3 mg dL-1). This may

suggest that despite the observed high supply of metabolizable protein, protein nutrition may quite accurately match cattle requirements on the farms under study.


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Table 3.3. N balance for lactating cows on farms from the Basque Country.

Mean SD 10th percentile 90th percentile

N intake, g cow-1 d-1 562.6 87.4 465.5 675.0

N output, g cow-1 d-1 Feces 214.7 43.1 162.3 273.5

Urine 215.5 63.3 140.1 295.9 Milk 144.9 24.5 110.8 173.6

N output, % N intake Feces 38.3 5.8 31.3 45.9

Urine 38.1 8.4 27.4 47.5 Milk 25.8 2.9 22.0 29.5

MUN, mg dL-1 10.4 2.7 7.2 13.8

Figure 3.1. Herd mean milk yield and nitrogen use efficiency (NUE).

The present estimates indicated that 76.4% of ingested N was excreted as fecal or

urinary N (Table 3.3). Recently, Yan et al. (2006) reported that 72% of the ingested N was excreted by Holstein lactating cows and the authors concluded that the amount of N

ingested could be a good predictor of excreted N. In this sense, the present data also indicated N ingestion as the best estimator of N excretion (P < 0.05; R2 = 0.7; positive

slope). Therefore, the amount of N ingested can be described as a more accurate predictor of N excretion than the CP content of the ration, which is usually considered

by dairy farmers. Previous studies have demonstrated a similar contribution by fecal and


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urinary N outputs to the total N excretion (Kebreab et al., 2001; Yan et al., 2006). Nevertheless, Kebreab et al. (2001) also suggested a mean N intake of 400 g d-1 as a

threshold value for an increasing urinary output. The present estimates of N excretion by the fecal and urinary pathways were similar to those reported in the above-mentioned

studies. In fact, we estimated that 38% of N was excreted in feces and 38% in urine. Univariate regression revealed the relationship between the total N ingested and the

estimation of excreted N in urine and feces (Table 3.4: Equation 1). Estimations were significant (P < 0.05) for fecal N (R2 = 0.44) and urinary N (R2 = 0.41) (Equations 2 and

3) (Table 3.4; Figure 3.2). Nevertheless, data from the present survey may overestimate the fecal N output regarding to urinary N output. In fact, the estimated mean fecal DM

excretion (7.5 kg d-1) in the present study was similar to that reported by Nennich et al. (2005) for high-producing Holstein cows (7.3 kg d-1) even though not all herds

comprised high producing cows in the present study (26.6% of the herds averaged fewer than 8000 kg milk year-1). Furthermore, the present data would overestimate fecal N

excretion by 22.3% when the mean N intake (569 g d-1) was used to predict the fecal N output with regression model developed in relation to the regression model established

by Kebreab et al. (2001). This overestimation may explain why the urinary N threshold was established at around 550 g d-1 in the current study, suggesting that the threshold of

400 g d-1 proposed by Kebreab et al. (2001) may be advisable even for dairy farmers in the Basque Country.

Table 3.4. Prediction equations for manure, fecal and urinary N output1.

Equation R2 SE Equation no.

Manure N output = 0.8(0.067) NI -19.8(38.0) 0.698 46.3 1 Fecal N output = 0.329(0.047) NI + 29.8(26.6) 0.444 32.4 2

Urinary N output = 0.164(0.178) NI1.13(0.172) 0.410 0.23 3

1Values in subscript parentheses are SE. NI = Nitrogen intake.


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Figure 3.2. Relationship between total N intake and fecal, urinary and milk N output.

From an environmental point of view, it is advisable to reduce N excretion per milk unit produced. In this sense, improved milk production reduces the partial contribution of

maintenance N requirements (Rotz, 2004), which directly helps to improve the NUE and reduce N excretion per litre of milk. The present data showed that NUE

improvement contributed to reducing N excretion per litre of milk produced (P < 0.05; R2 = 0.68). Thus, considering two of the quota levels on surveyed farms (low 268 t year-

1 and high 1150 t year-1), a reduction of 3.2 g N L-1 between two farms with 268 t year-1

milk quota would minimize the excreted N to 865 kg year-1. Meanwhile, N excretion

would be reduced by 7,337 kg year-1 considering the reduction of 6.4 g N L-1 observed between two farms with high milk quotas (1,150 t year-1). The overall reduction in N

excretion in lactating herds obtained by minimizing N excretion per milk litre would allow N excretion to be decreased in the herd from 17.2% to 35.5%.

3.3.3.2. Lactating cow P balance.

Mean P intake was 84.8 g d-1 (SD = 14.1), similar to that reported by Powell et al. (2002) for commercial farms in Wisconsin. On average, daily P intake exceeded the P

requirements estimated by CNCPS 5.0 by 63%. Previous studies have demonstrated P overfeeding as common practice on several commercial dairy farms (Dou et al., 2003;

Chapuis-Lardy et al., 2004; Hristov et al., 2006), although P overfeeding on sampled farms was higher than reported by other authors (Valk et al., 2000; Powell et al., 2002;


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Dou et al., 2003). Reduction of P overfeeding was the main strategy for improving PUE (P < 0.05; R2 = 0.78; negative slope) and the second was to increase milk yield (P <

0.05; R2 = 0.35; positive slope). Optimization of both parameters should be considered as the main strategies for decreasing fecal P excretion. Milk P content is fairly constant

(NRC, 2001), but urinary P was not consistent in previous studies. Urinary P variability ranges from negligible amounts (Valk et al., 2002) to 500 mg L-1 (Wu et al., 2000).

The mean PUE was 31.9% (SD = 4.5) and ranged from 19.3 to 44.7% (Table 3.5). No

correlation was observed between dietary P content and milk yield (P > 0.05). The highest PUE values were reached with rations containing 0.39% P, which allowed

production of more than 30 kg d-1 of milk. The high P overfeeding levels demonstrated that rations did not match P requirements because diets for lactating cow are nearly

always formulated to match energy and CP requirements. This meant that although farmers tried to improve NUE, the PUE was not usually considered. However, and

according to previous data (Jonker et al., 2002; Nennich et al., 2005), NUE improvement might contribute to optimizing PUE (P < 0.05; Figure 3.3). The above

mentioned P reducing strategies should be implemented on farms in order to improve PUE and make NUE a better predictor of PUE.

Table 3.5. N balance for lactating cows on farms from the Basque Country.

Mean SD 10th percentile 90th percentile

P intake, g cow-1 d-1 84.8 14.1 65.2 103.1

P output, g cow-1 d-1 Feces 59.3 11.6 45.1 73.9

Milk 26.7 4.4 20.7 31.8 P output, % P intake

Feces 69.9 12.3 54.5 80.3 Milk 31.9 4.5 27.0 37.1


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Figure 3.3. Relationship between nitrogen use efficiency (NUE) and phosphorus use efficiency (PUE).

Data from the present study indicate that 69.9% (SD = 12.3) of ingested P was excreted

as fecal P (Table 3.5). Daily fecal P excretion (Equation 4 in Table 3.6) and fecal P concentration (Equation 5 in Table 3.6) were dependent on the amount of daily P intake

or ration P content, as indicated Valk et al. (2002) and Powell et al. (2002), respectively. The endogenous fecal P excretion (the sum of P in microbial residues, sloughed cells

and digestive secretions as salivary P) (Valk et al., 2002) and the above mentioned fecal output overestimation from CNCPS 5.0 model may contribute to the weak relationship

observed between the ingested and excreted P (R2 = 0.30). In fact, previous studies showed higher coefficients of determination than observed in the present study (Dou et

al., 2003, Chapuis-Lardy et al., 2004). Nevertheless, the linear equation obtained was consistent with the equations obtained by the same authors (Dou et al., 2003, Chapuis-

Lardy et al., 2004).

Table 3.6. Prediction equations for manure, fecal and urinary P output1.

Equation R2 SE Equation no.

Fecal P output, g cow-1 d-1 = 0.46(0.09) PI + 19.9(7.8) 0.303 9.7 4

Fecal P content, g kg-1 DM= 0.75(0.37) Pcont + 5.21(1.5) 0.06 1.3 5 1Values in subscript in parentheses are SE. PI = Phosphorus intake, g cow-1 d-1; Pcont = Ration P content,

g kg-1 P.


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Taking into account the four quota levels on the surveyed farms, two low milk quotas (268 t year-1) with a reduction of 0.91 g P L-1 between farms could reduce P excretion

by 244 kg year-1. Similarly, a reduction of 0.82 g P L-1 for the two higher milk quotas (1,150 t year-1) may enable reduction to about 989 kg year-1. Therefore, excreted P at

herd level might be reduced from 18.0% to 30.8% by minimizing P excretion per litre.

3.3.4. Management Practices to Improve N and P Utilization Efficiency

St-Pierre and Thraen (2001) determined a positive effect of animal grouping on nutrient utilization efficiency and nutrient excretion in research dairy herds. Nevertheless, recent

data obtained on commercial farms (Jonker et al., 2002; Powell et al., 2006) did not demonstrate any improvement in NUE or PUE values through group feeding. Data from

our survey in which cows were divided into different feeding groups by days in milk did not improve NUE (P > 0.05), although PUE was improved by herd grouping (P < 0.05)

(Table 3.7). Improvement in P use may occur due to the higher milk yield of those herds fed in separate groups (P < 0.05) rather than to well matched P feeding. This strategy

was not observed in NUE because of the higher N intake recorded in farms with several feeding groups (P < 0.05).

Increased frequency of ration balancing has also been associated with higher milk yields

on commercial dairy farms (Jonker et al., 2002) although it was not related to the improvement of NUE. In the present study, 51% of farmers recognised that they

reformulated the diets in accordance with forage availability (grazing activity or grass and corn silage availability) and feedstuff prices. The present data showed that although N excess was reduced through diet reformulation (P < 0.05), there was no improvement

in NUE or milk yields (P > 0.05) (Table 3.7). Phosphorus overfeeding was not reduced by diet reformulation (P > 0.05) and PUE did not vary depending on this strategy.

Choice of different feeding systems may also help to improve nutrient use in milk

because TMR rations may improve milk yield or NUE compared with PCF (Jonker et al., 2002). Our data showed that the feeding system did not contribute to increasing

NUE and PUE (Table 3.7) although TMR and PCF provided higher milk yields (30.0 – 31.0 kg cow-1day-1) than CF system (26.5 kg cow-1day-1) (P < 0.05).


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Table 3.7. Effect of feeding groups, feeding systems, reformulation and degree of intensification on N and P utilization in milk.

Management Option NUE (%) PUE (%)

Feeding System TMR 26,6ª 32,6ª

CF 24,9ª 31,0a

PCF 25,6ª 32,1ª Group feeding Yes 26,6ª 34,7ª

No 25,6a 31,3b

Reformulation Yes 25,9ª 31,8ª

No 25,6ª 31,9ª a,b,Different superscripts within the same row indicate significant differences (P < 0.05).

3.3.5. Effect of Intensification on N and P Excretion

A multivariate regression model was used to determine the nutritional influence on

whole herd N and P excretion by considering intensification parameters. When N excreted by lactating herd per farmland hectare was considered as a dependant variable,

N intake was able to explain 11.2% of the variance (P < 0.05), and 60.4% of the variation was accounted for by farm intensification parameters (herd size and land

availability) (P < 0.05). When P excreted by lactating herd per farmland hectare was considered, fecal P excretion accounted for 16% of the variance and those parameters

related to farm intensification explained 57% of the variation. Phosphorus intake was not included in the regression model (P > 0.05) and the weaker relationship observed

between P intake and P excretion may explain the above observation. Therefore, the data indicated that nutritional management may not be able to reduce overall herd N and

P excretion per hectare to above 20% in commercial dairy farms in the Basque Country. Although highly intensified dairy farms were more efficient in terms of N use (26.5%)

(P < 0.05), N excretion of the herd per hectare of land was greater in highly intensified farms. The same trend was observed for P excretion (P < 0.05) taking into account that

PUE was not improved on highly intensified farms (P > 0.05). The mean daily and annual N and P excretion by the sampled herds in relation to farmland area is shown in

Table 3.8. On average, N excreted by the herds was 781 g ha-1 day-1 (317 kg ha-1 year-1

on annual basis) and the daily excretion ranged between 293.4 and 2225.0 g ha-1 day-1.


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In relation to the mean P excretion, it averaged 110 g ha-1 day-1 (45.4 kg ha-1 year-1 on annual basis), ranging the daily P excretion from 41.7 to 394.4 g ha-1 day-1.

Table 3.8. Daily and annual N and P excretion by herds concerning land area.

Mean Low3 Medium3 High3

N excreted, g ha-1 day-1 781.1 (424.7) 479.2b 635.4b 1,071.3a

P excreted, g ha-1 day-1 110.0 (64.9) 66.0b 98.3b 149.2a

N excreted,1 kg ha-1 year-1 317.8 (164.9) 187.2b 220.3b 402.0a

P excreted,1 kg ha-1 year-1 45.4 (29.2) 24.7b 31.4ab 58.4a

Purchased N excretion,2 g ha-1 day-1 647.8 (435.5) 337.9b 484.7b 950.1a

Purchased P excretion,2 g ha-1 day-1 89.4 (60.4) 46.8b 71.9b 129.3a

Purchased N excretion,1,2 kg ha-1 year-1 273.3 (165) 137.7b 181.9b 359.1a

Purchased P excretion,1,2 kg ha-1 year-1 38.0 (26.5) 17.5b 25.2b 50.9a

a,b,Different superscripts within the same row indicate significant differences (P < 0.05). 1Annual excretion was estimated for farms which did not change the diet during the year (n = 31). 2Purchased N and P excretion considering the purchased N and P intake relative to total N and P intake.3Low < 12,000 milk kg ha-1; 12,000 milk kg ha-1 < Medium > 15,000 milk kg ha-1; High > 15,000 milk kg

ha-1

These data confirmed that estimated annual N and P excretion per hectare were higher

than reference values for N and P slurry application on grassland (150 kg ha-1 and 40 kg ha-1, respectively). Even considering N gas losses of about 50% from housing and

storage (Rotz, 2004), the sampled farms would exceed the maximum annual threshold for N excretion. Highly intensified farms had the highest annual pollutant capacity for N and P with 402 and 58.5 kg ha-1 year-1, respectively (P < 0.05) (Table 3.8). Lactating

herds from farms with medium level of intensification had mean N and P excretion of 220 and 31.4 kg ha-1 year-1 and herds from farms with low levels of intensification

showed N and P excretion of 187 and 24.7 kg ha-1 year-1. Furthermore, the origin of excreted N and P may vary depending on the farm intensification level. Highly

intensified farms showed the lowest use of home-grown N with 10.7% compared with 17.4% and 26.4% for medium and low levels of intensification, respectively (P < 0.05).

Similarly, intake of home-grown P was also lower for highly intensified farms (12.8%) than for medium (19.7%) and low levels of intensification (29.1%) (P < 0.05).

Therefore, considering the proportion of home-grown N and P in rations and applying the same ratio to N and P excretion, we estimated the N and P excreted imputed to


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imported feedstuffs. The differences between high and low levels of intensification increased for daily and annual imported N and P excretion by the herd and farmland

hectare (Table 3.8). Annual imported N excretion was estimated as 359 kg ha-1 year-1

for highly intensified farms, and 181 and 137 kg ha-1 year-1 for medium and low levels

of intensification (P < 0.05). Phosphorus imported excretion was also higher for highly intensified farms (50.9 kg ha-1 year-1) than for medium (25.2 kg ha-1 year-1) and low

levels of intensification (17.5 kg ha-1 year-1) (P < 0.05).

3.4. Conclusions

Feeding N closer to recommendations together with an increase in milk yield may contribute to enhancing NUE and reducing N excretion. Nutritional P management may

be improved by no mineral supplementation of concentrates, low-P grass silage production through reducing P fertilization in grasslands and the use of feedstuffs

containing low amounts of P. Improvement of NUE contributed to improving PUE, despite the use of grass silage and commercial concentrates with high P content.

Increasing NUE and PUE in milk can lead to reductions in N and P excretion per liter of milk, and the observed differences in these on different farms allowed estimation of the

reduction in N and P excretion at herd level of 17% and 35%, respectively. Management strategies such as the use of different feeding groups, ration reformulation and selection

of feeding system did not result in a reliable improvement in NUE and PUE on commercial dairy farms. When nutrient excretion was considered for the whole lactating

herd and farmland availability, the level of intensification was the main factor found to affect farm N and P excretion. Dietary N manipulation may explain only 11% of total

variance, and dietary P manipulation could explain no more than 17 % of the variance in herd nutrient excretion per hectare. We conclude that the correct match between CP and

P quantity fed and that required by the animal, together with an increase in animal productivity are feasible strategies for reducing N and P excretion by lactating herds on

commercial farms. Nevertheless, the level of farm intensification may limit the effect of nutritional strategies on N and P excretion at farm level.


4 DIETARY MODIFICATION IN DAIRY CATTLE: FIELD MEASUREMENTS TO ASSESS THE EFFECT ON AMMONIA EMISSIONS IN THE BASQUE COUNTRY


99

Dietary Modification in Dairy Cattle: Field Measurements to Assess the Effect on Ammonia Emissions in the Basque Country

Abstract

Ammonia (NH3) derived from cattle, especially dairy cattle in the Basque Country, is one

of the main types of atmospheric pollution. The amounts of nitrogen (N) in excreta and subsequent emissions of NH3 can be reduced by manipulating cattle diets. Ten Holstein

cows were fed diets supplying different N intake and forage:concentrate ratio and the resulting slurries obtained were applied to grassland to study the emissions of NH3 to the

atmosphere. The forage:concentrate ratio was 2.5 in the lower N intake or high forage (HF) diet (405 g N d-1) and 1.0 in the higher N intake or low forage (LF) diet (498g N d-

1), respectively. Decreasing the N intake led to 11% decrease of N output (feces and urine). This affected the N composition of the slurry, and larger amounts of slurry from

HF diet were applied to the soil to provide the same amount of N than in LF diet. Slurries were applied to soil to provide 120 kg NH4

+-N ha-1. When equal amounts of N (120 kg

NH4+-N ha-1) were applied to soil via the slurry, there was no significant effect on NH3

emissions with no effect of the diet supplied, possibly because of the high variability in

emissions and the effect of edaphoclimatic conditions. Cumulative NH3 emissions over the 65 hours following application of the slurry to soil resulted in 5.7 kg NH3-N ha-1

emitted from soil treated with LF diet derived slurry, while for HF diet, 12.6 kg NH3-N ha-1 were emitted. In terms of NH3 volatilization per amount of slurry applied, no

statistical differences between treatments were found, with 142.2 and 100.6 mg NH3 kg-1

of slurry applied for treatments HF and LF, respectively. Thus, dietary N control does not guarantee that NH3 emissions would be reduced under the same N application doses,

nevertheless, the amount of slurry applied to reach the same N doses decreases in the lower N intake diet.

Key words: ammonia, forage: concentrate ratio, fecal nitrogen, efficiency, urine,

nitrogen, volatilization. Abbreviation key: CP = Crude protein, DM = Dry matter, DMI = Dry matter intake,

MP = Metabolizable protein, MUN = Milk urea nitrogen, NDF = Neutral detergent fiber, NFC = Non fibrous carbohydrates, RDP = Rumen degraded protein, RUP =

Rumen undegraded protein.


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4.1. Introduction

Livestock production is well recognised as a major source of NH3 emitted to the atmosphere. O n average, about 60% of the N in manure is volatilized (Muck and

Richards, 1983; Bussink and Oenema, 1998; Powers and Van Horn, 2001). Emission of NH3 is caused by the bacterial decomposition of urea and undigested N-containing

compounds in feces and urine. As a result of urease activity in fecal microbes, urea is rapidly converted into NH3, which volatilizes easily. Ammonia emissions have serious

consequences, such as acidification and eutrophication of natural ecosystems (Van der Eerden, 1982; Willers et al., 1996).

In the Basque Country, considerable efforts have been made to reduce the

environmental influence from the livestock sector (CBPA 1999; Pinto, 2005). Initially, the efforts were focused on dairy waste management and the use of chemical additives

in the farm to facilitate manipulation and distribution of dairy wastes. Some technical solutions have also been implemented, but their high cost means that they are not used

on most commercial farms. On the other hand, it is known that one of the greatest inputs of N to a dairy farm is from commercial feedstuff. The release of NH3 depends on the N

content of cow manure. In turn, the N content in the manure depends on the feed ration and the feeding strategy of the cow (Chase, 1999; Godden et al., 2001). Dairy cattle

excrete about 80% of their N intake through urine and feces (Tamminga, 1992). In terms of N pollution by dairy cows, urinary N is less desirable due to its greater

tendency to leaching (Pakrou and Dillon, 1995) and volatilization as NH3, the major source of which is urinary urea.

Previous dietary studies in the region have demonstrated that feeding amounts of protein closer to recommendations and increasing milk production help to improve the

efficiency of feed-derived N utilization and to reduce N losses from dairy farms (Arriaga et al., 2005). As mentioned above, the release of NH3 from slurry depends on

the N content. In this sense, James et al. (1999) investigated NH3 volatilization from manure from Holstein heifers and concluded that increased concentration of dietary

crude protein (CP) caused increased N intake, N excretion, urea-N excretion, and N excreted in the urine by the heifers. Reducing urinary N excretion should lead to

reductions in subsequent NH3 emissions, although Monteny et al. (1998) found that


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DIETARY MODIFICATION IN DAIRY CATTLE: FIELD MEASUREMENTS TO ASSESS THE EFFECT ON AMMONIA EMISSIONS IN THE BASQUE COUNTRY

losses from a dairy barn floor will depend on a number of variables including temperature, airflow, cleaning frequency, urease activity and urine puddle

replenishment rate.

Ammonia concentration monitoring is rare in Europe, probably because automatic optical measurements are not available, and wet chemistry in field measurements is

expensive and labour intensive (Dämmgen and Erisman, 2005). Most methods of estimating NH3 emissions use tables of N intake or N excretion to compute the N

available for volatilization (James et al., 1999). In the present study, urine and feces were collected from cows fed diets with different N intakes in one cross-over

experiment, and the resulting slurry was applied to soil for in situ measurements of volatilized NH3. The different diets were chosen in order to represent two different

feeding systems with respect to forage supply and thus, RDP:RUP ratio, which affects microbial growth and therefore the microbial N supply to the host animal (Fébel and

Fekete, 1996). Our aim was to study if mitigation of NH3 volatilization from dairy cattle slurry can be achieved by the reduction of N intake in the form of highly degradable

protein. Soil mineral N content and the availability of manure N for plant growth were also studied.


4.2.1. Animals, Diets and Manure Collection

Ten Holstein cows were each fed one of two diets consisting of concentrate and forage in different proportions in a crossover design (Table 4.1). Nitrogen intakes were 405 and 498 g N cow-1 in HF and LF diets, respectively. A 21-day dietary adjustment period

preceded a 5-day sampling period for each of the two diets.


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Table 4.1. Ration composition of HF and LF diets.

HF LF

Composition

Forage:concentrate 2.9 1.2

Concentrate, kg DM d-1 4.6 9.2

Triticale silage, kg DM d-1 11.7 9.4

Alfalfa hay, kg DM d-1 1.75 1.75

N intake, g d-1 405 498

Chemical composition of diets

DMI, kg d-1 18.1 20.4

CP, g kg-1 147 155

NDF, g kg-1 543 460

RDP/RUP 3.2 2.6

Soluble carbohydrate, g kg-1 6.1 8.6

Starch, g kg-1 116 185

Total NFC, g kg-1 175 264

Concentrate composition

Barley, g kg-1 400

Beet Pulp, g kg-1 150

Maize, g kg-1 150

Soy, g kg-1 140

Gluten feed, g kg-1 120

Minerals g kg-1 40

Separate collection of urine and feces from each cow started on the first day of the

balance week. Feces and urine were collected in buckets from each of the cows while in the stalls (excluding periods when the cows were being milked) and were immediately

stored in containers and kept refrigerated until required for the field trials. Subsamples of urine were collected in vessels and preacidified with 10% H2SO4 to adjust the pH of

the sample to below 3 to minimize NH3 losses. As no total collection of excreta was carried out, volumes of feces and urine were predicted as follows. Urine samples were

analyzed for N (macro-Kjeldahl), urea (diacetylmonoxime) and for creatinine, allantoin,


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uric acid, xanthine and hypoxanthine according to Balcells et al. (1992). The volumes of urine were estimated using the ratio between urinary creatinine concentration and daily

urine volume reported by Gonda et al. (1996). Volume of fecal excretion was estimated using the digestibility calculated for each cow taking into account the amount of lignin

excreted in feces.

The dry matter (DM) contents of the diets and feces were determined by drying at 105ºC until constant weight. Nitrogen content of diets and feces was determined by the

Kjeldahl method. The neutral detergent fiber (NDF) content of diets was determined using a modification of Van Soest et al. (1991). Starch was measured by the

amiloglucosidase technique followed by determination of total reducing substances and correction for water-soluble carbohydrates.

Total milk production was recorded daily from each milking (at a.m. and p.m. milking

times) and for each animal during the balance week. Milk samples were preserved with 2-bromo-2-nitropropane-1,3-diol. Although most urea is excreted in the urine, some

diffuses into the milk. Measurement of milk urea N (MUN) is simple and noninvasive, and may be used to monitor N excretion from lactating dairy cows (Jonker et al., 1998).

Milk samples were immediately shipped to the Milk Institute in Santander for routine analysis of milk components as well as MUN analysis. MUN analysis was measured

using a modified version of the method of diacetylmonoxime, based in Marsh et al. (1965).

4.2.2. Application of Slurries

The study was carried out in a cut grassland in the Basque Country (northern Spain) during the spring of 2005. The grass had been cut the day before in order to ensure

maximum presence of the slurry on the soil. A randomized complete block design with four replicates was established, and each experimental plot measured 2 x 1.5 m. The soil

was a poorly drained clay loam (34% fine sand, 3% coarse sand, 34% silt, 29% clay in the top 10 cm) with a pH (H2O, 1:2) of 6.6. A typical permanent pasture (Lolium


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perenne L. var. Herbus, 60%; Lolium hibridum L. var. Texi, 32%; Trifolium repens L. var. Huia, 8%) was sown at a density of 40 kg seeds ha-1.

After separation of subsamples of feces and urine for analytical purposes, slurry was

produced by mixing feces and urine at a representative rate obtained from the amount collected from each cow for each treatment. Slurry derived from HF and LF diets was

applied at a standard rate of 120 kg N-NH4+ ha-1 using 10L buckets to ensure an even

distribution of slurry. The amounts of slurry applied to soil were based on NH4+-N

concentration, which was measured 2 days prior to application on the soil and verified at the time of surface application. As the same doses of NH4

+-N were applied, larger

amount of HF diet-derived slurry were applied per square metre of soil. A treatment with no fertilizer was included as a control (C). Characteristics of the slurries are shown

in Table 4.2.

Table 4.2. Characteristics of the slurries applied to soil.

Treat DM N total

(% w fw-1)

NH4+-N

(% w fw-1)

C/N pH

HF 11.1a 0.38a 0.11a 17.0a 7.74a

LF 9.5b 0.37a 0.16a 15.3a 7.02a

a,bDifferent superscripts within the same column indicate significant differences (P < 0.05).

4.2.3. Measurement of NH3 Emissions

Ammonia emissions were measured for three days following application of the slurries

(Menéndez et al., 2006). Measurements were made at 2, 8 and 19 hours after slurry application on the first day. On days 2 and 3 only one sampling was carried out per day.

Ammonia emissions were measured using an open chamber technique with PVC chambers (volume 6.75 L, area 0.0314 m2) that fitted tightly on to a frame that was

inserted 3 cm into the soil. Inner walls of the chambers were covered with a polytetrafluoroethylene (PTFE) film to ensure minimal uptake of the soil-emitted NH3

by the walls (Hinz, 2005). One chamber was placed in each plot, and repositioned daily to account for spatial variation. In order to trap the NH3 in the surrounding air, the inlet

air to the chambers was taken from outside and pumped by an air compressor through


105


two 1 L glass flasks positioned in sequence and containing 500 mL of H3PO4 (10%) and 500 mL H2O, respectively. The air was passed through silicagel (free of CoCl2) to

absorb excess water. The filtered air was pumped through the chambers via PTFE tubing at a rate of 1 L min-1. Concentrations of NH3 were measured at the air inlet and

outlet of the chamber in 6 occasions per unit time using a photoacoustic infrared gas analyzer (Brüel and Kjaer 1302 Multi-Gas Monitor) for approximately 5 minutes, when

the steady-state value was reached. According to the technical specification of the instrument, the detection limit of the gas measurement was 0.2 ppm for NH3. Fluxes of

NH3 were calculated from the differences in concentration between inlet and outlet air, the air flow rate through the chamber, and the surface area covered by the chamber.

4.2.4. Cumulative Losses

Cumulative gas emissions during the sampling period were estimated by averaging the

rate of loss between two successive determinations, multiplying the average rate by the length of the period between the measurements, and adding that amount to the previous

cumulative total.

4.2.5. Soil Analysis

Eight soil cores (0-10 cm depth and 2.5 cm diameter) from each plot were used to determine gravimetric water content for calculating the soil water filled pore space

(WFPS) (Aulakh et al., 1991). The WFPS was calculated from the bulk density, assuming a soil particle density of 2.65 g cm-3. Soil ammonium (NH4

+-N) and nitrate (NO3

--N) contents were also measured. Fresh soil (100 g) was extracted in 200 ml 2M

KCl by shaking the soil suspension for 1 hour. The suspensions were filtered and the extracts frozen. Ammonium-N and NO3

--N in the extracts were determined by

segmented flow analysis (Alpkem 1986, 1987). Urease activity in soil sampled on day 1 after application of the slurry was measured following the methodology proposed by

Kandeler and Gerber (1988).


106



Results of the feeding trial were analyzed statistically using the GLM procedure of SAS 8.0. Measurement data, i.e., N intake, urinary N, fecal N, forage:concentrate ratio and

NFC were modelled with diet, period and the period by diet interaction as fixed effects. Prior to statistical analysis, NH3 emission rates were subjected to log-transformations

(as indicated by the normality test of Shapiro and Wilk) to homogenise variances. The LSD test was used for multiple comparisons of the instantaneous flux means, using the

SPSS (2004) software. Differences between cumulative emissions from the two slurries were compared by ANOVA and the separation of means between treatments by Duncan

test. The results of ANOVA as well as LSD and Duncan tests were considered significant at P < 0.05.

4.3. Results

The diets used in the study were designed to supply adequate quantities of all nutrients,

and contained significantly different amounts of metabolizable energy, corresponding to 167 and 194 MJ d-1 for diets A and B respectively. With respect to MUN content, there

were no significant differences between the treatments (Table 4.3). The MUN concentrations were positively correlated (P < 0.05) with ingested N and were not

associated with forage: concentrate ratio. Average efficiency of N utilization for milk was 21% in both diets, in consistency with data reported by Tamminga (1992) for Dutch

dairy cows.

4.3.1. Nitrogen Excretion

Manipulation of the diets by reducing the CP intake by 5.1% (DM basis) and increasing

RDP/RUP ratio resulted in a significant decrease in urine and fecal N, thus, 16.3% decrease was measured in urine N (Table 4.3 and Table 4.4), and a 11.1% decrease on

fecal N. On the contrary, NH4+-N content in the slurry did not significantly decrease

(Table 4.2). Urine urea was 13.1% higher in LF diet, although this difference was not

significant.


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Table 4.3. N use efficiency in milk and N excretion in urine and feces.

HF LF

Urinary N excretion, g d-1 128.5b 153.5a

Fecal N excretion, g d-1 144.8b 162.8a

Urine urea-N, g d-1 91.2a 104.9a

MUN, mg dl-1 7.7a 8.2a

1MP from bact, g d-1 962b 1207a

Milk N, g d-1 87.6b 103a

Milk yield, kg d-1 18.2b 21.4a

1 Based on CNCPS model 5.0 a,bDifferent superscripts within the same row indicate significant differences (P < 0.05).

Table 4.4. Daily N intake and excretion in lactating cows fed HF and LF diets.

N excretion (g d-1) N intake (g d-1)

Urine Feces

Day HF LF HF LF HF LF

1 418.2a 511.5a 106.9a 162.7a 140.5a 161.3a

2 391.8a 512.7b 137.,5a 143.3a 161.4a 212.9a

3 423.2a 488,2a 133.3a 131.5a 147.0a 160.2a

4 386.5b 481.0a 135.3a 182.0a 139.8a 146.8a

a,bDifferent superscripts within the same row indicate significant differences (P < 0.05).

With respect to the slurry collected during the experiment, NH4+-N was higher for LF

diet (Table 4.2), and represented a significantly greater proportion of total N, with

respective values of 29 and 43% for HF and LF diets. As slurry applications to soil were made on the basis of the concentration of NH4

+-N, the amount of slurry applied per square meter was higher in the slurry derived from HF diet. Nevertheless, volatilization

from HF diet-derived slurry did not differ when applied to soil. No significant differences in the urine urea excretion were found between treatments (Table 4.3),

because of the high variability in the results (coefficient of variation 28%).


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4.3.2. Ammonia Emissions from Slurry Applied to Soil

HF and LF diets contained 14.9 and 15.5% CP and, although the same doses of N were applied to soil, cumulative NH3 emission (over 65 hours) from the slurry applied to soil

were higher for HF diet than for LF diet, although the difference was not significant because of the high variability in emissions (coefficient of variation 110%). Besides, as

mentioned above, no significant differences in urine urea were measured between treatments. In this sense, Misselbrook et al. (2005) found that dietary manipulation may

not always result in a reduction in emissions proportional to the reduction in excreted urea-N. Thus, after 65 hours, the amounts emitted were 12.6 kg and 5.7 kg NH3-N ha-1

respectively from HF and LF diet-derived slurries respectively, with no other effect apart from the slurry N content, as the quantity of slurry applied was higher for

treatment HF due to the lower NH4+-N content (Table 4.2). With respect to NH4

+-N applied, these losses represented 13.2 and 5.7% for HF and LF diets respectively,

although these figures were not statistically significant because of the high variability and the lack of differences obtained for urease activity in soil on day 1 after slurry

application. Despite the differences in NH3 emission from the two slurries, the emissions from both peaked approximately 2 hours after application, and at least 81%

and 97% of the total NH3 measured in LF and HF diet-derived slurries respectively was lost within 40 hours (Figure 4.1).

Figure 4.1. Pattern of ammonia emissions from HF and LF diet derived slurry.

The vertical bars indicate LSD at 0.05 between treatments for each sampling time.


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During the first two hours, similar volatilization rates were obtained with LF and HF

diet-derived slurries, with maximum rates of 39.9 and 36.1 mg NH3-N m-2 h-1,

respectively. The NH3 emissions 20 hours after application of HF diet-derived slurry

increased sharply, but were only significantly higher than emissions from the LF diet-derived slurry 40 hours after application, and decreased to control levels after 65 hours.

Emissions from C plots remained constant during the trial, showing a slight increase after 20 hours.

4.3.3. Soil Mineral N

In the present study, the soil NH4+-N content decreased significantly in both slurries on

day 7 after application (Figure 4.2). Thus, at this time, 43.4 and 30.5% of the initial soil NH4

+-N content was found in HF and LF diets.

Figure 4.2. Evolution of soil ammonium content from HF and LF diet derived slurry.


4.4. Discussion

Milk urea nitrogen can be used to indicate a deficiency or an excess of CP in cattle diets (Klopfenstein et al., 2002) and to monitor N excretion from dairy cows (Jonker et al.,

1998). In the present study, MUN did not differ significantly for the two diets under


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study (Table 4.3), and the results indicated that CP was supplied in sufficient quantities. Similarly, Gonda et al. (1996) did not find any relationship between urea in body fluids

and efficiency of utilization of dietary N. Nevertheless, the amount of milk N increased (P < 0.05) in the higher concentrate diet (Table 4.3), as also found by Gonda et al.

(1996), although milk efficiency was similar in both cases. The present results showed that milk CP was negatively related to NDF, which is consistent with the results

reported by Emery (1978) and Spörndly (1986), although in other experiments no changes in milk CP concentration were found despite the difference in

forage:concentrate ratio (Broster et al., 1985; Sutton et al., 1987).

Fecal N was significantly higher in LF treatment (Table 4.3), representing 35.7% and 32.6% of ingested N for HF and LF diets, respectively. Aarts et al. (1992) found that the

fecal N yield was relatively constant over a very wide range of dietary CP concentrations. Similarly, Peyraud et al. (1995) and Castillo et al. (2001b) found that the

type of carbohydrate did not affect the total N in feces, as the amount of N excreted in feces in dairy cows is relatively constant because it mainly consists of undigested

material and metabolic fecal N. With respect to urine, we found significantly higher N content in urine of cows fed the diet with the higher N intake (Table 4.3). In this sense,

Kebreab et al. (2001) found that increasing N intake from 400 to 450 g N d-1 results in 50% of feed N increase appearing in urine, whereas a similar increase in feed N intake

at 500 g N d-1 would result in 80% of the increment being excreted in urine. Similarly, in our case, the increase from 405 to 498 g N d-1 resulted in 71.2% of the feed N

increase appearing in urine. Urine, rather than feces has been reported as the primary source of NH3 emissions (Paul et al., 1998). Nevertheless, Gonda et al. (1996) did not find any significant effect on the amounts or in the proportions of N excreted both in

feces and in urine by changing the roughage to concentrate ratio in the diet of lactating dairy cows.

The concentration of CP in the diet has a greater impact on urinary N excretion than on

fecal N excretion (Kebreab et al., 2001). With respect to the urine urea N content, there were no significant differences between the treatments, because of the high variability,

although LF diet produced considerably higher urine urea content than HF diet. There is evidence that N excretion, particularly as urea-N in the urine, can be affected through

manipulation of dietary CP (Gonda and Lindberg, 1994; James et al., 1999; de Boer et


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al., 2002) and carbohydrate degradability (Castillo et al., 2001b). As reported by Bristow et al. (1992), the percentage of urea in urinary N increases with higher RDP in

the diet. In the present study, HF and LF diets contained similar amounts of RDP. Kellems et al. (1979) showed a positive relationship between diet, urinary urea, and

volatilization of NH3 in beef cattle. Other studies (Moore and Beehler, 1980; Elzing and Monteny, 1997) have identified the urine component of manure as the source of N

volatilized as NH3 when the urine comes in contact with the feces. Likewise, Paul et al.(1998) found that urine, rather than feces, was the primary source of NH3 emissions.

Ammonia emission rates were quite similar between treatments up to 8 hours after

slurry application, but from then on, there were differences in the shape of the emission curves, with higher emissions from the soil treated with HF diet-derived slurry. Other

authors have reported lower NH3 emissions from manure obtained from dairy cattle fed 2% lower CP diets in laboratory experiments (Paul et al., 1998). These authors observed

a decrease in the proportion of NH3 in the total N excreted (from 44 to 35%) under the controlled conditions of their study. In the present study, due to the great variability of

results in NH3 emissions after having applied the same NH4+-N doses, the difference

between treatments was not significant, although NH3 emission rates were considerably

higher in HF diet-derived slurry. This variability could be due to the heterogeneity of soil, as the plot were not so large enough to refer uneven distribution of the slurry nor

the height of the sward could have interfered due to having been harvested on the day before. Volatilization from HF diet-derived slurry decreased more slowly (Figure 4.1)

probably because of the higher pH of the slurry (Table 4.2), which may have increased emission by shifting the NH4

+ NH3 equilibrium in favour of NH3. A slight increase in NH3 emission was observed in all slurries, even for C treatment, probably because of

the increase in temperature (from 20 to 30ºC) (Figure 4.1), which has been observed by other authors (Muck and Richards, 1983; Paul et al., 1998).

If we relate volatilized NH3 to the volume of slurry applied, we can see that

volatilization is significantly lower in HF diet-derived slurry, as the ratio of NH3-N ha-1

to weight of slurry applied was 1.53 times higher for HF diet and 7.33 times higher for

LF diet. Ammonia volatilization decreased to control levels after 65 hours, as reported in previous studies carried out in the study area (Menéndez et al., 2006).


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The significant decrease in soil NH4+-N content found on day 7 is consistent with

previous results obtained in the study area (Merino et al., 2002; Merino et al., 2005). It

is known that total NH4+-N in the surface-applied slurry decreases because of

volatilization and the high nitrification rates shown in our soils (Merino et al., 2002).

The concentration of soil NH4+-N remained constant between day 7 and day 33 after

slurry application and the lowest WFPS (average 40.6%) measured during the trial

corresponded to these days, which may explain the lower nitrification rate observed.

4.5. Conclusions

It is concluded that for up to 500 g N d-1 intake in dairy cattle, with an 11% decrease of N intake, N output was decreased significantly in feces and urine. This affects the N

composition of the derived slurry, with application of higher amounts of slurry required to apply the same doses of N from the lower N intake derived slurry. Once the same

doses of N was applied to soil from both diets, no significant effects on NH3 emissions were observed, possibly because of the high variability in emissions and the effect of

edaphoclimatic conditions, among other factors. With respect to milk yield, it was significantly higher in the lower forage to concentrate ratio, which makes this diet to be

better considered by the farmers due to the economic income and similar environmental impact on NH3 emission.


5 EFFECT OF DIET MANIPULATION IN DAIRY COW N BALANCE AND NITROGEN OXIDES EMISSIONS FROM GRASSLANDS IN NORTHERN SPAIN

______________________________________________________________________


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Effect of Diet Manipulation in Dairy Cow N Balance and Nitrogen Oxides Emissions from Grasslands in Northern Spain

Abstract

Dietary modifications in dairy cattle have been reported as a useful strategy to alter the

composition of manure. Many reports have been published on how changes in dietary crude protein content and forage-to-concentrate ratio reduce animal nitrogen (N)

excretion, but little information exists about the effect of diet modification on nitrous oxide (N2O) and nitric oxide (NO) emission when the subsequent slurry is applied on

grassland. Two diets differing in forage:concentrate ratio (high forage or HF diet, 75:25; low forage or LF diet, 55:45) were tested to detect the improvement of N use efficiency

in milk and the reduction of urinary and fecal N excretion. Triticale silage and barley grain were used as the main forage and concentrate sources in the diets. The subsequent

slurries were characterized for N and ammonium-N content (NH4+-N) and applied on

grassland in order to study the pattern of emission of N2O and NO.

The HF diet reduced the voluntary dry matter intake of the cows, N intake and urinary

and fecal N excretion. However, the reduction of N intake did not improve the N use efficiency in milk (NUE) (21.0 %) and did not reduce N excretion per unit of milk

produced (15 g N L-1) due to the lower milk yield. Slurries did not differ in N content but differed in NH4

+-N content, being lower in HF. Therefore, different slurry amounts

were applied on grassland to reach a usual fertilisation rate (120 kg NH4+-N ha-1). Total

emissions of N2O (5.8 and 5.0 kg N2O-N ha-1 for HF and LF, respectively) and NO (507.2 and 568.6 g NO-N ha-1 for HF and LF, respectively), and the pattern of emissions

were not affected by dietary treatments. When fertilisation management depends on the collected volume to empty the slurry pit, higher N2O and NO emissions per kg of slurry

could be expected from LF slurry. Nevertheless, if slurry is applied following recommended rates, N2O and NO emission per unit of milk produced might be slightly

lower from LF slurry. Grass yield (1.5 t dry matter ha-1) and N uptake (50 kg N ha-1) did not vary due to the applications of different slurries, and was attributed to low rainfall.

A judicious management of the slurries on grasslands may justify an adequate nutritional strategy of dairy herds from an environmental and productive point of view.


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Key words: Dairy nutrition, crude protein, forage:concentrate ratio, nitrous oxide, nitric oxide, grassland

Abbreviation key: ADF = Acid detergent fiber, ADIP = Acid detergent insoluble protein, BW = Body weight, CNCPS = Cornell Net Carbohydrate and Protein System,

CP = Crude protein, DIM = Days in milk, DM = Dry matter, DMI = Dry matter intake, EMPS = Efficiency of microbial protein synthesis, FCM = Fat corrected milk, ADL =

Acid detergent lignin, ME = Metabolic energy, MPS = Microbial protein synthesis, MUN = Milk urea nitrogen, NDF = Neutral detergent fiber, NFC = Non fibrous

carbohydrates, NH4+ = Ammonium, N2O = Nitrous oxide, NO = Nitric Oxide, NO3

-= Nitrate, NUE = Nitrogen use efficiency in milk, OC = Organic carbon, OM = Organic

matter, SOC = Soluble organic carbon, TMR = Total mixed ration, UUN = Urinary urea N, VDMI = Voluntary dry matter intake, VFA = Volatile fatty acids, WFPS = Water

filled pore space.

5.1. Introduction

Dairy farm activities have been described as contributors to N environmental pollution (Spears et al., 2003b), especially in intensively managed dairy farms in which N inputs

exceed by far the nutrient outputs in animal products (Oenema, 2006). The fate of the remaining N is not always easily accounted for due to multiple interactions, but one of

the most important is gaseous loss as N2O and NO (del Prado et al., 2006). Nitrous oxide is one of the main contributors to the greenhouse effect (Bouwman, 1990), and

together with NO, is involved in ozone layer depletion (IPCC, 1996). Nitric oxide also plays a major role in the chemistry of the tropospheric ozone (Bouwman, 1990) and in the formation of acid rain (Vos et al., 1994).

Manure application on grassland has been reported as the most notable N2O emission

source in dairy farming systems (Oenema et al., 2007) and is also considered as an important source of NO emission in other agricultural production systems (Meijide et

al., 2007). When available N from manure exceeds plant needs, emissions of N2O and NO occur through nitrification and denitrification processes (Granli and Bockman,

1994). The regulation of N2O and NO emission is described by the “hole-in-the-pipe” conceptual model (Firestone and Davidson, 1989), where the movement of N through


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EFFECT OF DIET MANIPULATION IN DAIRY COWN BALANCE AND NITROGEN OXIDES EMISSIONS FROM GRASSLANDS IN NORTHERN SPAIN

the pipe has been widely modelled in relation to soil features such as moisture, temperature, pH or nitrate and ammonium content (Granli and Bockman, 1994).

Dietary modifications in dairy cattle have been reported as a useful strategy to alter the

composition of manure in which dietary CP content is positively correlated with the fertilizer N value of manure (Reijs et al., 2007). From a nutritional point of view, the

efficiency of ammonia utilization in the rumen is the central factor determining the environmental impact of N (Hristov and Pfeffer, 2005). In this sense, matching the

quantity and quality of protein supplied and required by the animal is a valid option to minimize N excretion (Rotz, 2004; Yan et al., 2006). In addition to the reduction of CP

content, synchronization of energy and rumen-degradable N contributes to reduce N excretion (Hoover and Stokes, 1991). However, few data are available on the effect of

dietary strategies on N2O emission (Cárdenas et al., 2007). No available dataset exist for NO emissions.

Previous studies on N2O and NO emissions in the territory have focused on dairy waste

management and the addition of chemical additives to manure (Merino et al., 2002; Merino et al., 2005; Menéndez et al., 2006). Nevertheless, due to the high economical

cost of these options, farmers have not adopted them and nutrition might be a realistic alternative to reduce N2O and NO emissions in the Basque Country (Arriaga et al.,

2009).

The first objective of this study was to establish the effect of two forage:concentrate ratios on NUE, urinary and fecal N excretion, milk yield and milk quality parameters of two lactating Holstein herds. The second objective was to identify changes in slurry

composition through dietary manipulation. The third objective was to study N2O and NO emission pattern and cumulative emission on grassland from two types of manure

and to relate gas emissions to productive yields (milk and grass production).


5.2.1. Animals, Diets and Experimental Design.


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Ten Holstein cows (6 multiparous and 4 primiparous) averaging 108 DIM (SD = 8.3), 2.4 parity (SD = 1.8), 693 kg of BW (SD = 42.0) and 25.6 milk kg d-1 (SD = 3.7) were

blocked by DIM and milk yield into two groups and were assigned to treatments randomly in a two-period crossover design. The duration of each experimental period

was 26 days, with 21 days for diet adaptation and 5 days for data collection. Cow groups were held in separated free stalls and cows were weighed at the beginning and at

the end of each period. Experimental diets were initially formulated to contain two levels of forage and concentrate proportions, 77:23 for high forage diet (HF) and 45:55

for low forage diet (LF), respectively, and to supply about 500 g N d-1 to meet requirements for an average milk yield at 25 kg d-1 (CNCPS 5.0). Triticale silage was

supplied ad libitum and adjusted daily to produce 10% of orts. Individual triticale silage DMI was estimated through dietary and fecal ADL content. Concentrate and alfalfa hay

were supplied individually and twice daily for each cow after the milking periods. Feed composites were analyzed for DM at 105ºC (AOAC, 1990), CP (Kjeldahl N method),

NDF, ADF, ADIP, ADL (van Soest et al., 1991), ash (AOAC, 1990), starch (amiloglucosidase technique by Haissig and Dickson, 1979) and soluble carbohydrates

(Deriaz, 1961).

In situ OM and CP ruminal degradability was estimated for both diets. The assay was carried out in one ruminally fistulated cow, which was sampled twice for both diets

changing the diet between sampling periods (diet changed from HF to LF). The washout period was 15 days. HF and LF diet samples were weighed (3 g on fresh matter) in 11

cm x 16 cm nylon bags (pore size of 45 µm) and were placed in the rumen for 2, 4, 8, 16, 24, 48, 72 hours (5 replicates per hour). Bags were manually removed at each time and rinsed in four cycles of 5 min with cold water in a semi automatic washing

machine. Samples were dried at 60ºC over 48 hours for DM determination. Residues from 5 bags were grouped in a composite sample for CP and ash analysis. Ruminal fluid

samples were collected just after the morning feeding and at 2, 4 8, 9, 11 and 24 hours and were squeezed through a cheesecloth. Two subsamples (30 mL) were taken each

time and were centrifuged at 8000 x g for 10 minutes. Then, 10 mL were extracted and placed into tubes with pivalic and oxalic acid to avoid sample decomposition. Samples

were frozen at -20ºC until laboratory analyses for NH4+-N by spectrophotometry

(Nelson, 1983) and VFA by HPLC-UV chromatography (Waters 2690 Analytical


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HPLC with 996 PDA detector, SODEX RS Pak KC-811 column, 0.025% phosphoric acid as mobile phase at a flow rate 1 mL min-1, analysis temperature 40ºC ).

Cows were milked twice daily at 8:00 a.m. and 7:00 p.m and milk weights were

recorded at each milking. Individual milk samples (a.m./p.m. composite) were preserved with 2-bromo-2-nitropropane-1,3-diol and shipped to Milk Institute of

Santander (Cantabria, Spain) for analyses of CP and fat (Milkoscan 4000) and MUN (modified version of diacetylmonoxime method, Marsh et al., 1965). Nitrogen use

efficiency (NUE) was calculated as N in true milk protein multiplied by the daily milk yield, and expressed as a percentage of total consumed N.

Spot urine samples were collected daily from each cow during two sampling periods

(0.5-2.0 L d-1) through a non-invasive method using buckets. 250 mL of urine were acidified with 10% H2SO4 solution and distributed in 5 subsamples. Subsamples were

frozen at -20ºC until laboratory analysis for N (Kjeldahl N method), UUN (modified version of diacetylmonoxime method, Marsh et al., 1965), purine derivatives and

creatinine (Balcells et al., 1992). Urine volume was computed using creatinine as a marker assuming a daily creatinine excretion of 22.6 mg kg-1 BW (Gonda et al., 1996).

Purine derivatives (allantoin, uric acid, xanthine and hypoxanthine) were used for estimating the ruminal microbial protein synthesis (Chen and Gomes, 1992).

Freshly deposited fecal samples were collected daily from each cow (1-3 kg d-1) through

a non-invasive method. Samples (1 kg) were oven-dried at 105ºC for DM determination and at 60ºC for N (N-Kjeldahl method), ADL (van Soest et al., 1991), OM (Walkley, 1946) and C/N ratio analysis. Ration DM digestibility was estimated at 24 h through in

situ ruminal DM degradability or dietary and fecal ADL, and was used for estimating the daily fecal DM excretion.

Slurry cumulated from each group on the stall floor was collected into two 250 L

hermetically sealed containers (it included some washing water). The exceeding fecal and non-acidified urinary samples were mixed in 2:1 proportion and were also


120


introduced into containers. Slurry containers were kept sealed until its application on the field and were opened to take samples for chemical analysis. Fresh samples were

measured for pH and later on were oven-dried at 105ºC for DM determination and at 60ºC for N (N-Kjeldahl method), NH4

+-N (direct distillation with MgO), OM (Walkley,

1946), OC, SOC and C/N.

5.2.2. Field Experiment

The field experiment was carried out on a grassland in the Basque Country (northern Spain, 43º 18’ 20’’ N, 3º 53’ 0’’ W) during the spring of 2005. The region has a

temperate climate with typical annual mean temperature of 12ºC and rainfall of 1200 mm yr-1. During the current trial, mean air temperature was 20.5ºC (SD = 3.2) whereas

soil temperature was 20.1ºC (SD = 1.7). A typical permanent pasture (Lolium perenne

L. var. Herbus, 60%; Lolium hibridum L. var. Texi, 32%; Trifolium repens L. var. Huia,

8%) was sown at a density of 40 kg seeds ha-1 in the previous autumn. Before slurry applications, grass was cut the day before in order to ensure the maximum presence of

the slurry on the soil. Slurries were applied to surface using 10L buckets to ensure an even distribution of slurry. HF and LF derived slurries were randomly applied into plots

(2 x 1.5 m) in four-fold replication at a standard rate of 120 kg NH4+-N ha-1. A

treatment with no fertilizer was included as a control (C). Concentration of NH4+-N was

measured 2 days prior to application and verified at the time of surface application. As the same doses of NH4

+-N were applied, larger amount of diet HF-derived slurry were

applied per square metre.

5.2.3. N2O Emission Measurements

The N2O emissions were measured for 65 d. Fluxes were measured daily for the first 2

weeks and continued at a frequency of three times per week the following 2 weeks and twice per week during the last 2 weeks. Field measurements of N2O fluxes were

assessed using PVC chambers (volume 6.75 L and area 0.0314 m2) which were fitted tightly on to a frame. One frame per plot was inserted 3 cm into the soil and

repositioned daily to account for spatial variation. To minimize the effects of diurnal


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variation (Conrad et al., 1983), emissions were always sampled between 9:00 and 14:00 hours. Nitrous oxide emissions were measured using a closed air circulation technique

in conjuction with a photoacoustic gas analyser (Brüel and Kjaer 1302 Multi-Gas Monitor). Measurements were taken during 40 min after insertion of the chamber and

fluxes were calculated from the linear concentration increase in the headspace with time (R2 > 0.90).

5.2.4. NO Emission Measurements

Measurements of NO emissions lasted 29 d and were carried out using the open

chamber technique (Harrison et al., 1995). Charcoal filtered air was pumped through the chamber via polytetrafluorethylene (PTFE) tubing at a rate of 1 L min-1 to remove

ambient O3 from the air stream, thus eliminating reactions between ambient O3 and NO within the chamber. Concentrations of NO were measured at the air inlet and outlet of

the chamber using an NO-NO2-NOx chemiluminescence analyzer (Model AC31M, detection limit 0.08 mg kg-1; Environment SA, Poissy, France). Fluxes of NO were

calculated from the concentration differences between inlet and outlet air, the air flow rate through the chamber, and the surface area covered by the chamber.

5.2.5. N2O and NO Cumulative Losses

Cumulative N2O and NO emissions were estimated by averaging the rate of loss

between two successive determinations, multiplying the mean rate by the length of the period between measurements, and adding that amount to the previous cumulative total.

5.2.6. Soil Analysis

The soil was a poorly drained clay loam classified as a dystric gleysol (34% fine sand,

3% coarse sand, 34% silt, 29% clay in the top 10 cm). Soil pH was 6.6 in sampling area. During the trial, eight soil cores (0-10 cm depth and 2.5 cm diameter) from each plot

were taken when gaseous emissions were measured. Temperature was monitored every sampling day. Gravimetric water content was measured in soil samples for calculating


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the WFPS, assuming a soil particle density of 2.65 g cm-3 (Aulakh et al., 1991). Fresh soil (100 g) was extracted in 200 mL 2M KCl by shaking the soil suspension for 1 min

to determine the soil content of NH4+ and NO3

- by segmented flow analysis (Alpkem 1986, 1987). Organic carbon and SOC were analysed in soil samples collected the first

day and the last day of the trial.

5.2.7. Yield Production

After finishing N gas measurements, grass yield was assessed on one randomly chosen area of 1.8 m2 per sampling plot. Grass was dried in a forced-air oven at 70ºC for 48 h,

weighed, and ground. Nitrogen (N) concentrations were analysed by Macro Kjeldahl method. In addition, a representative subsample of each plot was taken and the botanical

composition for ryegrass, clover and other species determined.


Results of the feeding trial were analyzed by SAS 9.1 (SAS Institute, 2006). Data were analysed according to a crossover design, using the MIXED procedure. The model

included treatment, period, herd and treatment x herd interaction. Cow was considered a random effect within the herd factor. Overall treatment differences were examined using

least squares means. Nitrous oxide and NO emission rates were subjected to log-transformations (as indicated by the normality test of Shapiro and Wilk) to homogenise

variances. The LSD test was used for multiple comparisons of the instantaneous flux means and the soil mineral N evaluation. Differences between cumulative emissions from two slurries and the herbage yield were compared by ANOVA and the separation

of means between treatments by Duncan test. Differences among treatments were considered to be significant when P < 0.05.


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5.3.1. Composition of Diets and Ruminal Processes

The chemical composition of forages and the concentrate is presented in Table 5.1. Alfalfa hay had the highest CP content (197.9 g kg-1 DM) of selected ingredients

although triticale silage was the main CP source (129.9 g CP kg-1 DM) in HF, while concentrate (171.0 g CP kg-1 DM) was in LF. Concentrate, which was barley based to

increase the energy availability in the rumen, had a mean starch content of 378.6 g kg-1

DM. This concentration agreed with the starch content of the concentrate that Castillo et

al. (2001b) formulated for similar milk yields. Triticale silage was the main fiber source with 660.9 g NDF kg-1 DM and 420.5 g ADF kg-1 DM in HF and LF diets. The ADF

content was above the recommended 390 g ADF kg-1 DM for triticale silages (Baron et al., 2000) and therefore it might be reported as forage with a slight energy limitation.

Table 5.1. Mean (SD) chemical composition of ingredients.

Triticale Silage Alfalfa hay Concentrate

DM, g kg-1 FM 246.4 (43.5) 878.3 (2.1) 922.9 (0.04) Ash, g kg-1 DM 118.6 (21.3) 112.0 (13.0) 67.5 (15.6)

OM, g kg-1 DM 881.4 (12.9) 888.0 (1.7) 932.5 (1.9)

CP, g kg-1 DM 129.9 (2.1) 197.9 (9.0) 171.0 (10.6)

ADIP,% CP 6.47 (0.06) 9.04 (0.09) 3.27 (0.08)

NDF, g kg-1 DM 660.9 (21.8) 565.5 (19.9) 287.5 (6.4) ADF, g kg-1 DM 420.5 (5.0) 401.4 (1.8) 156.0 (0.1)

ADL, g kg-1 DM 59.6 (16.0) 117.9 (0.5) 12.3 (0.8) CNF, g kg-1 DM 68 (22.1) 96.9 (10.5) 461.8 (64.3)

Starch, g kg-1 DM 23.9 (0.8) 63.0 (2.2) 378.6 (24.6)

Nitrogen intake for LF diet was similar to the formulated level (498 g d-1) but the mean N intake for HF was 405 g d-1 because triticale silage intake was 3.3 kg d-1 lower than

initially expected. Thus, the mean CP content of diets varied and was 147 g kg-1 DM in HF and 155 g kg-1 DM in LF (Table 5.2). The high NDF content of the triticale silage

could have limited the VDMI of the silage, reducing the expected DMI in HF. In fact,


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Khorasani et al. (1993) reported that the high NDF concentration in the triticale silage (whose NDF content was lower than ours) might reduce feed intake.

Table 5.2. Ingredients and chemical composition of HF and LF diets. HF LF

Composition

Forages

Triticale silage, kg DM d-1 11.70 9.40 Alfalfa hay, kg DM d-1 1.75 1.75

Concentrate

Barley, kg DM d-1 1.84 3.68

Beet Pulp, kg DM d-1 0.69 1.38

Corn grain, kg DM d-1 0.69 1.38

Soybean Meal, kg DM d-1 0.64 1.29

Gluten Feed, kg DM d-1 0.55 1.10

Dicalcium phosphate, kg DM d-1 0.14 0.29

Sodium bicarbonate, kg DM d-1 0.04 0.08

Chemical composition

CP, g kg-1 DM 147 155 NDF, g kg-1 DM 543 460

NFC, g kg-1 DM 175 264

Soluble carbohydrate, g kg-1 DM 6.1 8.6

Starch, g kg-1 DM 116 185 CP:starch ratio 1.27 0.84

ME, MJ d-1 DM 167 194

As result of the variation in DMI, actual forage:concentrate ratio intake was 55:45 in LF

and 75:25 in HF diet. Nevertheless, this slight alteration did not change the initial hypothesis that the correct match between a rapid energy source (barley) (Foley et al.,

2006) and a high degradable protein source (triticale silage) (Khorasani et al., 1993)


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might contribute to improve the capture of ruminal ammonia and increase MPS in LF diet.

The in situ OM degradability trial indicated that the extent of OM degradability was

very similar in both treatments after 72 hours of incubation (845.2 g OM kg--1 DM). Nevertheless, OM degradability pattern was different within the first 4 hours of ruminal

incubation. The OM degradability was higher in LF, ranging from 463.7 to 514.0 g OM kg-1 DM, whereas in HF ranged from 406.6 to 457.2 g OM kg-1 DM. Barley grain,

which is characterized by the rapid and high ruminal starch degradability (80 and 90% of barley starch is digested in the rumen) (Nocek and Tamminga, 1991), could have

contributed to the higher OM degradability. In addition to OM degradability, the increasing ruminal VFA synthesis may be used as an indicator of ruminal fermentation.

Mean total VFA was slightly higher in LF diet with 521.8 mg mL-1 (SD = 41.2) than in HF with 458.0 mg mL-1 (SD = 61.7). We suggest the higher DMI and OM degradability

as responsible for higher total VFA synthesis in LF. Nevertheless, acetate:propionate ratio did not vary due to the diet modification (higher propionate synthesis was expected

in high starch LF diet), finding 3.64 and 3.62 ratios in LF and HF diets, respectively.

In relation to ruminal CP degradability, the extent and pattern did not differ between diets neither after 72 hours nor within the first 4 hours of incubation. Although higher

ruminal CP degradability was expected for HF diet due to the higher triticale silage intake, the VDMI limitation together with the higher soybean meal intake in LF could

have minimized the initially expected differences. Nevertheless, and despite the scarce difference between diets, higher amounts of CP degradability were found in HF diet. After 4 hours of incubation, CP degradability ranged from 286.9 to 473.1 g CP kg-1 DM

in HF whereas LF ranged between 315.4 and 386.9 g CP kg-1 DM. Therefore, data recorded from OM and CP degradability rates pointed out that LF diet might be related

to a lower ruminal NH4+-N concentration and an enhanced MPS. However, ruminal

liquid samples had lower NH4+-N concentration in HF (P < 0.05), due to the lower N

intake. Ruminal NH4+-N content ranged between 5.42 and 6.68 mmol L-1 for LF

whereas ranged from 1.68 to 3.67 mmol L-1 for HF. In relation to ruminal MPS, the

initial scope of getting a higher MPS was partially achieved because the MPS was slightly higher in LF (1.68 kg day-1) than in HF diet (1.59 kg day-1) (P > 0.05).

Available evidence indicates that rumen ammonia-N concentrations of 5 to 11 mmol L-1


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are needed to maximize flows of microbial N from the rumen (Hume et al., 1970). Thus, MPS might be limited in HF diet due to the low ruminal NH4

+-N concentration. In

addition to the ruminal N availability, MPS is often limited by energy supplied from OM fermentation (Oba and Allen, 2003). In this sense, fermented OM was lower in HF

(11.6 kg MO d-1) than in LF (14.3 kg MO d-1), which might lead to a reduced energy supply for MPS. However, despite the low ruminal NH4

+-N and OM availability in HF

diet, daily EMPS (expressed as grams of microbial N per kg of fermented OM) was slightly higher in HF (21.9 g microbial N kg-1 fermented OM) than in LF (18.8 g

microbial N kg-1 fermented OM). Apart from the amount of ingested degradable protein and energy, the synchrony at which nutrients become available is also important for

MPS (Bach et al., 2005) and might explain the current results. The nutrition management in this trial (no TMR rations with ad libitum forage offer) could contribute

to an imbalance between energy and protein availability and therefore MPS improvement may have not been as good as expected for LF.

5.3.2. Milk Yield, N Use Efficiency and N Excretion

LF diet reached higher milk yield (3.2 kg d-1 more) and 4% FCM (3.3 kg d-1 more)

during the current trial (P < 0.05) (Table 5.3). Khorasani et al. (1993, 1996), who formulated two triticale silage based rations with 50:50 forage concentrate ratio and 3.0

kg d-1 CP intake, obtained higher milk yields (about 25 kg d-1) than the ones found in our study. The higher fiber content of triticale silage in the current trial might have

limited milk production although feed efficiency ratio (expressed as milk yield DMI-1) was not improved through concentrate use (P > 0.05). Milk quality parameters (CP, fat) and MUN were not affected by treatments (P > 0.05) (Table 5.3). Milk CP content was

3.07% in HF and LF, which might be explained by the scarce differences in ruminal CP degradability and MPS (Firkins et al., 2006). The high milk fat contents (4.10% and

4.13% in HF and LF, respectively) would be related to the high NDF content in forages (Zebeli et al., 2006). Milk urea N did not differ between treatments (7.7 and 8.2 mg dL-1

in HF and LF, respectively) in spite of the differences in dietary CP intake and the protein-energy ratio between diets (Rajala-Schultz et al., 2003). The MUN content was

positively related to N intake (P < 0.05) but was not associated to the forage:concentrate ratio (P > 0.05). In this sense, Broderick and Clayton (1997) indicated that MUN is


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more closely related to changes in dietary CP content than to the ratio of dietary CP to energy intake. However, all relationship derived from dairy nutrition and MUN may not

be universally applicable due the physiological variability of dairy cattle (Nousiainen et al., 2004), especially when a small number of dairy cows is considered.

Increase in milk yield has been related to higher milk NUE and lower N excretion per

litre of milk produced (Rotz, 2004). In this study, mean milk NUE was 21.0% and was not affected by treatments (P > 0.05) (Table 5.3). Although the mean NUE was

consistent with data reported by Tamminga (1992) for Dutch dairy cows (20%), our efficiency data were largely lower than those reported more recently for high-producing

Holstein cows (Ipharraguerre et al., 2005; Olmos-Colmenero and Broderick, 2006). Castillo et al. (2001b) also reported milk NUE values around 25% for similar CP intake

levels due to higher mean milk yields (24 kg d-1). The unexpected low milk production during the trial, especially for LF, might have contributed to reach these lower NUE

values. Similarly, as a result of limited milk yield, N excretion per unit of milk produced was also similar between diets (Table 5.3) (P > 0.05), and was above the

excretion level observed in commercial dairy farms in the Basque Country (Arriaga et al., 2009), in which mean N intake (562 g d-1) and milk yields (29.7 kg d-1) were higher.

Castillo et al. (2000) and Yan et al. (2006) reported that, on average, 72% of the N

consumed is excreted in feces and urine. In the current trial excreted N averaged 67.5% and 63.5% for HF and LF diets, respectively (Table 5.3). The lower proportion of

excreted N in the current trial might have been due to the estimations assumed through the experiment. Urinary N output accounted for 31.7% and 30.8% of N intake for HF and LF (P > 0.05), whereas fecal N output accounted for 35.8% and 32.7% of ingested

N for both diets (P > 0.05). Results disagree with Castillo et al. (2001b) who reported an exponential increase of urinary N excretion when N intake exceeded 400 g d-1. Urinary

urea N content, which is the main N compound in the urine, did not differ between treatments (P > 0.05) with 3.88 g L-1 in LF and 3.22 g L-1 in HF, respectively.

Considering the daily N excretion data from both treatments (Table 5.3), and according to previous NUE data, either HF or LF had similar N excretion per unit of milk

produced. Mean excretion was about 15 g L-1 in both treatments (Table 5.3) and was above the excretion level observed in commercial dairy farms in the Basque Country

(Arriaga et al., 2009).


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Table 5.3. Effect of dietary manipulation on N metabolism from HF and LF diets.

HF LF SE P

DMI, g d-1 18.1 20.4 0.54 < 0.05

N intake, g d-1 405 498 10.5 < 0.05 Milk output

Milk yield, kg d-1 18.2 21.4 0.61 < 0.05 4% FCM, kg d-1 18.5 21.8 0.64 < 0.05

Milk CP, % 3.07 3.07 0.07 0.89 Milk fat, % 4.10 4.13 0.09 0.80

MUN, mg dL-1 7.7 8.2 1.2 0.62

Milk protein N, % of N intake 21.6 20.7 1.6 0.82 Urinary excretion

Urinary N excretion, g d-1 128.5 153.5 9.6 < 0.05 Urine N, % of N intake 31.7 30.8 4.2 0.38

Urea N, g L-1 3.22 3.88 0.28 0.08 Microbial Protein, kg d-1 1.60 1.68 0.09 0.29

Fecal excretion Fecal N excretion, g d-1 144.8 162.8 38.5 < 0.05

Fecal N, % of N intake 35.8 32.7 0.76 0.96 Total N excretion

Total N excretion, % of N intake 67.5 63.5 - - N excretion per milk, g L-1 15.0 14.8 1.2 0.76

5.3.3. Slurry Composition

Nitrogen content of slurries did not differ between treatments (Table 5.4) due to the

high N content variability of fecal and urinary samples (Table 5.3). On the contrary, NH4

+-N was higher in LF diet-derived slurry (Table 5.4). The lower pH of LF diet-

derived slurry (Table 5.4) might partially explain the higher NH4+-N content due to a

lower NH3 volatilization loss when containers were periodically ventilated. As slurry

was applied to soil on the basis of the concentration of NH4+-N (120 kg NH4

+-N ha-1), the amount of slurry applied per square meter was higher in HF treatment. Organic


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matter and OC content of the two slurries were similar between both slurries (Table 5.4) and soluble OC percentage over OC content was about 5.8% in both slurries.

Table 5.4. Chemical composition of HF and LF diet derived slurries. HF Slurry LF Slurry

DM, % 11.1 9.5

pH 7.74 7.02

N, % w fw-1 0.38 0.37

NH4+-N, %w fw-1 0.11 0.16

MO, %DM 56.0 54.9

Organic Carbon, g kg-1 369.2 375.0

Soluble Organic Carbon, mg kg-1 26.7 21.6

C/N 17.0 15.3

5.3.4. N2O Fluxes

Mean daily N2O emission from control plots was 10.2 g N2O-N ha-1 day-1 (SD = 9.3). Nitrous oxide fluxes from zero-N control plots were expected below 100 g N2O-N ha-1

day-1 due to previous results obtained from the same grassland through in situ N2O emission measurements (Menéndez et al., 2006) or N2O fluxes from incubated soils (del

Prado et al., 2006). Slurry application on grassland increased significantly N2O emissions from both treatments compared with control plots (P < 0.05), but the daily

mean N2O fluxes did not differ between the two treatments (P > 0.05). Mean daily emissions from HF and LF slurries were 84.7 g N2O-N ha-1 day-1 (SD = 36.4) and 75.8

g N2O-N ha-1 day-1 (SD = 42.4), respectively. The pattern of N2O flux between treatments did not differ along the experimental period (P > 0.05) (Figure 5.1). The

spatial variability in fluxes was notoriously high (Ball et al., 2000) which contributed, together with the temporal variability, to large sources of uncertainty in N2O fluxes at

field scale (Velthof et al., 2000). Soil C availability and the heterogeneity of anaerobic “microspace” distribution have been described as main variability sources of N2O

emission (van Cleemput, 1998). Soil samples measured for OC content did not differ between treatments (P > 0.05), as OC and SOC content in the slurries were similar (P >

0.05) (Table 5.4). Mean values for soil OC were 21.7 and 23.2 g kg-1 for plots amended


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with HF and LF slurries, respectively, at the beginning of the trial and 18.6 and 17.2 g kg-1 at the end. As expected, control plots had lower soil OC content during the two

periods (16.7 g kg-1).

Figure 5.1. Nitrous oxide emission pattern from HF and LF treatments.


The highest emission values were 150.5 g N2O-N ha-1 day-1 for plots amended with slurry from HF diet and 177.4 g N2O-N ha-1 day-1 in LF diet-derived slurry after 34 d of

trial. Nitrous oxide emissions are closely related to WFPS (Davidson, 1991) and the highest N2O emission peaks were observed at 60-70% of WPFS (Figure 5.1). This fact

was remarkable because the trial was carried out in a dry spring-summer period in 2005 when rainfalls were limited (daily mean rainfall = 0.6l mm) and WFPS was usually

below 60%. Cumulative rainfalls in sampling grassland were 32.8 mm, distributed in 18 d. Based on the “Hole-in-the pipe” conceptual model proposed by Firestone and

Davidson (1989), the main N2O fluxes from nitrification happen between 30% < WFPS < 60% and from denitrification between 60% < WFPS < 90%. In addition to soil WFPS

content, NH4+ availability for nitrifier and NO3

- and OC availability for denitrifier microbes are essential factors to control N2O fluxes to the atmosphere (Firestone and

Davidson, 1989). Figure 5.2 and Figure 5.3 show the evolution of NH4+-N and NO3

--N.


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Figure 5.2. Soil ammonium content in the grassland soil.


Figure 5.3. Soil nitrate content in the grassland soil. The vertical bars indicate LSD at 0.05 between treatments for each sampling time.

Thus, soil NH4+-N content in HF and LF slurries reached values found in control plot

after 40 days (10 kg NH4+-N) (Figure 5.2). In addition to NH3 volatilisation losses

which removed 12.6 and 5.7 kg NH3-N from soils amended with HF and LF slurries, respectively (Merino et al., 2008), the disappearance of NH4

+-N from the soil was

mainly dependant on the nitrification process. Soil WFPS was below 60% during the whole trial except for the first week after slurry fertilisation, and therefore, nitrification

process might have been favoured. As a result of the microbial nitrifier activity, the


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maximum values of soil NO3--N content reached 38.5 kg NO3

--N from HF slurry and 55.7 kg NO3

--N in LF slurry 33 days after slurry application, but did not differ between

treatments due to the high variability (Figure 5.3) (P > 0.05). Although better soil conditions could have taken place for nitrification during the field trial, nitrification and

denitrification can happen simultaneously in the soil (Abbasi and Adams, 1998), since aerobic and anaerobic microsites can exist within the same soil aggregate (Kuenen and

Robertson, 1994). Peaks of N2O might be due to the contribution of both processes in this trial. In fact, the high N2O peaks observed during the first week happened when 15

kg NO3--N had already produced by nitrification microbes and soil WFPS achieved a

value above 60%, which was optimum for denitrification processes (Davidson, 1991).

Similarly, the second N2O peak occurred after 30 d, when nitrification lead to maximum NO3

--N levels and at the same time soil WFPS was above 60%. In this sense, del Prado

et al. (2006) confirmed through a laboratory experiment carried out on these soils that both processes were likely to happen simultaneously, depending on N-NH4

+-N and NO3-

-N availability. The cumulative N2O emission was 5.8 kg and 5.0 kg N2O-N ha-1 for HF and LF slurries, respectively, and was not significantly different (P > 0.05). Mean

emission from control plots was 0.8 kg N2O-N ha-1 (P < 0.05) (Table 5.5).

Table 5.5. Cumulative N2O and NO emissions from HF and LF treatments.

N2O-N NO-N

kg N ha-1 g N kg-1

slurry ha-1

g N milk kg-1

kg N ha-1 g N kg-1

slurry ha-1

g N milk kg-1

Control 0.8b - - 0.17b - - HF Slurry 5.8a 0.06 7.93 0.51a 0.006 0.70

LF Slurry 5.0a 0.08 5.79 0.57a 0.01 0.66 a,bDifferent superscripts within the same column indicate significant differences (P < 0.05).

Because slurry application on grasslands is usually carried out by farmers by discharging onto the soil high volume tanks to empty the slurry pit, the difference of

N2O emission related to the amount of applied slurry should be considered from a environmental point of view. In this sense, differences between treatments were not

significant (P > 0.05), although N2O flux per applied slurry unit (kg) was slightly higher for LF slurry (0.08 g N2O-N kg-1 ha-1) compared with HF slurry (0.06 g N2O-N kg-1 ha-

1) after 65 d of measurements (Table 5.5). Similarly, considering that the main purpose


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of the dairy activity is the production of milk, it would also be advisable to relate N2O emissions to the herd milk production. Data from the current trial showed a lower

emission per kilogram milk produced from LF slurry (5.79 g N2O-N ha-1 milk kg-1) than from HF slurry (7.93 g N2O-N ha-1 milk kg-1) (Table 5.5). Therefore, a responsible

management of the slurries on grasslands justifies an adequate nutritional strategy for dairy herds from an environmental and productive point of view.

5.3.5. NO Fluxes

Mean NO emission from control plots was 6.1 g NO-N ha-1 day-1 (SD = 3.9). Nitric oxide emission data from the previous trials carried out in the same grassland never

exceeded 15 g NO-N ha-1 day-1 for control plots (Menéndez et al., 2006). The slurry application on grasslands increased the mean NO emissions compared with control

plots, with daily averages of 17.2 g NO-N ha-1 day-1 (SD = 11.3) and 19.1 g NO-N ha-1

day-1 (SD = 11.5) for HF and LF slurries, respectively. The increase of NO emission

due to the application of organic fertilizers supports previous data in which fertilised soils had greater NO emissions than unfertilised soils (del Prado et al., 2006). However,

dietary treatments did not affect mean daily NO emission (P > 0.05) nor NO flux patters (Figure 5.4).

The NO emissions from soils are affected by changes in soil water content (Skiba et al.,

1992). In this sense, del Prado et al. (2006) and Menendez et al. (2006) reported that WFPS and NO-N emission in the same grassland were negatively related. When soil

WFPS increase, anaerobic sites in soil increase, and nitrification, the main process involved in NO production, is decreased (Davidson, 1991). Therefore the higher NO peaks increased in both treatments between Day 9 and 14 when WFPS contents

decreased between 40 and 52%. Maximum NO flows reached values around 40 g NO-N ha-1 day-1 for both treatments (P > 0.05) (Figure 5.4). The fact that in fertilised soils,

NH4+-N (nitrifiable N) content was higher than NO3

--N (denitrifiable N) content during the initial 12 days after fertiliser application (Figure 5.2 and Figure 5.3) suggest that

nitrification, and not denitrification, was the main process of NO formation (Slemr and Seiler, 1984). This is consistent with previous reports where maximum NO emissions

coincided with maximum nitrification rates (Pinto et al., 2004). Cumulative NO


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emissions from both treatments did not differ between treatments (P > 0.05). Accumulated NO emission was slightly lower in HF slurry (0.51 kg NO-N ha-1) than in

LF (0.57 kg NO-N ha-1) (Table 5.5). Emission from applied kg of LF slurry was numerically higher (0.01 g NO-N kg-1 ha-1) than emission from HF slurry (0.006 g NO-

N kg-1 ha-1) and differences in NO emission related to herd milk production averaged 0.70 and 0.66 g NO-N ha-1 milk kg-1 for HF and LF diets, respectively (Table 5.5).

Figure 5.4. Nitric oxide emission pattern from HF and LF treatments.


5.3.6. Grass Yield and N Uptake

Mean grass yield in plots fertilized with slurry was not improved compared with control plots (P < 0.05) (Table 5.6). Cumulative grass yield was 1.8 t dry matter ha-1 and 1.2 t

dry matter ha-1 for HF and LF slurries, respectively, and control plots reached 1.6 t dry matter ha-1. These results were totally unexpected when the trial was designed, but the

strong rainfall limitation for the spring and summer in 2005 reduced the grass yield in slurry amended plots. Although the annual rainfall was 1294 mm in the sampling site,

close to the average annual amounts, rainfalls during the trial period were very scarce (32.8 mm in 18 d). In fact, Estavillo et al. (1996) reported higher grass yields in the

same grassland for similar periods, when annual rainfall was 1234 mm, and more than 7.0 t dry matter ha-1 were recovered from April to June. Grass N uptake did not differ


135


between treatments due to the high variability observed among plots (P > 0.05). The total N uptake was 54.5 and 43.6 kg N ha-1 for HF and LF slurries, respectively,

representing 45.4% and 36.3% of applied NH4+-N. Botanical composition of the

grassland did not differ (P > 0.05) and ryegrass and white clover were the main species

in the grasslands (63.6%) in spite of the significant presence of other plant species. The lack of fertilisation resulted in slightly higher white clover proportion in the untreated

zero-N control plots.

Table 5.6. Grass yield, botanical composition and N uptake from HF and LF slurries.

Composition Grass yield

(t DM ha-1)

N uptake

(kg N ha-1)

Ryegrass

(%)

Clover

(%)

Others

(%)

Control 1.6a 56.4a 10.8a 40.4a 48.8a

HF Slurry 1.8a 54.5a 25.3a 26.3a 48.4a

LF Slurry 1.2a 43.6a 19.9a 37.3a 42.7a


5.4. Conclusions

Dietary strategies such as the change of forage:concentrate ratio or N intake variation

were able to modify the mean milk yield of the herds, the amount of excreted N and the subsequent slurry composition. Slurry NH4

+-N content decreased in the high forage and

low N content diet. Nevertheless, the application of 120 kg NH4+-N ha-1 on grassland

did not affect the amount and pattern of N2O and NO between treatments. The judicious

management of the slurries on grasslands may justify an adequate nutritional strategy of dairy herds from an environmental and productive point of view.


6 DIETARY CRUDE PROTEIN MODIFICATION ON AMMONIA AND NITROUS OXIDE CONCENTRATION ON A TIE-STALL DAIRY BARN FLOOR

______________________________________________________________________


139

Dietary Crude Protein Modification on Ammonia and Nitrous Oxide Concentration on a Tie-Stall Dairy Barn Floor

Abstract

Dietary CP reduction is considered a useful strategy to minimize cow N excretion, and NH3 and N2O emissions. The aim of the current work was to relate dietary CP

modification with the whole animal N balance and the subsequent NH3 and N2O concentration on a tie-stall barn floor. The effect of temperature on NH3 and N2O

concentration was also studied. Three Holstein mid-late lactating cows were confined in separate tie-stalls and randomly assigned to three diets with varying CP content (LP:

14.1%; MP: 15.9%; HP: 16.9%). Increasing N intake (from 438.6 to 522.8 g N d-1) improved milk yield (from 22.1 to 24.2 kg d-1). However, N use efficiency tended to

decrease with increasing dietary CP, as showed milk NUE (from 23.9% to 22.6%), MUN (from 15.4 to 18.7 mg dL-1) and excreted N per milk yield unit (from 14.7 to 16.4

g N kg-1 milk). As a result of the higher N excretion, NH3 concentration on the dairy barn floor increased (LP: 7.1 mg NH3 m-3; MP: 10.4 mg NH3 m-3; HP: 10.8 mg NH3 m-

3). On the contrary, N2O concentration did not respond to dietary manipulation (mean 1.1 mg N2O m-3). Temperature, which ranged between 12.6 and 18.0ºC, did not affect

NH3 and N2O concentrations at stall level. However, when fecal and urinary samples were incubated at 4ºC, 19ºC and 29ºC in the laboratory, all dietary treatments increased

NH3 concentration in relation to incubation temperature, especially MP and HP diets. On the contrary, N2O concentration was negatively related to increasing temperatures. As conclusion, data from the current trial demonstrate that lowering dietary CP

minimizes NH3 concentration on dairy stall floors although temperature will control the rate of NH3 volatilisation. On the other hand, N2O concentration is not affected by

dietary treatments on tie-stall floors.

Key words: Dairy nutrition, crude protein, ammonia, nitrous oxide, stall. Abbreviation key: ADF = Acid detergent fiber, BW = Body weight, CP = Crude

protein, DM = Dry matter, DMI = Dry matter intake, FCM = Fat corrected milk, MPS = Microbial protein synthesis, MUN = Milk urea nitrogen, NDF = Neutral detergent fiber,

NFC = Non fibrous carbohydrates, NH3 = Ammonia, NH4+ = Ammonium, N2O =


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Nitrous oxide, NUE = Nitrogen use efficiency in milk, OM = Organic matter, TMR = Total mixed ration, UUN = Urinary urea N.

6.1. Introduction

Animal husbandry results in considerable N losses to the atmosphere in terms of

ammonia (NH3) and nitrous oxide (N2O) emissions (Amon et al., 2001; Amon et al., 2006). Overall, animal husbandry contributes to about 40% of the global NH3 emission

(van Aardenne et al., 2001) with dairy farming activity being the largest source of NH3

emission (Bussink and Oenema, 1998). On the other hand, 63% of global N2O

emissions can be attributed to agricultural production, where dairy production systems are the main contributors (Weiske et al., 2006). At dairy farm level, the main sources of

NH3 and N2O fluxes are the dairy barn, the manure stored in slurry pits and the manure applied on grasslands (Amon et al., 2001). Although less studied than NH3 fluxes from

grasslands, the large contribution of dairy stalls to NH3 emissions (around 20%, Frank et al., 2002) has promoted several studies (Monteny and Erisman, 1998; Powell et al.,

2008). On the contrary, there are fewer studies on N2O fluxes from dairy stalls (Amon et al., 2001; Külling et al., 2003).

According to Gothenburg Protocol (1999) and Kyoto Protocol (1997), European

countries have been obliged to adopt practices which contribute to minimize national NH3 and N2O emissions from different activities, including animal husbandry. Many

strategies have been already tested to affect NH3 fluxes from barn floors (animal management, animal nutrition, bedding, manure management, ventilation, etc)

(Monteny and Erisman, 1998). Among all these strategies, dietary manipulation is considered an effective and easy strategy to minimize the impact of NH3 fluxes in stalls

(de Boer et al., 2002; Frank et al., 2002). On the contrary, little information is still available about the effect of dietary modification on N2O emission (Cárdenas et al., 2007). Dietary composition of livestock has an effect on total N, ammoniacal-N and

soluble organic fraction of manure (Velthof et al., 2005), and therefore modifications on N2O emission might be expected from diet manipulation.


141

DIETARY CRUDE PROTEIN MODIFICATION ON AMMONIA AND NITROUS OXIDE CONCENTRATION ON A TIE-STALL DAIRY BARN FLOOR

This work aimed to study the effect of CP content in the ration on N balance of lactating dairy cows and to relate it with the accumulation of NH3 and N2O on a barn floor. A

second objective was to study the effect of temperature on NH3 and N2O concentrations at different CP content in the ration.


6.2.1. Animals, Diets and Experimental Design

Three first lactation Holstein cows (210 ± 12 days in milk, 644 ± 8 kg BW and 27 kg of

milk per day) were selected from the experimental dairy herd of I.E.S. “La Granja” (Cantabria, Spain). Cows were randomly assigned to three diets in a 3 by 3 Latin Square

design. The duration of each experimental period was 15 d, with 11 d for diet adaptation and 4 d for data collection. Cows were held in separated metabolic tie-stalls to control

the individual feed intake and collect the whole fecal excreta. Cows had free access to drinking water and mineral supplements.

The experimental diets were formulated to contain three levels of CP on DM basis (LP:

14%; MP: 16%; HP: 17%), being isoenergetic and with a forage:concentrate ratio of 52:48. Corn silage and alfalfa hay were used as forage sources and concentrates were

formulated with varying amounts of soybean meal to fit diet CP content (Table 6.1). Diets were fed twice daily as a total mixed ration (TMR) at 09:00 and 17:00, and

provided to produce at least 10% refusals. Total refusals were weighed the next morning to determine the daily dry matter intake (DMI) for each cow. Samples of individual feedstuffs were taken for laboratory analyses twice weekly during the adaptation period,

and during 2 days after finishing the experimental period. Feedstuffs were analyzed for dry matter content (DM) at 105ºC (AOAC, 1990), ash (AOAC, 1990), organic matter

(OM) (AOAC, 1990), fat (Tecator Soxtec System), CP (Kjeldahl N method by KjeltecTM 2300), neutral detergent fibre (NDF), acid detergent fibre (ADF) (van Soest

et al., 1991) and non fibrous carbohydrates (NFC) was estimated as 100 – (fat + CP + ash + NDF).


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Table 6.1. Composition of LP, MP and HP diets.

LP MP HP

Ingredients

Corn silage, kg DM d-1 5.27 5.42 5.41

Alfalfa hay, kg DM d-1 5.33 4.60 3.75

Beet pulp, kg DM d-1 0.92 0.89 0.89

Barley grain, kg DM d-1 4.52 2.73 1.84

Corn grain, kg DM d-1 0.00 0.91 1.84

Beans, kg DM d-1 0.84 1.07 0.45

Soybean Meal, kg DM d-1 0.00 1.18 2.73

Cottonseed, kg DM d-1

Tallow, kg DM d-1

Dicalcium phosphate, kg DM d-1

1.05

1.05

0.10

1.01

1.01

0.09

1.03

1.03

0.08

Limestone, kg DM d-1 0.18 0.17 0.16

Sodium bicarbonate, kg DM d-1 0.10 0.09 0.08

Vit-Min, kg DM d-1 0.10 0.09 0.08

Chemical composition

CP, g kg-1 DM 141 159 169

NDF, g kg-1 DM 377 373 368

ADF, g kg-1 DM 206 208 211

Fat, g kg-1 DM 37 39 39

NFC, g kg-1 DM 346 335 343

Ash, g kg-1 DM 99 94 79


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Daily deposited whole fecal samples were collected from each cow after the morning milking and weighed for their quantification. A subsample (300 g) was taken for

laboratory analyses and incubations, and remaining was flushed with some water into the slurry pits located behind each metabolic stall under a slatted floor (0.51m3).

Samples were stored at -20ºC until analysis. Samples were oven-dried at 105ºC in a forced air oven for DM determination, and at 55ºC for nitrogen (N) and carbon (C)

analysis (LECO CNS 2000 analyser, LECO Corporation, MI, USA). Fecal pH was also measured (Schott CG-843 pH-meter). Apparent digestibility of DM was calculated as

the amount of digested DM (ingested DM – excreted DM in feces) per unit of ingested DM. Daily fecal N excretion was calculated multiplying the whole fecal amount and N

content in each subsample.

Spot urine samples were collected from each cow through a non-invasive method using buckets. Individual urine samples (two or three per cow) were composited to provide

one urine sample per day (0.5-2.0 L d-1). A 200 mL subsample was acidified with 10% H2SO4 solution to analyze for N (Kjeldahl N method), urea N (UUN) (Douglas and

Bremner, 1970), purine derivatives and creatinine (Balcells et al., 1992). Non acidified urine subsamples (100 mL) were separated for laboratory incubations and were

analyzed for pH (Schott CG-843 pH-meter), N and urea using the same methods. Urine volume was computed using creatinine as a marker assuming a daily creatinine

excretion of 22.6 mg kg-1 of BW (Gonda et al., 1996). Daily urinary N excretion was calculated multiplying computed urine volume and N content in each sample. Data from

purine derivatives (allantoin, uric acid, xanthine and hypoxanthine) were used for estimating the ruminal microbial protein synthesis (MPS) (Chen and Gomes, 1992).

Cows were milked twice daily at 08:00 and 18:30 and milk weights were recorded at each milking. Individual milk samples (a.m./p.m. composite) were placed in a sealed

container and preserved with 2-bromo-2-nitropropane-1,3-diol. Milk samples were shipped to the Milk Institute of Santander (Cantabria, Spain) and analyzed for CP and

fat (Milkoscan 4000), and milk urea N (MUN) (MilkoScan FT6000, Foss Analytical). Nitrogen use efficiency (NUE) was calculated as N in true milk protein (conversion

factor, 6.38) multiplied by the daily milk yield, and expressed as a percentage of total N intake.


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6.2.2. On-Farm Trace Gas Measurements

Concentrations of NH3 and N2O were measured using a photoacoustic infrared gas analyzer (Brüel and Kjaer 1302 Multi-Gas Monitor; detection limit 0.2 ppm for NH3

and 0.03 ppm for N2O) equipped with an internal pump. Gas measurements were always carried out between 10:00 and 13:30 to minimize the effects of diurnal variation

(Conrad et al., 1983) and began 3 hours after cleaning the excreta cumulated the day before. Gas measurements were always conducted on freshly deposited feces and urine.

Measurements were carried out from LP to HP diet to prevent any carryover effect between treatments. Gases were measured in each stall through a Teflon tube at 15 cm

distance over the barn floor, which was continuously repositioned 30-50 cm behind each cow until the steady-state value was reached for NH3 (approximately 20 minutes).

Gas sampling from nearby slurry pits was avoided if possible. Atmospheric temperature (HI 935005 thermometer, Hanna Instruments) was recorded at the beginning and at the

end of each sampling. One control point was established next to the farm (200 m) but free of dairy cow influences to record NH3 and N2O at atmospheric concentrations.

Stall (30 m2) was naturally ventilated, where air flow was passing from an air opening

on the western wall (2 m2) to the stall access door (5 m2) located on the eastern wall. The door was always closed when gases were measured to avoid lateral air flow through

metabolic tie-stalls, which might promote carryover effects among treatments. Tie-stalls were physically separated through 1 m height cemented walls to prevent lateral air

contamination.

6.2.3. Laboratory Incubations

A slurry incubation trial was carried out in the laboratory at 3 temperatures (4ºC, 19ºC

and 29ºC) to simulate the effect of temperature on NH3 and N2O concentration from different CP diet-derived slurries. Minimum temperature represented the mean temperature in winter in the Basque Country (northern Spain), 19ºC was the mean

temperature in summer and 29ºC represented a typical hot summer day in the territory. Incubations were conducted inside a plant growth chamber (Sanyo MLR-351H), whose

temperature variability was: 4 ± 1.3ºC, 19 ± 0.2ºC and 29 ± 0.2ºC. Slurry was created


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mixing 60 g of fresh fecal samples and 30 mL of non acidified urine samples (2:1 ratio) into a 1-L glass jar. This ratio was established according to the fecal and urinary

excretion dataset obtained previously in commercial dairy farms from the Basque Country (Arriaga et al., 2009). The jars were sealed with lids fitted with rubber stoppers

in which one Teflon tube was inserted and connected to a photoacoustic infrared gas analyzer (Brüel and Kjaer 1302 Multi-Gas Monitor). A polyethylene tube was used to

transport the outflow gas from the photoacoustic analyzer into the jars, avoiding therefore the disturbance of jar atmosphere. Before starting measurements, jars and

slurries were adapted to the corresponding temperature. Measurements were carried out for 40 min to consider the concentration within the linear increase of the gas. Non-

incubated slurry subsamples were dried at 105ºC for DM analysis (AOAC, 1990) and at 55ºC for N and C analysis (LECO CNS 2000 analyser, LECO Corporation, MI, USA).


On-farm and laboratory dataset were analyzed by SAS 8.0 (SAS Inst., Inc., Cary, NC, 1999), using the PROC MIXED procedure. Data were previously analyzed for

normality test of Shapiro Wilk to comply with the “equal variances” assumption. Data from manure incubation were base-10 log transformed to fit the normality test. Two

model sets were used for statistical analysis. First, a model was carried out to test the effect of the diets on NUE, N excretion, milk parameters and on-farm NH3 and N2O

concentrations. In this model, diet was the fixed effect and animal was used as a random effect. The interaction of diet and day was studied. The second model was used for

laboratory NH3 and N2O concentrations and included diet and temperature as fixed effects, and animal as a random effect. The interaction between diet and temperature was also analyzed. Differences were analyzed by least squares means, and when

differences were relevant, LSD test was used to determine significant differences among treatments. Univariate and multivariate regressions were carried out to test the effect of

nutritional parameters on NH3 and N2O concentration on dairy barn floor and to study the effect of manure source on NH3 and N2O accumulation in laboratory incubations.

Colinearity tests were carried out in multivariate regressions. Regression models for on-farm dataset included N intake, dietary CP content, fecal N content, urinary N content,

UUN, MUN and milk yield. Regression model for incubations included fecal N, pH and


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N content, urinary pH, N, and UUN content, and slurry OM and N content. Temperature was also introduced in this model to test its effect on NH3 and N2O accumulation.

Differences among treatments were considered significant at P < 0.05


6.3.1. Feeding Trial

Table 6.1 shows the ration and chemical composition of the diets. Feed intake did not

differ among treatments and averaged 19.4 kg DM d-1 (P > 0.05). Forage:concentrate ratio was established around 50:50 for all diets. As expected, CP levels were different

between treatments (P < 0.05) (Table 6.1). This range of dietary CP concentrations was selected to represent target CP contents in lactating dairy cow nutrition. Thus, HP diet

represented dietary CP recommended for high yielding dairy cows (Ipharraguerre and Clark, 2005; Olmos-Colmenero and Broderick, 2006). Medium protein diet agreed with

some authors who reported that 16.0% CP did not compromise the performance of midlactation cows (Castillo et al., 2001). Finally, LP diet represented dietary CP content

able to maintain milk productivity in mid and late lactation cows (Frank et al., 2002).

6.3.2. Milk Yield and Composition

Milk yield and MUN differed as a result of dietary treatment (P < 0.05) while fat-corrected milk (FCM), milk CP and fat contents, and feed efficiency were not affected

(P > 0.05) (Table 6.2). Milk yield increased with higher CP content of the rations and averaged 24.2 kg d-1 in HP, 23.7 kg d-1 in MP diet and 22.1 kg d-1 in LP diet. When milk

yields were corrected by fat content (4% FCM), data did not show any difference between treatments because milk fat % tended to increase with the lower CP (P < 0.10)

(Table 6.2). Feed efficiency, defined as FCM/DMI, was not affected by dietary treatment (P > 0.05) and averaged 1.14 kg FCM kg -1 DMI. Milk protein content was slightly higher in HP diet in relation to MP and LP treatments (Table 6.2). Similarly,

MPS increased with higher CP rations (Hoover and Stokes, 1991), differing between LP diet (1.17 kg MPS), and MP and HP treatments, which averaged 1.48 and 1.52 kg MPS,


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respectively (P < 0.05). Milk urea N increased with higher N intake treatments (P < 0.05) and LP diet had the lowest MUN value (Table 6.2).

Table 6.2. Effect of dietary CP content on N metabolism. LP MP HP SE P

N intake, g d-1 438.6 490.8 522.8 7.5 < 0.05

Milk output

Milk yield, kg d-1 22.1 23.7 24.2 0.5 < 0.05

%4 FCM, kg d-1 21.9 22.6 22.5 0.6 0.66

Milk CP, % 3.01 3.02 3.10 0.03 0.15

Milk fat, % 3.95 3.70 3.60 0.1 0.06

MUN, mg dL-1 15.4 18.7 17.8 0.4 < 0.05

NUE, % 23.9 22.9 22.6 0.6 0.24

Urinary excretion

Urinary N excretion, g d-1

Urine volume, kg d-1

197.8

25.0

255.0

34.3

255.2

32.6

18.1

2.9

0.05

0.08

Urine N, % of N intake 46.0 52.4 48.3 4.0 0.51

Urea N, g L-1 5.77 6.22 7.31 0.5 0.08

Microbial CP, kg d-1 1.17 1.48 1.52 0.1 < 0.05

Fecal excretion

Fecal N excretion, g d-1 117.6 129.6 112.0 3.7 < 0.05

Fecal DM, kg d-1 4.53 3.75 3.79 0.1 < 0.05

Fecal N, % of N intake 29.0 22.9 22.5 0.7 < 0.05

Total N excretion

Total N excretion, % of N intake 71.5 75.3 71.0 3.9 0.64

N excretion per milk, g N kg-1 milk 14.7 15.5 16.4 1.4 0.09


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6.3.3. Cow N Balance

As expected, daily N intake varied among treatments (P < 0.05) and ranged from 522.8 g d-1 in HP treatment to 490.8 g d1 for MP and 438.6 g d1 for LP diet. These N intake

levels have been reported as capable to maintain recorded milk yields without compromising any physiological feature (Castillo et al., 2001). Milk NUE values did

not differ between treatments (P > 0.05), and averaged 23.9% in LP diet, 22.9% in MP and 22.6% in HP diet. Recorded NUE values were similar to those observed by Castillo

et al. (2001) for midlactating cows which were fed with similar N intakes. Efficiency values also coincided with NUE data collected in commercial farms from the Basque

Country within tested N intake range (25.1%, SD 3.1%) (data not published). Data showed that decreasing N intake for mid-late lactating cows might increase NUE as

lactation period may better limit milk responses from high protein content diets (Huhtanen and Hristov, 2009).

As Yan et al. (2006) reported from an extensive review, around 70-75% of the N

consumed by dairy cows was excreted in feces and urine (Table 6.2). Nutritional CP manipulation is known to be more effective controlling urinary N excretion than fecal N

output, as the urinary pathway is reported as the main N excretion source above 400 g N ingested (Kebreab et al., 2001). Urinary N output accounted for 46.0%, 52.4% and

48.3% of N intake in LP, MP and HP diets, respectively (P > 0.05). These percentages might be questioned as urinary N excretion was estimated through creatinine

concentration in spot urine samples, which has been reported to show diurnal variations (Gonda et al., 1996). However, a recent publication has concluded a constant creatinine

excretion (0.212 mmol kg1 BW) through the day in Holstein dairy cows (Chizzotti et al., 2008). In addition, if urinary N excretion had been calculated as the difference between

ingested N and the sum of milk and feces N, results would have overestimated 3.2% (Table 6.2). Increasing CP intake has been related to higher amounts of UUN, which is the main N compound in urinary samples (Bristow et al., 1992). Urinary urea N and

excreted urinary volume tended to increase with higher CP diets (P < 0.10) (Table 6.2). Nitrogen excreted per unit of milk yield did not differ between treatments (P > 0.05)

(Table 6.2).


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6.3.4. On-Farm NH3 and N2O Measurements

6.3.4.1. Ammonia.

On-farm NH3 concentrations showed that minimizing dietary CP content resulted in lower NH3 concentrations on tie-stall floors (P < 0.05). Decreasing CP content from

16.9% to 14.1%, mean NH3 concentration was reduced by 36.5% from 10.8 mg NH3 m-

3 in HP to 7.1 mg NH3 m-3 in LP. This result meant that NH3 concentration is reduced

13% per each unit of dietary CP reduced. Such a reduction rate agreed with previous data published in the literature (Külling et al., 2001; Frank et al., 2002).

Ammonia emission begins in dairy stalls when fecal urease rapidly converts UUN to

NH3 on barn floor (Powell et al., 2008). Ammonia is in an equilibrium state with NH4+

ion as the result of chemical and (micro)biological features of manure (pH, DM, urease

activity) (Monteny et al., 2002) or physical parameters such as air temperature (van der Stelt et al., 2007). Fecal pH (7.4-8.5) and DM content (16.5%-22.4%), and urinary pH

(8.2-8.8) did not show any response to nutritional treatments (P > 0.05), and therefore differences in NH3 concentration were not related to these parameters. On the contrary,

UUN showed an increasing trend from LP to HP treatment (P < 0.1) (Table 6.2), even though diurnal patterns in the concentration of urea in urine of lactating dairy cows were

previously observed (Burgos et al., 2007). The same authors reported that UUN responded linearly to dietary CP content in different lactating stages. Besides, they

confirmed that UUN was significantly related to MUN in mid-late lactating cows (R2 > 0.95). In this sense, MUN was the main diet-derived parameter related to NH3

concentrations through univariate and multivariate regressions (P < 0.05; R2 = 0.20). Milk urea N ranged between 15.4 and 18.7 mg dL-1 in LP and MP, respectively. These data were within the range established by the linear relationship determined between

dietary CP and MUN by Nousiainen et al. (2004).

Although different authors have reported the influence of temperature on NH3 emission (van der Stelt et al., 2007; Powell et al., 2008), no relationship was established between

air temperature and NH3 concentration in the current study (P > 0.05). Mean temperature in the stall was 14.6ºC, ranging from 12.6ºC to 18.0ºC. According to Braam

et al. (1997a), temperature did not limit urease activity on the floor. Thus, we suggest


150


that the narrow range of temperatures recorded during the sampling period might have hidden the effect of temperature on NH3 concentration.

6.3.4.2. Nitrous Oxide

On-farm N2O concentrations did not respond to the different dietary CP levels (P >

0.05) in this trial. LP treatment averaged 1.21 mg N2O m-3, while MP and HP showed concentrations around 1.10 mg N2O m-3. Recorded concentrations were slightly higher

than those estimated by Jungbluth et al. (2001) in dairy tie-stalls, who measured N2O emissions between 1.4 and 10.0 g h-1. Although some authors concluded that N2O

emission rate may not be significantly different from zero in dairy barns (Sneath et al., 1997), our data supported that N2O emission may be detected on-farm, as control N2O

concentration was 0.55 mg N2O m-3. These data suggest that more research will be necessary on-farm considering that more detailed data are still required to associate

default emission factors in defined livestock classes to reduce uncertainties in national N2O inventories (Gac et al., 2007).

Limited published studies exist, but suggest that N2O emissions may be reduced by

lowering dietary CP content (Külling et al., 2001). However, our results did not show the same trend concerning dietary CP content and N2O concentration. Although

reducing CP of diets has an effect on total N and NH4+-N content of the manure

(Velthof et al., 2005), high dietary CP content may also be associated with an increased

digestibility of the diet, particularly of fibre (Külling et al., 2001). This fact is to some extent negatively related to manure fibre content and the subsequent surface crust

formation, which enables N2O synthesis (Sommer et al., 2000; Külling et al., 2003). Külling et al. (2003) reported that N2O formation was related to the thick surface crust

formed on liquid manure due to the relatively high fibre content of the forage-based rations. This effect might be possible in our trial because N2O measurements might be

partially affected by slurry cumulated in storages, which were located close to air sampling area in dairy barn. Apparent digestibility of our rations decreased by reducing dietary CP content as fecal daily excretion increased (P < 0.05). Fecal daily excretion

was 4.5 kg DM d-1 in LP diet and averaged 3.7 kg DM d-1 in MP and HP diets. In addition, fecal samples also differed in their C:N ratio (P < 0.05), ranging from 12.6 in

HP to 14.1 in LP diet. The higher alfalfa hay intake (1.5 kg DM d-1) together with the


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lower daily N intake (84 g d-1) in LP treatment, explained the differing C:N between diets. In the present study, the contrasting effects of lower CP content of the diet and

lower digestibility may have resulted in no net difference in N2O concentration.

6.3.5. Effect of Temperature on NH3 and N2O

6.3.5.1. Ammonia

Ammonia concentration increased in the incubation jars with higher temperatures for all dietary treatments (P < 0.05) (Table 6.3). Ammonia accumulation rate increases with

higher temperatures because temperature will result in an increase in the NH3/NH4+

ratio, a decrease in the solubility of NH3 in liquid phase of slurry, and the increase of the

activities of micro-organisms present in slurry (van der Stelt et al., 2007). Incubation data showed that NH3 concentration increased 2.7 mg NH3 m-3 per degree in LP diet,

and the increase was 4.1 mg NH3 m-3 in MP and HP diets. Similarly, van der Stelt el al. (2007) demonstrated that increasing temperature from 4ºC to 20ºC NH3 volatilization

increased 2.6-5.8 times, while when temperature was increased to 35ºC, NH3

concentration increased a further 3.0-6.0 more times. In the present study we assume

that NH3 concentration might have increased further with a longer incubation period, especially for higher CP and incubation temperatures.

Table 6.3. Effect of temperature on NH3 concentration (mg m-3) in incubated jars.

Temperature LP MP HP

4ºC 15.2c 14.2c 19.4c

19ºC 39.6b 48.9b 53.3b

29ºC 82.7a 116.1a 121.0a

a,b,cDifferent superscripts within the same column indicate significant differences (P < 0.05).

Dietary treatments did not affect NH3 concentration at 4ºC and 19ºC, but at 29ºC NH3

concentration was significantly greater for the medium and high CP diet than low CP (P< 0.05), with an increase of 13.7 mg m-3 per increased CP unit. Direct comparison of

NH3 data between incubation and on-farm fluxes may not be appropiate as NH3

emission includes both diffusion and convection processes (van der Stelt et al., 2008).


152


The small size of the incubation jar facilitates the high NH3 concentration and may alter the diffusion process because of NH3 saturation. A stepwise regression with NH3

concentration as the independent variable showed that those parameters related to nutritional features, such as slurry organic matter (OM), urinary pH and UUN,

explained 22.8% of the variance. Some reports related the increasing dietary CP with higher manure pH values (Misselbrook et al., 2005), probably related with the higher

urinary pH. Similarly, several reports recorded the effect of increasing UUN on NH3

concentration (Monteny et al., 2002; Burgos et al., 2007). When temperature was

included in the regression model, the coefficient of determination of the model increased to 89%. Thus, in accordance with previous results (Amon et al., 2001; van der

Stelt et al., 2007), NH3 flux was positively related to temperature.

6.3.5.2. Nitrous Oxide

As Table 6.4 shows, temperature influenced N2O concentration (P < 0.05) although this

relationship was in contrast to NH3 concentration in incubated jars. Samples incubated at 4ºC showed the highest N2O concentration of all dietary treatments, averaging 1.11

mg N2O/m-3. Slurry incubated at higher temperatures averaged 0.86 mg N2O/m-3. Nevertheless, such concentrations were low according to data reported by other authors

(Amon et al., 2001; Jungbluth et al., 2001; Külling et al., 2003). Similar to on-farm data, dietary treatments did not differ in N2O concentration at different temperatures (P >

0.05). Small effect of dietary CP on N2O accumulation was observed when the multivariate regression (N2O concentration as dependant variable) included urinary and

fecal pH in the model, explaining just 6.7% of variance. On the contrary, Cárdenas et al. (2007) reported a significant role of livestock diet in slurry composition and

consequently in the emissions of N2O from slurry amended incubated soils. They reported that reducing the NH4

+ and soluble organic C added with the slurry mitigated

the N2O emission. However, the lack of nitrifier and denitrifier microbial genera in dairy fecal samples (Dowd et al., 2008) might explain the scarce effect of dietary CP on N2O concentration in our results. Therefore, unless allowing the physical contact of

excreted N and nitrification and dentrification microbial sources (soil pollution, bedding materials, feedstuffs), N2O accumulation may be limited from barns (Sneath et al.,

1997).


153


Table 6.4. Effect of temperature on N2O concentration (mg m-3) in incubated jars

Temperature LP MP HP

4ºC 1.13a 1.17a 1.04a

19ºC 0.88b 0.86b 0.84b

29ºC 0.85b 1.10a 0.87b


6.4. Conclusions

Reducing dietary CP content from 17% to 14% DM in mid-late lactating cows

minimized N excretion and the subsequent NH3 concentration in tie-stall floors. On the contrary, tie-stall floor N2O concentration was not affected by dietary CP manipulation. Different CP diet-derived slurries incubated in the laboratory demonstrated that NH3

concentration increased with temperature, especially in high CP diets. In contrast, N2O concentration did not show the same positive relationship with increasing temperature.

We conclude that limiting dietary CP content minimizes NH3 concentration on dairy stall floors although temperature will control the rate of NH3 volatilisation. On the other

hand, N2O concentration on tie-stall floors is low and is not affected by dietary CP manipulation.


7 GENERAL DISCUSSION


157

GENERAL DISCUSSION

The environmental pollution detected in high density dairy production areas in the Basque Country and the increasing environmental awareness of European agricultural

policies demanding an environmental-friendly farming sector (van Passel et al., 2007), make it necessary to consider environmental issues in dairy production either at farm or

regional level. Besides, considering that economical incomes of dairy farmers have dramatically dropped in recent times and European agri-environment payments have

been integrated in the farm economy, the consideration of environmental issues in dairy farming may also be economically relevant for dairy farmers.

Before the proposal of this PhD-thesis, most research conducted in the Department of

Ecotechnologies at NEIKER-Tecnalia concerning dairy production was related to an inventory of slurry production at regional level (Lekuona et al., 2002), the study of N

balances at farm level (del Hierro et al., 2006) or the emission of NH3, N2O and NO gases after grassland fertilisation. In relation to gaseous N losses, the effect of the

application of different types of fertilisers (dairy cow slurry, ammonium-nitrate, urea), doses, agricultural practices or commercial products added on fertilisers (additives,

urease and nitrification inhibitors) have been tested (Merino et al., 2001a; Merino et al., 2001b; Merino et al., 2002; Pinto et al., 2004; Merino et al., 2005). Results showed that

fitting N fertilisation rate to plant requirements and/or adding chemical products on fertilisers may contribute to reduce gaseous N losses from dairy farming. However, the

scarce farmland availability in the territory for the larger herds in commercial dairy farms (EUSTAT, 2009), and the high economical cost of adopting chemical products on

fertilisers reduce the suitability of slurry management as a strategy to reduce gaseous N emission in dairy farms. In addition, the study of gaseous N emission from fertilised grasslands just represent a partial approach to gaseous N pollution in dairy farming as a

dairy farm is a complex system with several interacting subsystems (animals, housing, slurry storages and grasslands/crops) (Olesen et al., 2006). Field gaseous N losses

represent one of the last steps of N losses in a dairy farm and up to 75% of excreted N by cows may be emitted into the atmosphere before manure/fertiliser application on

grassland (van Horn et al., 1994; Rotz, 2004). Therefore, the current PhD-thesis was planned to test the effectiveness of dairy cow nutrition to reduce gaseous N losses (NH3,

N2O and NO), integrating features of dairy production (nutrition, milk yield, ruminal processes, milk N use efficiency) with gaseous N losses in different subsystems of a

dairy farm.


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7.1. Is it feasible to reduce farm N surplus through dietary strategies in commercial dairy farms from the Basque Country?

Efficient use of N is one of the major objectives of sustainable production systems as its

inefficient use results in harmful losses to the environment and affects the economic performance (Oenema and Pietrzak, 2002). Minimizing dietary CP overfeeding

contributes to improve milk N use efficiency and the economical profitability of dairy farms (Jonker et al., 2002). In accordance with all these statements, the first objective of

the current PhD-thesis was to ascertain whether dietary manipulation might be a feasible strategy to reduce N accumulation in commercial farms in the Basque Country, where

no information was available.

Dietary dataset recorded during on-farm surveys showed that 69.7% of rations exceeded recommended N intake in lactating dairy herds, as estimated by Cornell Net

Carbohydrate and Protein System (CNCPS 5.0). In this sense, metabolizable protein intake exceeded by 7.4% the average protein requirements. These results might be

expected because N overfeeding has been a common practice among dairy farmers because the cost associated with the risk of underfeeding protein (reduced milk yield)

was greater than the cost of overfeeding (vandeHaar and St-Pierre, 2006). Despite protein overfeeding estimated during the survey, mean CP content of sampled rations

(16.4% CP) was far from 18.0% CP reported by the same authors as usual for overfed lactating cows. Two reasons might have contributed to the moderate N overfeeding in

these commercial farms: the technification of dairy farmers and the increasing prices of high CP content feedstuffs in national and international markets. Farmers’ technification let improve cattle nutrition as they are more conscious of its importance on farm

profitability (milk production, cow welfare, incidence of diseases, etc). Thus, above mentioned overfeeding level was similar to N excess reported by Jonker et al. (2002) in

specialized commercial dairy farms from US (estimated as 6.6% more N than recommended by NRC). On the other hand, the price of ingredients such as soybean

meal, which is the main imported N source in cow rations, increased up to 80% for the last decade. Alfalfa hay, high CP-content forage which is produced and sold in national

markets, also increased the price by 25% during the last years.


159

GENERAL DISCUSSION

Rotz (2004) summarized that two general strategies may be used to reduce N pollution in dairy farms through animal management. The first one is to reduce the protein fed by

improving the match between the protein quality and that required by the animal. The second one is to improve cow productivity, because as more milk is produced per cow,

the maintenance requirement of protein per unit of production is reduced. Among these strategies, Rotz (2004) suggested that reduction of N excretion through improvement of

milk N use efficiency (NUE) is more effective using strategies which improve protein-feeding efficiency. Our data also suggested the same conclusion. Nitrogen intake was

the best predictor of N excretion in commercial dairy farms (R2 = 0.7 in Table 3.4), which was in accordance with previous studies (Yan et al., 2006). On the contrary,

increasing milk yield (mean, 29.7 kg-1 milk cow-1) did not show so strong relationship in relation to milk NUE in sampled farms (R2 = 0.21; Figure 3.1). Mean NUE in

surveyed farms (25.8%) was lower than reported by other authors for grass silage based rations, which reached about 28% (Castillo et al., 2001b; Yan et al., 2006; Huhtanen et

al., 2008). The heterogeneity of sampled farms, which included highly specialized and small familiar farms, might have contributed to this difference in mean milk NUE. In

addition to breed, genetics, nutrition and management heterogeneity of sampled herds, it was noteworthy (data not published in previous chapters) the variability found in the

quality of grass silages. Being grass silage the main forage in the rations (Table 3.2), improving the overall quality of grass silages might be a good option to enhance milk

NUE of lactating herds.

In addition to fitting dietary N intake or improving milk yield of herds, other nutritional strategies have been reported in the literature as feasible to minimize cow N excretion. Some authors have reported that improving the quality of ingested protein (Santos et al.,

1998; Børsting et al., 2003) or using strategies such as animal grouping, increasing frequency of ration reformulation or the selection of different feeding systems (St-Pierre

and Thraen, 2001; Jonker et al., 2002) may contribute to enhance milk NUE and reduce farm N surplus. However, data from this survey did not show reported effectiveness.

The quality of the protein fed (estimated as ruminally undegradable protein or ruminal microbial protein), the management of herds in different feeding groups by lactation

state, the periodical reformulation of diets or the use of different feeding systems (total mixed rations, purchased complete feed or component feeding) did not affect milk NUE


160


(P > 0.05). As previously mentioned, the heterogeneity of sampled diets and lactating herds might have reduced the likely effect of these nutritional measures on milk NUE.

Therefore, these strategies might be complementary alternatives to fitting N intake and improving milk yield.

Despite dietary manipulation may improve milk NUE and reduce therefore N excretion

in lactating cows, the next question might be: how far dietary measures may reduce N excretion from lactating dairy herds at farm level? Considering the milk quota allocated

to each farm and estimating cow N excretion per milk unit produced, we estimated that up to 35.5% of N excreted might be reduced through the optimisation of N intake. This

percentage highlights that a significant amount of N may be removed from dairy farms. However, data also demonstrated that farms which showed an accurate CP fitting and

high milk productivity were mainly classified as medium or high intensified (more than 12,000 milk kg ha-1 in accordance with Berentsen and Tiessink, 2003). So, if among

samples farms, those more intensified show the best milk NUE values, how far can cow nutrition reduce N excretion when intensification parameters (herd size and farmland

availability) are included in the study? A multivariate regression model showed that N intake was just able to explain 11.2% of the variance when N excreted by lactating

herds per farmland hectare was considered as a dependant variable. More than 60% of the variation was accounted for by farm intensification parameters (herd size and land

availability). Thus, the effect of dietary manipulation on farm N excretion may be particularly limited in highly intensified dairy farms.

As summary, data recorded in commercial farms from the Basque Country highlighted that fitting dietary CP and improving milk yield of lactating herds are feasible strategies

to reduce N pollution at cow and farm level. However, the suitability of dietary manipulation may be particularly limited in highly intensified dairy farms (high animal

density per hectare).

In addition to mentioned N-derived pollution, dairy farm activities also contribute to P pollution (Spears et al., 2003a). However, few scientific contributions have reported a

whole perspective of N and P pollution from dairy production. A complementary study


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GENERAL DISCUSSION

carried out on animal P balance showed that P intake exceeded P requirements by 63%, as estimated by CNCPS 5.0, demonstrating that dairy farmers do not formulate rations

on P basis. Nevertheless, the positive relationship found between NUE and milk P use efficiency (PUE) values (P < 0.01; R2 = 0.35) suggested that measures for reducing N

excretion might contribute to reduce P excretion as well. However, the variability of such relationship also suggests that rations should be balanced for P content, especially

when high P-content feedstuffs (high-P forages and concentrates) are used for lactating dairy cow nutrition.

7.2. Dietary strategies to improve milk NUE and reduce N excretion in lactating cows.

In accordance with the linear relationship reported by Yan et al. (2006) between N intake and N excretion, reducing N intake to minimum values would lead to minimize N

excretion at maximum. However, this strategy would solve environmental pollution in dairy farming but it would not guarantee milk production. In fact, the main aim of dairy

farming activity is to produce milk, and therefore N pollution should be related to milk production. Thus, the conversion of N in animal production should begin by improving

N use efficiency of animals (Rotz, 2004). Ruminants make efficient use of diets as ruminal microbes are able to synthesise a large proportion of the protein required by

cows. However, despite this physiological adaptation, dairy cows excrete 2-3 times more N in manure than they secrete in milk. As previously mentioned, milk NUE in

commercial farms from the Basque Country was 25.8%, which was slightly higher than values reported by Tamminga (1992) for Dutch dairy cows and lower than NUE reported by Castillo et al. (2000) and Huhtanen et al. (2008) (mean, 28.0%) from

extensive reviews of published studies. Milk NUE has been significantly improved in research trials conducted under controlled conditions (values around 35.0%), in which

cow nutrition, genetics and management may be considered as nearly optimum (Broderick, 2003; Ipharraguerre and Clark, 2005; Olmos-Comenero and Broderick,

2006). In spite of this low conversion of dietary N in valuable milk N, the improvement of milk NUE should be considered as a key strategy to reduce N surplus in dairy farms

and to increase farm economic profit. Measures like fitting dietary CP or increasing milk yield reduce N pollution and have a direct impact on farm economy.


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As the following equation shows, dietary N intake, milk yield and milk CP content are the three parameters involved in determining milk NUE value.

NUE = (Milk yield, kg d-1 x Milk protein, %) x 100

N intake, g d-1

Despite managing any of the three parameters that define NUE may contribute to improve milk NUE, Rotz (2004) concluded that reduction of N excretion through

improvement of NUE is more effective using strategies which improve protein-feeding efficiency. Data collected in our survey, and previously reported, agreed with this

statement. In fact, from high NUE variability showed in Figure 3.1 might be understood that other factors different from milk yield were largely affecting milk NUE in

commercial dairy farms. However, as different herds (varying genetics and management), rations (different ingredients and feeding systems) and stalls (tie-stalls

and free-stalls) were included in the survey, the likely effect of nutrition (especially CP fitting) might be masked on such milk NUE variability. Thus, two experiments were

conducted under controlled conditions during the current PhD-thesis, in which the effect of two nutritional strategies were tested on milk NUE, N excretion by fecal and urinary

pathways and N excretion per unit of milk produced. The first study tried to highlight the importance of balancing energy and protein supply of diets to improve milk NUE,

while the second one emphasized on the importance of fitting dietary CP to improve milk NUE. These nutritional experiments were afterwards related to slurry N content

and gaseous N losses from a dairy stall and from slurry amended grassland

During the first experiment, we tried to test the effect of forage:concentrate ratio in milk

NUE, N excretion and diet-derived slurry N content. Varying ratios were selected as the synchronization of energy (from concentrates) and rumen-degradable N (from forages)

contributes to improve ruminal N balance through trapping ruminal NH4+ by microbes

(Hoover and Stokes, 1991). Selected forage:concentrate ratios were 45:55 in low forage

diet (LF diet) and 75:25 in high forage diet (HF diet). LF diet represented approximately the mean ratio found in commercial farms from the Basque Country (Table 3.2). HF

represented a feeding system which might be closer to the traditional nutrition system in


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GENERAL DISCUSSION

the Basque Country. Triticale silage (homegrown forage) was the main forage source in the diets and corn and barley grain constituted the base of the concentrates (Table

4.1/Table 5.2). According to the rations surveyed in commercial farms, grass silage should have been selected as the main homegrown forage (Table 3.2). However, we

were not able to get required grass silage before the trial. Thus, triticale silage was selected as an alternative forage source (Mergoum et al., 2009). Anyway, this alteration

did not change the initial hypothesis that the correct match between a rapid energy source (barley) (Foley et al., 2006) and a high degradable protein source (triticale

silage) (Khorasani et al., 1993) might contribute to optimize the capture of ruminal NH4

+, increase the ruminal protein synthesis and improve milk yield and milk NUE in

LF diet.

Ten Holstein cows from the experimental dairy herd from I.E.S. La Granja (Cantabria, northern Spain) were blocked into two groups and were assigned randomly to two diets

in a crossover design. Rations were formulated to be isonitrogenous (500 g N cow-1) but differing in their forage:concentrate ratio. The objective was to determine whether HF

ration, usually considered feed and environmentally sustainable, or LF ration, more energetic and usually related to high N input:output intensified farms, are more efficient

enhancing milk NUE and reducing N excretion. Although HF and LF diets were formulated to be isonitrogenous, the high NDF content in HF (54.3% DM) reduced 3.3

kg cow-1 day-1 voluntary intake of triticale silage. As a consequence, N intake was 405 g N cow-1 in HF and 498 g N cow-1 in LF diet. The initial scope of improving the ruminal

protein synthesis in LF was partially achieved in this trial. Although LF diet averaged higher microbial protein synthesis (P > 0.05) (Table 5.3), the difference was lower than initially expected. Ruminal NH4

+-N concentrations in HF (1.68 to 3.67 mmol NH4+-N

L-1) were below concentrations needed to maximize flows of microbial N from the rumen (Hume et al., 1970). Thus, microbial protein synthesis should have been

seriously reduced in HF diet. However, the efficiency of microbial protein synthesis (g microbial N per kg of fermented OM) was not reduced in HF diet. Bach et al. (2005)

reported that apart from the amount of ingested degradable protein and energy, the synchrony at which nutrients become available is an important factor for microbial

protein synthesis. Thus, we suggest that ration management in this trial (no TMR rations with ad libitum forage offer) could have contributed to an imbalance between energy


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and protein availability and therefore the improvement of ruminal protein synthesis was not as good as expected for LF.

As a consequence of lower N intake in HF diet, fecal and urinary N excretion decreased

(Table 4.2/Table 5.3), which might seem the perfect strategy to reduce N pollution in dairy farms. However, milk NUE (21.6% and 20.7% in HF and LF, respectively) and N

excreted per milk yield (15.0 and 14.8 g N kg-1 milk in HF and LF, respectively) was not improved by reducing N intake in HF. Results demonstrated that feeding lactating

herds with HF diets (lower voluntary dry matter intake, lower N intake, lower energy content) N excretion per cow is reduced but if N excretion is analyzed together with

milk parameters (milk yield or milk quota), no significant differences may exist between HF and LF diets. In contrast, available literature reports that difference in milk

NUE between herds fed with HF and LF diets may be more significant than the one observed in our study. Milk NUE values from low forage diets may average values

around 35% (Broderick, 2003; Ipharraguerre and Clark, 2005; Olmos-Colmenero and Broderick, 2006) while NUE from high forage or grazing diets may averaged 25%

(Bargo et al., 2002; Tas et al., 2006). In our study, the increasing milk yield in LF diet (18.2 and 21.4 kg milk cow-1 in HF y LF diets, respectively) did not increase NUE

significantly but with respect to HF diet. However, and according to previous studies which used triticale silage as the main forage, higher milk yields might have been

expected in LF. Khorasani et al. (1993, 1996) formulated two triticale silage based rations with 50:50 forage concentrate ratio and 3.0 kg d-1 CP intake and obtained higher

milk yields (about 25 kg d-1) than the ones found in our study. However, the higher fibre content of triticale silage in the current trial might have limited the milk productivity.

Despite the suggested imbalance between energy and protein availability and milk yield limitation in LF diet, results reported in our experiment highlighted the importance of

fitting dietary CP and balancing energy content of rations to increase herd milk yields and to improve milk NUE (or reduce N excreted per milk yield). Further discussions

will have to elucidate whether the use of low forage diets is environmentally sustainable (increasing N inputs from concentrates) or economically profitable (high production


165

GENERAL DISCUSSION

costs), whether NUE may be improved using energetic homegrown forages (maize silage) and many other questions related to cow nutrition and N pollution in dairy farms.

The second trial tried to demonstrate the importance of fitting dietary CP in isoenergetic

diets. Three first lactation Holstein cows (mid-late lactation period), which were held in separated metabolic tie-stalls, were randomly assigned to three diets in a 3 by 3 Latin

Square design. Rations varied in their CP content with 17.0% in high protein diet (HP), 16.0% in medium protein diet (MP) and 14.0% in low protein diet (LP), and were

formulated on corn silage basis with forage:concentrate ratio of 50:50. Dietary CP contents were selected to represent target CP values in lactating dairy cow nutrition.

High protein diet represented dietary CP content recommended for high yielding dairy cows (Ipharraguerre and Clark, 2005; Olmos-Colmenero and Broderick, 2006). Medium

protein diet agreed with some authors who reported that 16.0% CP did not compromise the performance of midlactation cows (Castillo et al., 2001b). Finally, LP diet

represented the dietary CP content capable to maintain the milk productivity for mid and late lactation dairy cows (Frank et al., 2002). Mean N intake ranged from 438.6 g N

d-1 in LP to 522.8 g N d-1 in HP diet, and as expected, N excretion decreased by minimizing N intake (Table 6.2). Milk yield also decreased with lower N intake (from

22.1 to 24.2 kg milk d-1 in LP and HP, respectively) (P < 0.05). As milk yield decreased with lower N intakes, milk NUE and N excreted per unit of milk was not improved

significantly by reducing N intake (Table 6.2). However, N excreted per unit of milk tended to be lower in LP (from 14.7 g N kg-1 in LP to 16.4 g N kg-1 milk in HP) (P <

0.10). Milk NUE also averaged higher value in LP diet (23.9%) in relation to MP and HP diets (22.9% and 22.6%, respectively) (P > 0.05). Data demonstrated that fitting dietary CP to animal requirements (in this case low protein diets to mid-late lactating

cows) in isoenergetic diets may contribute to improve milk use N efficiency of lactating cows. These results have practical on-farm implications as lactating herds might be

separated in different feeding groups according to their lactation state, which would lead to reduce N excretion at farm level and increase farm profitability by reducing N

overfeeding.


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7.3. Dietary strategies to alter slurry composition.

Dietary modifications in dairy cattle have been reported as a useful strategy to alter the composition of manure in which dietary CP content is positively correlated with the

fertiliser N value of manure (Reijs et al., 2007). During the current PhD-thesis, the first nutritional experiment related dietary modification to changes in manure composition,

especially concerning N compounds. As Table 4.2 and Table 5.4 show, slight variations were reported overall in HF and LF slurries during the trial. Nitrogen content of HF and

LF diet-derived slurries did not vary between them (P > 0.05). Although higher N concentration might be expected from LF diet-derived slurry (because of higher N

intake), fecal and urine N concentration usually show high variability as concentration is affected by excretion volume as well (dilution or concentration effect). Although data

of fecal and urine N concentration were not reported in our papers (samples from two experiments), high variability was recorded among different samples. In addition, some

flushing water was also added to diet-derived slurries during the first experiment, which might have diluted original concentrations. Nevertheless, slurry of commercial dairy

farms is not just a sum of feces and urine. It also includes flushing water, bedding materials or feedstuffs debris, which all together contribute to an heterogeneous slurry.

Anyway, although no statistically significant, the higher NH4+-N concentration

measured in LF slurry might be directly related to the higher N intake in LF diet. As

previously mentioned, ruminal NH4+-N content was higher in LF, and urinary urea also

tended to be higher in the same treatment (Table 5.3) (P < 0.10) In fact, nutritional CP

manipulation is known to be more effective controlling urinary N excretion than fecal N output (Kebreab et al., 2001). Increasing CP intake is related to higher amounts of UUN, which is the main N compound in urinary samples (Bristow et al., 1992) and

closely related to NH4+-N content of slurry. As a result of urease activity in fecal

microbes, urinary urea is rapidly converted into ammonia-ammonium equilibrium. This

equilibrium may be modified by different factors, such as slurry pH. In this sense, the lower pH of LF diet-derived slurry (Table 4.2 and Table 5.4) might have also partially

contributed to this higher NH4+-N content due to lower NH3 volatilisation.

As slurry was applied to soil at a standard rate of 120 kg NH4+-N ha-1, the amount of

slurry applied per square meter was different in HF and LF treatments. In addition to


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GENERAL DISCUSSION

environmental issues, altering slurry content by dietary manipulation may produce changes on slurry management of dairy farms as reducing NH4

+-N content might favour

emptying more frequently slurry storages (preventing slurry accumulation, odorous gases, etc) to reach recommended N application doses. On the contrary, it might also

increase the economical cost of slurry management as more slurry tank applications to the field would be necessary. Thus, the responsible management of slurries may account

for an adequate nutritional strategy of dairy herds from an environmental and economical point of view.

7.4. Dairy cow nutrition and ammonia emissions from barn floors and grasslands.

According to Gothenburg Protocol (1999), European countries are obliged to adopt

practices which contribute to minimize national NH3 emissions from different activities, including animal husbandry. Three subsystems constitute NH3 sources in dairy farms:

stalls (Monteny and Erisman, 1998), the slurry storage system (Rumburg et al., 2008b) and slurry application on field (Misselbrook et al., 2000). Among referred subsystems,

dairy stalls and slurry applications are remarkable NH3 sources as between 50% and 75% of N excreted by herds may be lost in dairy stalls (van Horn et al., 1994; Rotz,

2004), and 10% of total NH3 emission in EU was accounted for slurry applications on the field. In accordance with these data, the current PhD-thesis was focused on the study

of NH3 losses from barn floors and slurry amended grassland.

Reducing N excretion through dietary manipulation represents a feasible option to minimize NH3 volatilisation in dairy farming (Paul et al., 1998; James et al., 1999; Misselbrook et al., 2005). However, dietary strategies do not affect on NH3 emission in

the same way in the different subsystems which integrate the dairy farm. In this sense, the principle conclusions extracted from our experiments were: a) Data recorded at stall

level showed that increasing CP intake enhanced NH3 concentration from barn floor; b) dietary strategy is subordinated to manure management in NH3 emission when slurry is

applied on grassland. Ammonia concentration averaged 7.1 mg NH3 m-3 in LP diet (14% CP), 10.4 mg NH3 m-3 in MP (16.0% CP) and 10.8 mg NH3 m-3 in HP (17.0% CP)

(atmospheric concentration, 0.4 mg NH3 m-3). Nevertheless, as atmospheric conditions


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were very similar in the stall, differences in NH3 concentration might suggest different NH3 emission. As mentioned above, NH3 emissions occur when urea excreted in urine

reacts with urease produced by microbes in feces (Burgos et al., 2005). Thus, such relationship might be explained by urinary urea N, which tended to increase in relation

to higher CP intake (Table 6.2) (P < 0.10). Mean NH3 concentration reduction between LP and HP treatments meant to reduce 13% NH3 concentration per each dietary CP unit

reduced in the ration. This rate agreed with previous data reported in the literature (Külling et al., 2001; Frank et al., 2002).

It is noteworthy the effect of temperature on NH3 volatilisation at stall level (van der

Stelt et al., 2007; Powell et al., 2008). Ammonia accumulation rate increases with higher temperatures because temperature will result in an increase in the NH3/NH4

+

ratio, a decrease in the solubility of NH3 in liquid phase of slurry, and the increase of the activities of micro-organisms present in slurry (van der Stelt et al., 2007). The narrow

range of temperatures recorded during the sampling period (12.6ºC-18.0ºC), together with the daily fecal and urinary N content variability might have hidden the effect of

temperature on NH3 concentration. However, when diet-derived fecal and urine samples were incubated at three temperatures (4ºC, 19ºC and 29ºC), NH3 concentration

increased with higher temperatures for all dietary treatments (P < 0.05) (Table 6.3). These results demonstrated that limiting dietary CP content minimizes NH3

concentration on dairy stall floors although temperature may control the rate of NH3

volatilisation.

As mentioned above, dairy cow nutrition was subordinated to manure management concerning field NH3 emissions. That means that despite nutritional strategy (HF or

LF), NH3 emission will eventually depend on the use of slurry in grassland applications. Nutritional experiment conducted during this thesis demonstrated that the choice

between HF and LF diet was able to change slurry characteristics (Table 4.2 and Table 5.4). As a result of this slurry modification, especially if NH4

+-N content is changed,

NH3 emissions might be regulated by ration manipulation. However, NH3 emission pattern and cumulative emission will depend on slurry management in the field. How

can slurry management affect the selection of a proper nutritional strategy from the gaseous N loss point of view? Farmers who apply slurry on fresh matter basis should

firstly minimize slurry NH4+-N content through reducing dietary CP. On the contrary, if


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GENERAL DISCUSSION

farmers try to fit NH4+-N doses on grassland, as N gas emissions may be similar among

diets, they should check their nutritional strategy in relation to herd productivity.

Further discussions may be inferred from data recorded in this experiment whether HF

or LF feeding strategies are more or less pollutant when grassland emissions are corrected by applied slurry volume or produced amount of milk. Because different

amounts of slurry were applied from each treatment, NH3 emissions per kilogram of slurry was slightly higher in HF (0.14 g NH3-N kg-1 slurry) compared with LF (0.10 g

NH3-N kg-1 slurry). This highlights the potential risk of having higher NH3 emission, even from HF diets (lower N intake), if slurry is not fitted to plant N requirements.

Relating total milk yield of each diet to NH3 emission cumulated in each treatment, HF also showed higher emission (17.3 g NH3-N ha-1 kg-1 milk) than LF diet (6.7 g NH3-N

ha-1 kg-1 milk). This result suggests that LF diets (higher N intake) may emit less NH3

per unit of milk produced if slurry is amended by fitting N load on grassland.

Data demonstrated that NH3 losses constitute the main source of gaseous N losses to

the atmosphere when dairy cattle slurry is applied on grassland. After one month since slurry application, around 75% of total gaseous N losses were accounted for NH3

volatilisation, belonging to N oxides the rest 25% (discussed in the next chapter). Ammonia volatilisation represented 60% of total gaseous N losses after two months of

field measurements.

7.5. Dairy cow nutrition and N oxides emissions from stalls and grasslands.

Manure application on grassland has been reported as the most notable N2O emission

source in dairy farming (Oenema et al., 2007). When available N from manure exceeds plant needs, emissions of N2O occur through nitrification and denitrification processes

(Granli and Bockman, 1994). On the contrary, there are fewer studies on N2O fluxes from dairy stalls (Amon et al., 2001; Külling et al., 2003), which has been related to the

difficulty of quantifying the low N2O concentrations by continuous gas analyzers (Jungbluth et al., 2001). In addition to N2O, nitric oxide (NO) emission was studied in

the experiment conducted on grassland. Field manure application is also considered as


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an important source of NO emission in agricultural production systems (Meijide et al., 2007). Nitric oxide is involved in ozone layer depletion (IPCC, 1996), also plays a

major role in the chemistry of the tropospheric ozone (Bouwman, 1990) and in the formation of acid rain (Vos et al., 1994).

Overall, few data are available on the effect of dietary strategies on N2O and NO

emissions (Cárdenas et al., 2007). From the literature it may be extracted that lowering dietary CP content of rations, reduced N2O emission rates might be achieved (Külling et

al., 2001). However, high dietary CP content may also involve an increasing digestibility of the diet, particularly of fibre (Külling et al., 2001). This fact is

negatively related to manure fibre content and the subsequent surface crust formation, which enables N2O synthesis (Sommer et al., 2000; Külling et al., 2003). As previous

results were not too clear, the current PhD-thesis aimed to clarify the relationship between dairy cattle nutrition and N2O emissions in stall and after slurry application on

grassland. When this thesis was planned, we hypothesised that reducing the CP content of diets would have an effect on N2O and NO emissions from dairy production As

NH4+-N is required by nitrifiers to carry out the nitrification process, dietary

manipulation might modify. In addition, if nitrification process was modified,

denitrification could be also altered. The regulation of N2O and NO emission is described by the “hole-in-the-pipe” conceptual model (Firestone and Davidson, 1989),

where the movement of N through the pipe has been widely modeled in relation to ammonium and nitrate, in addition to soil features such as moisture, temperature, pH

(Granli and Bockman, 1994).

Results recorded through two nutritional experiments (stall and grassland) highlighted:

a) reducing dietary CP had low effect on reducing N2O concentration in tie-stall; b) dietary strategy is also subordinated to field manure management in relation to N2O and

NO emissions. According to these statements, reduction of N oxide emission from ration manipulation seems to be more difficult than minimizing NH3 volatilisation.

Stall N2O concentrations did not respond to the different dietary CP levels (P > 0.05).

Low protein (LP) diet averaged 1.21 mg N2O m-3, while MP and HP showed


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GENERAL DISCUSSION

concentrations around 1.10 mg N2O m-3. In accordance with a previous comment, the contrasting effects of higher CP content and higher digestibility of LF diet may have

resulted in no net difference in N2O concentration during the present study. Apparent digestibility of rations decreased by reducing dietary CP content as fecal daily excretion

increased (Table 6.2) (P < 0.05). Nevertheless, it was noteworthy that N2O concentrations recorded in the experiment were slightly higher than those estimated by

Jungbluth et al. (2001) in dairy tie-stalls. In addition, diet-derived emissions were also significantly different from zero, in contrast to data reported by (Sneath et al., 1997). In

fact, despite the low concentrations measured our data suggested that N2O emission may be detected on-farm, as control N2O concentration was 0.55 mg N2O m-3. This

difference among emission from treatments or control supports that more research will be necessary on-farm considering that more detailed data are still required to associate

default emission factors in defined livestock classes to reduce uncertainties in national N2O inventories (Gac et al., 2007).

As commented for NH3, ration formulation was subordinated to manure management in

field NH3 emission. In accordance with data reported by Velthof et al. (2005) nutritional experiment conducted during the experiment demonstrated that the choice between HF

and LF diet was able to change slurry NH4+-N content (Table 4.2 and Table 5.4).

However, N2O and NO emission pattern and cumulative emission will depend on slurry

management in the field. Reasons are those exposed for NH3: farmers who manage slurry on fresh matter applications may promote to higher N2O and NO emissions. On

the other hand, environmentally conscious farmers, who fit slurry NH4+-N doses to

grassland requirements, will likely reduce N oxides emissions.

Nitrous oxide and NO emissions during the field experiment (slurry application rate, 120 kg NH4

+-N ha-1) demonstrated that emission did not differ between HF and LF diets

(Figure 5.1 and Figure 5.4) (P > 0.05). Nitrous oxide emissions from HF and LF slurries averaged 84.7 g N2O-N ha-1 day-1 and 75.8 g N2O-N ha-1 day-1, respectively. Nitric

oxide averaged 17.2 g NO-N ha-1 day-1 in HF and 19.1 g NO-N ha-1 day-1 in LF slurry. The spatial variability in fluxes was notoriously high, which contributed, together with

the temporal variability, to large sources of uncertainty in N2O and NO fluxes at field


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scale (Velthof et al., 2000). Such variability sources add a major difficulty to ascertain the effect of nutritional manipulation on N2O and NO gaseous losses. Nitrous oxide and

NO emissions are closely related to WFPS (Davidson, 1991; Skiba et al., 1992). The highest N2O and NO emission peaks were observed at 60-70% of WPFS (Figure 5.1 and

Figure 5.4). As nitrification and denitrification can happen simultaneously in the soil since aerobic and anaerobic micropores can coexist in the soil (Abbasi and Adams,

1998), N2O peaks might be due to the contribution of both processes in this trial. On the contrary, nitrification has been described to be the main process of NO formation

(Slemr and Seiler, 1984).

The cumulative N2O emission was 5.8 kg and 5.0 kg N2O-N ha-1 for HF and LF slurries, respectively, and was not significantly different between them. Emission factor was

4.8% in HF and 4.2% in LF diet, similar to previous results reported by our research group for dairy slurry (Merino et al., 2001a; Merino et al., 2002; Merino et al., 2005).

Accumulated NO emission was slightly lower in HF slurry (0.51 kg NO-N ha-1) than in LF (0.57 kg NO-N ha-1). Nitrous oxide was the main source of N oxides emission after

slurry application on grassland. Because slurry application on grasslands is usually carried out by farmers by discharging onto the soil high volume tanks to empty the

slurry pit, the difference of N2O and NO emission related to the amount of applied slurry should be considered from an environmental point of view. As similar trend was

observed between both gases, N2O data are commented in the next lines. In this sense, differences between treatments were not significant (P > 0.05), although N2O flux per

applied slurry unit (kg) was slightly higher for LF slurry (0.08 g N2O-N kg-1 ha-1) compared with HF slurry (0.06 g N2O-N kg-1 ha-1) after 65 days of measurements. Similarly, considering that the main purpose of the dairy activity is the production of

milk, it would also be advisable to relate N2O emissions to the herd milk production. Data from the current trial showed a lower emission rate per kilogram milk produced

from LF slurry (5.8 g N2O-N ha-1 milk kg-1) than from HF slurry (7.9 g N2O-N ha-1 milk kg-1). Therefore, a responsible management of the slurries on grasslands justifies an

adequate nutritional strategy for dairy herds from an environmental and productive point of view. As previously mentioned in NH3 section, further discussions will have to

be planned through data recorded in this experiment whether HF or LF feeding


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GENERAL DISCUSSION

strategies are more or less pollutant when grassland emissions are corrected by applied slurry volume or produced amount of milk.

7.6. Gaseous N losses in the future and other comments

The evolution of gaseous N losses derived from dairy farming will be related to the

future of dairying activity itself in next years. In this sense, the liberalisation of milk quotas agreed by EU member states to 2015 will be a key factor on the future of

European dairying activity and the subsequent gaseous N losses. Its effect on European dairy farming is not still very clear among dairy community (farmers, technician,

advisors, milk industry, etc). However, it seems that the price of milk will decrease after 2015 because milk production will increase above milk demand. In this context, two

strategies may predominate in the next future: highly intensified and specialized large dairy farms or smaller organic dairy farms.

As reported in the current thesis, dairy farming is conducted in the northern side of the

Basque Country, which is characterized by the mountainous orography and high social and industrial pressure in valleys. This situation make it difficult to extensify farming

activity by increasing farmland availability (the lack of land availability increases the price of land). According to the prevision by 2015, dairy farmers in the territory should

prepare their strategy for the future: a) increase dairy herds size above farmland availability, producing milk as much as they are able to do in highly intensified and

specialized farms; b) try to adapt their farm to an organic farming system, maybe completing their activity/economy with other farming activities (organic crop production, etc). Previsions point out medium size dairy farms (specialized dairy farms

with 50 lactating dairy cows) may have survival problems unless rethinking their future strategy. How can this situation affect gaseous N losses in dairy farms? In the first case,

increasing gaseous losses will be expected at farm level. Larger stalls will loss more NH3, larger slurry storages will be required with subsequent higher NH3 and N2O

emissions and finally more slurry units will be applied on grassland, increasing therefore NH3, N2O and NO emissions. In addition, this system will be maintained

through high N inputs by concentrate and fertiliser incorporations. On the other hand, organic farming would be friendlier from gaseous N losses point of view. Maybe, milk


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NUE would be lower in grazing cows but overall dairy herd would be fitted to land availability and therefore whole gaseous N losses would be lower than recorded in

highly intensified farms. How might mentioned trends affect N losses in the territory? Although future scenarios are uncertain, according to the evolution of dairying activity

for the last years and predictions in the next future, gaseous N losses would increase in areas with high farm density (mainly Karrantza and Asteasu-Aia in the Basque

Country). On the contrary, it is not sure that emissions will increase in the territory, especially if dairy farmers continued abandoning the activity.

Although it was not the aim of the current PhD-thesis to discuss on what production

systems is more sustainable (intensification vs organic or familiar production), data recorded during the experiments and the future scenario of dairy farming make us

wonder ourselves some questions. Sustainability is the adjective that all human economical activities must obligatorily fit. Sustainability must include economical,

environmental and social items derived from the activity (Brundtland report, Anonymous, 1987). Although this PhD-thesis was based on environmental issues, and

environment is an essential part of whole sustainability, it must be remarked that dairy farming is basically an economical activity. In fact, the main aim of dairy farming is to

provide economical income for dairy farmers and their families. Therefore, environmental and social sustainability must be primarily based on the economical

sustainability of the activity. Perhaps, gaseous N pollution or social discussions derived from dairy farming should be considered as good news by themselves because their

existence means that economical sustainability may be guaranteed. Perhaps decreasing N pollution from dairy farming to minimum levels would be the worst news for dairy producers and milk consumers. This option would be also negative from feed

sovereignty point of view because having a well-established dairy farming in a territory is a pathway to guarantee feed sovereignty in same way. The challenge for the future

will be to establish where the minimum gaseous N pollution level is and what production systems will guarantee in economical and social sustainable dairy farming to

reach territorial sovereignty. In addition, the likely effect of the climate change should be also taken into account.

Maybe, in another PhD-thesis…


8 CONCLUSIONS


177

The different trials conducted in the current PhD-thesis let us to conclude that:

1. Protein overfeeding (metabolizable protein intake exceeded 7.4%) is a common practice among lactating herds of commercial dairy farms (milk yield, 5,713 to

12,165 kg cow-1 year-1) from the Basque Country (northern Spain). 2. The correct match between dietary CP fed and that required by lactating cows is

a valid option to reduce N excretion in commercial and experimental farms. In addition to fitting dietary CP content, balancing the energy content of rations

may increase milk N use efficiency (or reduce N excreted per milk yield) through improving milk yield.

3. Dietary N manipulation may alter diet-derived slurry composition, especially in relation to urinary N compounds (N content and urea-N). Modifications in slurry

N composition may afterwards affect slurry management at farm level (whether slurry is applied on field to fit properly plant N requirements or is applied on

fresh matter basis to empty slurry storages), which has environmental and economical implications.

4. Ammonia concentration is reduced in dairy tie-stalls when dietary CP content is fitted to animal requirements. Air temperature will control NH3 concentration in

barn floors. Nitrous oxide concentration, which may be classified as low, is not affected by dietary manipulation at stall level. Nevertheless, N2O concentration

may be significantly different from background concentration. 5. Dietary strategy is subordinated to slurry management on grassland in relation to

gaseous N losses (NH3, N2O and NO). Appling equal NH4+-N doses (120 kg

NH4+-N ha-1), gaseous N losses do not differ between diet-derived slurries. The

high spatial variability of fluxes may also limit the effect of nutrition in gaseous

N emissions. Between 10% and 15% of applied NH4+-N is lost as gaseous N

(NH3, N2O and NO) when slurry is applied on grassland. Most of gaseous N

losses occur as NH3 (NH3 represented more than 60% of volatilised N after two months of measurements). A proper nutritional strategy should be considered

together with the subsequent management of slurry in relation to gaseous N losses per kilogram of milk produced or kilogram of slurry applied.

6. The success of dietary manipulation to minimize N excretion at farm level is seriously limited in highly intensified dairy farms. Intensification parameters

(high animal density and low farmland availability) are main factors affecting


178


farm N excretion per farmland hectare (dietary N manipulation explained 11.2% of total variance on farm N excretion per hectare).

7. Rations which improve milk N use efficiency may indirectly reduce cow P excretion through the improvement of milk P use. Nevertheless, cattle fed with

high P-content feedstuffs will require major attention in P formulation.


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TESIS DOCTORALES PUBLICADAS

Nº 1. La raza Latxa: Sistemas de producción y características reproductivas. EDUARDO URIARTE EGURCEGUI

Nº 2. Estudio y puesta a punto de un método simplificado de control lechero cualitativo en la raza ovina Latxa y su inclusión en el plan de selección. GUSTAVO ADOLFO MARIA LEVRINO

Nº 3. Implicaciones tecnológicas de la composición química del pescado con especial referencia a los lípidos. ROGELIO POZO CARRO

Nº 4. Estudio de suelos de Vizkaia. MARGARITA DOMINGO URARTENº 5. El Maedi o neumonía progresiva en el conjunto de las enfermedades respiratorias crónicas del

ganado ovino en la Comunidad Autónoma Vasca. LORENZO GONZÁLEZ ANGULONº 6. Estudio experimental de las fases iniciales de la paratuberculosis ovina. RAMÓN A. JUSTE JORDANNº 7. Identificación, origen y factores fisicoquímicos que condicionan la contaminación por elementos

metálicos de sedimentos de ríos. ESTILITA RUIZ ROMERANº 8. Análisis financiero de proyectos de inversión en repoblaciones forestales. ÁLAVARO AUNOS GÓMEZNº 9. Desarrollo y evaluación del sistema integrado de diagnóstico y recomendación (DRIS) para la

fertilización de las praderas permanentes. Marta Rodríguez JuliaNº 10. Estudio de las mieles producidas en la Comunidad Autónoma del País Vasco. MARÍA TERESA

SANCHO ORTIZNº 11. La biomasa microbiana como agente de las transformaciones de nitrógeno en el suelo tras el ente-

rrado de la paja de cereal. JESÚS ÁNGEL OCIO ARMENTIANº 12. Análisis jurídico y económico de la implementación de la política agraria comunitaria en la

Comunidad Autónoma del País Vasco. BEATRIZ PÉREZ DE LAS HERASNº 13. Nemátodos formadores de quistes (Globodera spp.) en patata (Solanum tuberosum L.): caracteri-

zación taxonómica, reproducción y actividad de las formas juveniles. AZUCENA SALAZAR BAYONANº 14. Ensayo comparativo de tres métodos de tratamiento antihelmítico estratégico en rebaños de ove-

jas latxas. ANA LUISA GRACIA PÉREZNº 15. Estudio sobre una encefalitis vírica similar al Louping-ill en el ganado ovino de la Comunidad

Autónoma Vasca. DANIEL FERNÁNDEZ DE LUCO MARTINÉZNº 16. Análisis de caracteres involucrados en la selección y mejora de Lupinus hispanicus Boiss. et

Reuter. VERÓNICA ARRIETA PICONº 17. Contribución al estudio de fermentaciones artesanales e industriales de Rioja Alavesa. MILAGROS

VIÑEGRA GARCÍANº 18. Estudio del manejo de la alimentación en los rebaños ovinos de raza Latxa y su influencia sobre

los resultados reproductivos y de producción de leche. LUIS Mª. OREGUI LIZARRALDENº 19. El sector pesquero vizcaíno, 1800-1960. Análisis de la interacción de los elementos ambiental,

extractivo y comercial en la pesquería. JOSÉ AGUSTÍN MAIZ ALCORTANº 20. Epidemiología, diagnóstico y control de la paratuberculosis ovina en la Comunidad Autónoma

del País Vasco. J. J. ADURIZ RECALDENº 21. Agrupación de poblaciones locales de maíz (Zea mays L.) mediante caracteres morfológicos y

parámetros ambientales. JOSÉ IGNACIO RUIZ DE GALARRETA GÓMEZNº 22. Estudio del potencial melífero de Bizkaia. AMELIA CERVELLO MARTÍNEZ Nº 23. Influencia de los procesos de salado y ahumado sobre las características fisicoquímicas del queso

Idiazabal (compuestos nitrogenados). FRANCISCO C. IBAÑEZ MOYANº 24. El Euskal Artzain Txakurra (el perro pastor vasco) descripción y tipificación racial. MARIANO

GÓMEZ FERNÁNDEZNº 25. Evaluación de diferentes ciclos de selección recurrente en dos poblaciones sintéticas de maíz.

GOTZONE GARAY SOLACHINº 26. Valoración agronómica de la gallinaza: Compostaje. ADOLFO MENOYO PUELLESNº 27. Relación clima-vegetación en la Comunidad Autónoma del País Vasco. AMELIA ORTUBAY FUENTES


208

Nº 28. Influencia de los procesos de salado y ahumado tradicional sobre las características microbiológi-cas y organolépticas del queso Idiazabal. FRANCISCO J. PÉREZ ELORTONDO

Nº 29. Mastitis en la oveja Latxa: epidemiología, diagnóstico y control. JUAN C. MARCO MELERONº 30. Contribución al conocimiento anatomopatológico y diagnóstico de la tuberculosis caprina y ovina

por Mycobacterium bovis. M.ª MONTSERRAT GUTIÉRREZ CANCELANº 31. Estudio de factores que pueden influir en la calidad de la pluma de gallos Eusko-oiloa (Variedad

Marradune) para la fabricación de moscas artificiales utilizadas en la pesca de la trucha. ROSA M.ª ECHARRI TOMÉ

Nº 32. Estudio de la fracción lipídica durante la maduración del queso Idiazabal. Influencia de los pro-cesos tecnológicos del tiempo de permanencia en salmuera y ahumado. ANA ISABEL NÁJERA ORTIGOSA

Nº 33.- Influencia del tipo de cuajo y adición de cultivo iniciador sobre los compuestos nitrogenados durante la maduración del queso Idiazabal. M.ª SOLEDAD VICENTE MARTÍN

Nº 34. Estudio de la infección por Borrelia burgdorferi, grupo Ehrlichia phagocytophila y virus de la encefalitis ovina en las poblaciones de ixódidos de la Comunidad Autónoma Vasca. MARTA BARRAL LAHIDALGA

Nº 35. Lipolisis en el queso Idiazabal: efecto de la época de elaboración, del cultivo iniciador, de la pas-teurización y del tipo de cuajo. FELISA CHAVARRI DÍAZ DE CERIO

Nº 36. Aspectos inmunopalógicos de la paratuberculosis de los pequeños rumiantes. Respuesta inmune asociada a la vacunación. JUAN MANUEL CORPA ARENAS

Nº 37. Desarrollo y evaluación de nuevas técnicas de diagnóstico del Maedi-Visna. ANA BELÉN EXTRAMIANA ALONSO

Nº 38. Estudios sobre Patogenia y Diagnóstico de la Adenomatosis Pulmonar Ovina. MARÍA MERCEDES GARCÍA GOTI

Nº 39. Análisis de los factores de explotación que afectan a la producción lechera en los rebaños de raza Latxa de la CAPV. ROBERTO J. RUIZ SANTOS

Nº 40. Crecimiento y producción de repoblaciones de Pinus radiata D. Don en el Territorio Histórico de Gipuzkoa (País Vasco). LUIS MARIO CHAUCHARD BADANO

Nº 41. Puesta a punto de técnicas PCR en heces y de Elisa para el diagnóstico de la Paratuberculosis. Estudio de prevalencia en ganado bovino. JOSEBA M. GARRIDO URKULLU

Nº 42. Epidemiología y diagnóstico de la leptospirosis y la neosporosis en explotaciones de bovino lechero de la CAPV. RAQUEL ACHAERANDIO GALDOS

Nº 43. Relaciones aire-agua en sustratos de cultivo como base para el control del riego. Metodología de laboratorio y modelización. VALENTÍN TERÉS TERÉS

Nº 44. Zonas endémicas de enfermedad de Lyme en la CAPV: estudio del papel de los micromamíferos en el mantenimiento de Borrelia burgdorferi sensu lato en el medio natural. HORACIO GIL GIL

Nº 45. Optimización del esquema de mejora de la raza Latxa: análisis del modelo de valoración e intro-ducción de nuevos caractéres en el objetivo de selección. ANDRÉS LEGARZA ALBIZU

Nº 46. Influencia de las condiciones de almacenamiento, reimplantación y lluvia ácida en la viabilidad de Pinus radiata D. Don. MIREN AMAIA MENA PETITE

Nº 47. Estudio sobre encefalopatías en peces: patogenicidad del nodavirus causante de la enfermedad y retinopatía vírica (ERV) y transmisión experimental del prión scrapie a peces. RAQUEL ARANGUREN RUIZ

Nº 48. Enfermedades transmitidas por semilla en judía-grano (Phaseolus vulgaris L.): detección, control sanitario y mejora genética. ANA MARÍA DÍEZ NAVAJAS

Nº 49. Pastoreo del ganado vacuno en zonas de montaña y su integración en los sistemas de producción de la CAPV. NEREA MANDALUNIZ ASTIGARRAGA

Nº 50. Aspectos básicos de la mejora genética de patata (Solanum tuberosum L.) a nivel diploide. LEIRE BARANDALLA URTIAGA

Nº 51. El cuajo de cordero en pasta: preparación y efecto en los procesos proteolíticos y lipolíticos de la maduración del queso de Idiazabal. Mª. ÁNGELES BUSTAMANTE GALLEGO

Nº 52. Dinámica de la población de atún blanco (Thunnus alalunga Bonnaterre 1788) del Atlántico Norte. JOSU SANTIAGO BURRUTXAGA


209

Nº 53. El pino radiata (Pinus radiata D.Don) en la historia forestal de la Comunidad Autónoma de euska-di. Análisis de un proceso de forestalismo intensivo. MARIO MICHEL RODRÍGUEZ

Nº 54. Balance hídrico y mineral del pimiento de Gernika (Capsicum annuum L., cv Derio) en cultivo hidropónico. Relaciones con la producción. HUGO MACÍA OLIVER

Nº 55. Desarrollo de métodos moleculares y su aplicación al estudio de la resistencia genética y patoge-nia molecular del Scrapie. DAVID GARCÍA CRESPO

Nº 56. Estudio epidemiológico y experimental de la transmisión y control del virus Maedi-Visna en ovino lechero de raza Latxa del País Vasco. VEGA ÁLVAREZ MAIZTEGUI

Nº 57. Desarrollo y aplicación de técnicas de diagnóstico serológico para el estudio de la transmisión calostral y horizontal del virus Maedi-Visna (VMV) en ovino. MARA ELISA DALTABUIT TEST

Nº 58. Integral Study of Calving Ease in Spanish Holstein Population. EVANGELINA LÓPEZ DE MATURANA LÓPEZ DE LACALLE

Nº 59. Caracterización Molecular, Detección y Resistencia de Mycobacterium avium subespecie paratu-berculosis. IKER SEVILLA AGIRREGOMOSKORTA

Nº 60. Desarrollo de un sistema de fertilización nitrogenada racional en trigo blando de invierno bajo condiciones de clima mediterráneo húmedo. M.ª ARRITOKIETA ORTUZAR IRAGORRI

Nº 61. Estructura y dinámica de la materia orgánica del suelo en ecosistemas forestales templados: de los particular a lo general. NAHIA GARTZIA BENGOETXEA

Nº 62. Análisis sensorial del vino tinto joven de Rioja Alavesa: descripción y evaluación de la calidad. IÑAKI ETAIO ALONSO

Nº 63. Biología del gusano de alambre (Agriotes spp.) en la Llanada Alavesa y desarrollo de estrategias de control integrado en el cultivo de la patata. ANA ISABEL RUIZ DE AZÚA ESTÍVARIZ

Nº 64. La sucesión en la ganadería familiar: el ovino de leche en el País Vasco. GUADALUPE RAMOS TRUCHERO

Nº 65. Identificación molecular de las especies de piroplasmas en las poblaciones de Inóxidos de la Comunidad Autónoma del País Vasco. Distribución y prevalencia de babesia y theileria en los ungulados domésticos y silvestre. MIREN JOSUNE GARCÍA

Nº 66. Estudio de variables inmunológicas y bacteriológicas en relación con la inmunización frente a paratuberculosis en los rumiantes. MARÍA V. GEIJO VÁZQUEZ

Nº 67. Bacterias lácticas de sidra natural: implicación en alteraciones y potencial probiótico de cepas pro-ductoras de (1,3)(1,2)-ß-D-glucanos. GAIZKA GARAI IBABE

Nº 68. Influencia de los sistemas de producción ovina en la calidad y las propiedades tecnológicas de la leche y el queso. EUNATE ABILLEIRA CILLERO

Nº 69. Los carnívoros silvestres como reservorios de enfermedades de interés en sanidad animal y salud pública. XEIDER GERRIKAGOITIA SAGARNA


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