Nitrate as a fermentable nitrogen supplement for goats fed forage based diets low in true protein

Hao Trinh Phuc, Do Ho Quang, T R Preston and R A Leng

Cantho University,
Cantho, Vietnam
hqdo@ctu.edu.vn
* UTA-TOSOLY, AA 48 Socorro, Colombia
preston@lrrd.org
** Emeritus Professor, University of New England, Armidale NSW 2351 Australia,
PO Box 361, Coolum Beach 4573
Queensland, Australia
rleng@ozemail.com.au
 

Abstract

The adoption of management practices for ruminants which achieve major reductions in methane release is an issue of high priority to the amelioration of global warming. The biochemical changes when nitrate is converted to ammonia in the rumen of cattle and sheep is known to lower methane production. The present study aimed to demonstrate that nitrate salts could be included safely in low-protein diets fed to goats and that the efficiency of N utilization would be the same as in goats fed urea as the non-protein N source.

In Experiment 1, twelve young goats (initial weight 11± 2 kg), housed in individual cages, were fed a basal diet of rice straw and molasses and allocated in a completely random design to two treatments. Eight goats (K-N) received increasing amounts of potassium nitrate; the other four goats (CTL) were fed the same basal diet but without any additional fermentable N. The goats on the K-N diet were step-wise adapted to increasing levels of potassium nitrate, beginning with a concentration of 0.33% KNO₃ in diet DM in week 1, and increasing to 5.33% at the end of week 5.  In Experiment 2, the CTL goats were fed iso-nitrogenous levels of N from urea and all goats received additional foliage of Sesbania grandiflora. In Experiment 3, ammonium nitrate was given to 4 of the goats previously fed potassium nitrate. The nutritional density of the diets was increased by treating the rice straw with sodium hydroxide and including cottonseed meal as a source of bypass protein. Overall the study took place over a continuous period of 22 weeks.

Nitrogen balance in the goats improved as the amount of nitrate in the diet was increased stepwise at weekly intervals. At zero nitrate in the diet, the N balance was negative but then increased linearly as nitrate was increased reaching positive values by week 3 when the KNO₃ concentration was over 1% in the diet. Growth rates and N retention tended to be higher for the goats receiving nitrate compared with those not having fermentable N in the diet. There were no differences in growth rate and N retention between the nitrate and urea diets at the same level of feeding of the Sesbania.

 Ammonium nitrate gave similar results as potassium nitrate and both were comparable with urea in providing rumen fermentable N and supporting acceptable growth rates when the goats were fed diets of higher nutritional density.  

Key words: Ammonium nitrate, bacteria, cottonseed, growth rate, N retention, rice straw, rumen fermentation, Sesbania

Nitrato como suplemento de nitrógeno fermentable para cabras con dietas bajas en proteína verdadera basadas en forraje

Resumen

La implementación de prácticas de manejo de rumiantes que permitan alcanzar una mayor reducción en la liberación de metano es un tema de alta prioridad para disminuir el calentamiento global. Los cambios bioquímicos que se suceden cuando el nitrato es convertido a amonia en el rumen del ganado y las ovejas, reducen la producción de metano. El presente estudio intenta demostar que las sales de nitrato pueden ser incluídas en forma segura en las dietas bajas en proteína de cabras y que la eficiencia de la utilización de N puede ser similar a la obtenida en cabras consumiendo urea como fuente de N no proteico.

En el Experimento 1, doce cabras jóvenes (peso inicial 11± 2 kg) alojadas en jaulas individuales, con una dieta basal de paja de arroz y melaza se asignaron en dos tratamientos con un diseño completamente al azar. Ocho cabras (K-N) recibieron cantidades incrementales de nitrato de potasio; las otras cuatro cabras (CTL) recibieron la dieta basal sin adición alguna de N fermentable. Las cabras con la dieta K-N fueron adaptadas paulatinamente a niveles incrementales de nitrato de potasio, iniciando con una concentración de 0.33% KNO3 de la MS de la dieta en la semana 1, e incrementando a 5.33% al final de la semana 5. En el Experimento 2, las cabras CTL se alimentaron con niveles iso-nitrogenados de N proveniente de la urea y todas las cabras recibieron follaje adicional de Sesbania grandiflora. En el Experimento 3, nitrato de amonio se suministró a 4 de las cabras que habian recibido previamente nitrato de potasio. La densidad nutricional de las dietas fue incrementada mediante el tratamiento de la paja de arroz con hidróxido de sodio e incluyendo harina de algodón como fuente de proteína sobrepasante. En total el estudio se realizó a lo largo de 22 semanas continuas.

El balance de nitrógeno de las cabras mejoró a medida que la cantidad de nitrato de la dieta fue incrementado paulatinamente en intervalos semanales. Con una dieta con cero nitrato, el balance de N fue negativo, pero incrementó linealmente a medida que el nitrato ascendia, alcanzando valores positivos en la semana 3 cuando la concentración de KNO3 estaba sobre el 1% de la dieta. La tasa de crecimiento y la retención de N fueron mayores en las cabras recibiendo nitrato, al compararlas con aquellas sin N fermentable en la dieta. No hubo diferencias en la tasa de crecimiento y en la retención de N entre las dietas con urea vs. nitrato con el mismo nivel de suministro de Sesbania.

El nitrato de amonio mostró resultados similares al nitrato de potasio y ambos fueron comparables con la urea en cuanto al suministro de N fermentable en el rumen y también en permitir tasas de crecimiento aceptables cuando las cabras recibieron dietas de mas alta densidad nutricional.

Palabras clave: nitrato de amonio, bacteria, harina de algodon, tasa de crecimiento, retención de N, paja de arroz, fermentación ruminal, Sesbania

Introduction

On basal diets low in crude protein the availability of ammonia is often a primary deficiency in diets fed to ruminants, limiting microbial growth in the rumen and therefore digestibility and feed intake. Agro-industrial by products, low in protein such as rice straw are the primary source of feed given to ruminants in most developing countries (see Preston and Leng 1987). Providing supplements that contain urea (and a source of deficient minerals) are now common ways of increasing animal productivity (Loosli and McDonald 1968; Leng 1990). In ruminants fed rice straw,  grazing dry standing pastures or on grain based diets, urea is often added to the diet to correct a deficiency of ammonia in the rumen and increase the efficiency of rumen microbial growth; feed digestibility and feed intake (Leng 2005).  A source of  rumen ammonia, usually as urea but potentially replaced by nitrate, is essential in high energy diets low in true protein which have great potential as basal diets for ruminant production in tropical countries (eg: molasses [Preston  1972] or sugar cane [Preston and Leng 1978]) that support production levels close to genetic capacity of ruminants.

Nitrate could potentially replace urea in low protein diets to provide a source of rumen ammonia and at the same time provide a hydrogen sink and reduce enteric methane production. However, research in this area has been discouraged or stifled by the incidence of a poisoning syndrome in ruminants associated with nitrate in feed that largely occurs on heavily fertilized, temperate pastures (Crawford et al 1966; Kemp et al 1977) but also on heavily fertilized tropical pastures (eg: Kikuyu grass [Marais 1998]) that are already high in true protein. On such diets it would be illogical to supplement with any non-protein N source

Nitrate is not toxic to ruminants but  nitrite is detrimental to well being and at times accumulates in rumen fluid when nitrate is suddenly introduced by either an intra-ruminal injection (Lewis 1951) or inadvertently when animals consume forages containing high levels of both protein and nitrate (Kemp et al 1977). Many temperate pastures growing under high fertility conditions contain low levels of nitrate (0 to 0.4% of the dry matter depending on the level of fertilizer application and protein content of the forage (Lovett et al 2004). Nitrate content can increase by 10 fold these values when the plant’s photosynthetic rate is slowed or inhibited by environmental factors such as reduced illumination, a period of low temperatures or the drying out of the roots (Hibbard et al 1994; Pfister 1988; Krejsa et al 1987).

Early research demonstrated that nitrite was not released in significant amounts in the rumen in  sheep slowly acclimated to dietary nitrate by stepwise increase in nitrate in the feed over a period of 21 days (Allison and Reddy 1984; Alaboudi and Jones 1985).

Leng (2008) has recently reviewed the literature on nitrate metabolism and concluded that nitrite accumulation in the rumen when nitrate is administered is associated with:

•  abnormally high and sudden intake of nitrate in a meal  
•  lack of adaptation of rumen organisms to nitrate
• diets excessively high in rumen degradable protein.

These are the same limitations that could be applied to urea toxicity. Nevertheless, urea is widely used as a non protein N source throughout the world, where the aim is to increase ruminant live stock production from low-protein feeds.

It is hypothesised that nitrate will be used efficiently and safely as a major source of rumen ammonia in the diet when the three conditions listed above are taken into account. The diets used widely to support ruminant production in non-industrialised countries are based mainly on agro industrial by products that require supplementation with sources of fermentable nitrogen and bypass protein (Preston and Leng 1987). Thus there is considerable potential to use nitrate to replace urea as the major fermentable nitrogen source with potentially substantial benefits, if enteric methane production is also reduced.

In the study reported here it is shown that nitrate, at 50-90% of the total dietary N, supported normal growth of young goats on diets that are typically fed in most developing countries in the tropics.

Materials and Methods

Location, animals and treatments

The trials were carried out in the experimental farm of Can Tho University, Can Tho, Vietnam.

Experiment 1: Adaptation of goats to nitrate (6 weeks)

Twelve young goats (initial weight 11± 2 kg) were fed a basal diet of rice straw and molasses (Table 1). They were housed in individual cages and allocated in a completely random design to two treatments. Eight goats (K-N) received increasing amounts of potassium nitrate; the other four goats (CTL) were fed the same basal diet but without any additional fermentable N. The goats on the K-N diet were step-wise adapted to increasing levels of potassium nitrate, beginning with a concentration of 0.33% in diet DM in week 1, and increasing to 5.33% at the end of week 5.

N balance was measured daily over each week and the nitrate levels were adjusted every 7 days. The potassium nitrate was mixed in the molasses portion of the diet and sprayed on the straw prior to it being given to the goats.

Experiment 2: Comparison of potassium nitrate and urea as a fermentable N source

The 8 goats from Experiment 1 that had received nitrate were re-allocated at random to two treatments:

• K-NS1: Potassium nitrate + rice straw + molasses + foliage of Sesbania grandiflora at 1% of LW (DM basis)
• K-NS0.5: Same as KN-S1 but with Sesbania foliage at 0.5% of LW (DM basis)

The 4 goats in treatment CTL in Experiment 1 were changed to:

• US1: Urea (iso-nitrogenous with potassium nitrate) + rice straw + molasses + Sesbania grandiflora at 1% of LW (DM basis)

 Experiment 3: Comparing potassium nitrate, ammonium nitrate and urea as the fermentable N source in low protein diets fed to goats

12 goats were used in this experiment. The 8 goats from the potassium nitrate treatments in Experiment 2 were re-allocated to two treatments:

K-N: Potassium nitrate

A-N: Ammonium nitrate

The 4 goats on US1 continued  with urea as the fermentable N source

The basal diet for all the goats was changed to NaOH-treated rice straw + molasses + Sesbania grandiflora  + CaCO₃  + cottonseed meal. These diets were fed over a period of 10 weeks.

Feeding and management

Dry rice straw, molasses and foliage of Sesbania grandiflora were purchased from local farmers; cottonseed meal was bought from the market (Table 2). The rice straw was offered ad libitum with fresh straw being given at 7:00, 11:00, 15:00 and 18: 00h every day. Water was freely available. In experiment 2, fresh foliage of Sesbania grandiflora was given on 2 occasions each day at a level of 10 kg or 5 kg for each 100 kg live weight of goats. In experiment 3, the rice straw was treated with treated with 3% sodium hydroxide (15.7 g NaOH in 1 litre  of water sprayed on the straw  at 1.92 litres/kg dry straw). The goats were also fed cottonseed meal at a level of 10 g for each 1 kg live weight at 7: 00h every morning. In all experiments, the nitrate/urea was mixed in the molasses, which was then sprayed on the treated rice straw prior to feeding.

In experiments 1 and 2, faeces and urine were collected and weighed every morning. Samples of each were kept in the deep freezer. H₂SO₄ was added to the urine bucket in quantities sufficient to maintain the pH below 4.0.

In Experiment 2 rumen samples were taken by stomach tube before and 3 hours after feeding the Sesbania foliage.

Measurements

The goats were weighed periodically. In Experiment 1 they were weighed weekly whereas in Experiments 2 and 3 they were weighed before and at the end of each experiment. Feed intakes were recorded each day in all experiments.  Representative samples of feeds were taken for analysis of DM, N and ash at weekly intervals (Table 2). During the collection period in Experiments 1 and 2, samples of rice straw were taken once per week, while samples of Sesbania grandiflora foliage were taken every day and pooled weekly. DM content was determined on pooled samples. Feed refusals were collected from individual animals and weighed every day.  

The feeds and refusals in the digestibility study were analyzed for DM, ash and crude protein (N) according to AOAC (1990).  Organic matter (% OM) was calculated as 100-ash.

Samples of rumen fluid were acidified prior to analysis of ammonia by steam distillation. Samples for protozoa were preserved in formal-saline prior to counting at “x10” magnification. pH was determined on fresh rumen fluid using a glass electrode.

Statistical analysis

The data from each experiment were analysed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (version13.2). Sources of variation in the model were: treatments and error.

Results

Experiment 1: Adapting the goats to K- nitrate

Nitrogen balance in the goats improved as the amount of nitrate in the diet was increased stepwise at weekly intervals (Figure 1). At zero nitrate in the diet the N balance was negative but then increased linearly as nitrate was increased, reaching positive values by week 3 when the KNO₃ concentration was over 1% in the diet. Growth rates (P=0.079) and N retention (P=0.055) tended to be higher for the goats receiving nitrate compared with those not having fermentable N in the diet (Table 3).

Experiment 2: Comparison of potassium nitrate and urea as N sources in a diet of rice straw and molasses supplemented with foliage of Sesbania grandiflora

From observations made in Experiment 1 there appeared to be no ill effects in the goats from feeding nitrate as a major fermentable N source in a low protein diet. To confirm this observation, a second experiment was undertaken in which the nutritive value of the basal diet was improved by supplementing it with foliage of Sesbania grandifloraw, a foliage known to support very high growth rates in goats when fed as the sole diet (Nguyen Thi Hong Nhan 1998; Dahlanuddin 1991).  In this experiment urea was added to the control (CTL) diet so that all treatments had the same concentration of  non-protein N.

There were no differences between the nitrate (K-N1S) and urea (U1S) diets at the same level of feeding of the Sesbania (Table 4). Sesbania at 1% of live weight (DM basis) supported higher feed intake, growth rate and N retention with better fed conversion than when  Sesbania was fed at 0.5% of live weight. This is strongly indicative of the Sesbania protein having good rumen bypass characteristics.  

There were no differences in rumen pH, ammonia and protozoan numbers between goats fed potassium nitrate and those fed urea (Table 5). Lower levels of rumen ammonia in goats on treatment K-NS0.5 reflected the lower level of feeding the Sesbania foliage.

Experiment 3: Comparison of ammonium nitrate, potassium nitrate and urea as N sources in a diet of rice straw treated with sodium hydroxide and supplemented with foliage of Sesbania grandiflora and cottonseed meal

The final experiment examined ammonium nitrate as an alternative source of nitrate, which is more widely available, being the basis of many nitrogen-rich chemical fertilizers. In this experiment the nitrate/urea provided slightly over 50% of the dietary N (Table 6). The diets were fed over a total period of 70 days.  

Growth rates were higher on the diet with ammonium nitrate compared with the urea control (Table 7). Feed conversion was better on the nitrate diets compared with the urea control.

Discussion

Earlier research on nitrate utilisation by ruminants was largely aimed at investigating the toxicity syndrome in grazing animals that developed with increasing use of nitrogenous fertilisers to improve pasture production in temperate areas.  Nitrate metabolism has been mainly studied in ruminants fed diets high in crude protein mainly in the form of temperate forages. The research has focussed attention on the risks of poisoning in ruminants that result from the accumulation of nitrite in the rumen. When nitrite is absorbed it binds haemoglobin to form methaemoglobin in the red blood cells, limiting oxygen supple to essential organs and results in lowered production, abortion in pregnant animals and at times death (Hibberd et al 1994).

 To allow the rumen ecosystem and the animal to adapt, good nutritional management requires slow adjustment to any inclusion of nutrients in a diet. Adaptation is a requirement for the inclusion of high levels of urea in a diet (Coombe  and Tribe 1958) and sudden inclusion of  urea in a diet in high concentrations can be highly toxic. In early work there was considerable discussion about the use of urea in low protein diets for ruminants, which has similarities to the present concepts regarding nitrate toxicity. Without prior adjustment, 35g of urea would kill sheep but with adjustment and addition of urea slowly to the feed, 100g of urea were not fatal (Coombs and Tribe 1958).  

In the present study, a cautious approach was taken initially. In young goats, increasing the K-nitrate concentrations in a low protein feed in steps every 7 d converted a negative N balance at low concentrations (0.33 and 0.66% K-nitrate) to a positive increasing N balance at 1.33, 2.64 and 5.33% of the diet N (Figure 1). This demonstrated that nitrate was efficiently used as a fermentable nitrogen source for microbial growth in the rumen. The diet of molasses and rice straw supported a relatively low feed intake and N balance. Growth of young goats was improved in the second experiment when legume tree foliage was introduced into the same basal diet, supplemented with either urea or nitrate at iso-nitrogenous concentrations as the main fermentable nitrogen source. The comparison of urea or nitrate as the major N source in the diet showed that both could provide the fermentable N and therefore both were assimilated through ammonia generated by microbial action in the rumen and provided sufficient essential amino acids to support a positive N balance. It appeared that irrespective of the nitrogen source, ruminal ammonia concentrations were equal to, or above, optimal concentrations for efficient microbial growth (Table 5). Efficient rumen function on all of the iso-nitrogenous diets was indicated by the pH and ammonia content in rumen fluid.

In order to obtain further support for the concept that nitrate can safely replace urea in basal low protein diets given to live stock, a third study with goats compared three iso-nitrogenous sources of fermentable N in a higher quality diet of caustic soda-treated rice straw with molasses supplemented with a bypass protein source (cottonseed meal). Feed intake and growth rates did not differ between the two sources of nitrate. It even appeared that ammonium nitrate was slightly better than urea as the main fermentable N source in the diet.

Tillman et al (1965) demonstrated that nitrate could be used as the sole source of nitrate in a highly purified diet that supported low growth rates and without any toxic symptoms. However, the results reported here are the first to demonstrate that nitrate can be the fermentable N source in basal diets used in a large proportion of the world’s domestic ruminant population that are fed on low protein mature forages, particularly in developing countries.

The low protein diets used in these studies supported growth rates in the local breed of goats of slightly over 50% of their genetic potential, which is about 100g/day. These growth rates are likely to be acceptable in the subsistence agricultural systems that more then 1 billion people are locked into, and where their own diets are largely based on cereals  that are deficient in essential amino acids and other micro nutrients such as vitamin B₁₂ and zinc. In such situations,   availability of small amounts of meat protein will bring abut major improvements in human nutrition.

The use of higher energy diets based on sugar cane and nitrate holds great promise for the production of meat, milk and fibre with minimal emissions of methane.

Conclusions

• Potassium nitrate was fed safely to goats at levels that supplied all the requirements for rumen microbial protein synthesis in diets based on rice straw and molasses
  
  
• Growth rates and N retention were similar or slightly better when nitrate was the non-protein N source rather than urea.
  
  
• Over a 70 day period it was shown that both ammonium and potassium nitrate were  effectively used by growing goats as a high proportion of the total dietary crude protein.

References

Alaboudi R and Jones G A 1985 Effects of acclimation to high nitrate intake on some rumen fermentation parameters in sheep. Canadian Journal of Animal Science 65: 841-849

Allison M J and Reddy C A 1984 Adaptations of gastrointestinal bacteria in response to changes in dietary oxalate and nitrate. In “Current Perspectives in Microbial Ecology”. Proceedings of the 3^rd International Symposium on Microbial Ecology. 7-12 August 1983. M J Klug and C A Reddy (editors) American Society of Microbiology. Washington DC.

AOAC 1990 Official methods of analysis. Association of official Analysis (15^th edition). Washington, D.C, USA

Coombe J B and Tribe D E 1958  Toxicity of Urea to Sheep. Nature 182:116 – 117

Crawford R F, Kennedy W K and Davison K L  1966  Factors influencing the toxicity of forages that contain nitrate when fed to cattle. Cornell Veterinary 56: 3-17

Dahlanuddin 2001 Forages commonly available to goats under farm conditions on Lombok Island, Indonesia.  Livestock Research for Rural Development. Volume 13, Article #10.  http://www.lrrd.org/lrrd13/1/dahl131.htm

Hibberd C A, Rehberger T G, Swartzlander  J and Parrott T  1994  Utilization of high nitrate forages by beef cows, dairy cows and stocker calves. Conference proceedings Management of High Nitrate Forages for Beef and Dairy Cattle http://www.priorityiac.com/ace-files/Utilize_nit.pdf

Kemp A, Geurink J H, Haalstra R T and Malestein A 1977  Nitrate poisoning in cattle. 2. Changes in nitrate in rumen fluid and methaemoglobin formation in blood after high nitrate intake. Netherlands Journal of Agricultural Science 25: 51-62

Krejsa B B, Rouquette F M Jr, Holt E C, Camp B J, Nelson L R 1987 Alkaloid and nitrate concentrations in pearl millet as influenced by drought stress and fertilization with nitrogen and sulphur. Agronomy Journal  79:266

Leng R A 1990  Factors affecting the utilisation of 'poor quality' forages by ruminants particularly under tropical conditions. Nutrition Research Reviews. 3: 277-303 http://journals.cambridge.org/download.php?file=%2FNRR%2FNRR3_01%2FS0954422490000178a.pdf&code=be8c3a6567c6963c4579326df0a7aa58

 Leng R A  2005 Drought and dry season feeding strategies for cattle sheep and goats. Penambul Books . Australia

Leng R A 2008  The potential of feeding nitrate to reduce enteric methane production in ruminants. A Report to The Departmernt of Climate Change Commonwealth Government of Australia. ACT Canberra Australia For paper and PPT presentation see http://www.penambulbooks.com

Lewis D 1951 The metabolism of nitrate and nitrite by the sheep. 1. Reduction of nitrate in the rumen of the sheep. Biochemistry Journal 48: 175-179

Loosli J K and McDonald I W 1968  Non protein nitrogen in the nutrition of ruminants. FAO Agricultural studies No 75 Rome.

Lovett D K, Bortolozzo A, Conaghan P,  O’Kiely P O and O’Mara F P  2004 In vitro total and methane gas production as influenced by rate of nitrogen application, season of harvest and perennial rye grass cultivar.  Grass and Forage Science 59,227-232

Marais J P 1998  Nutritive Value of Kikuyu:Anti Quality Factors. In Proceedings Kikuyu Technology Day ( editor P E Bartholomew) (http://agriculture.kzntl.gov.za/portal/publications/books/kikuyu_technology_day/kikuyu_page6.htm )

Nguyen Thi Hong Nhan 1998 Effect of Sesbania grandiflora, Leucaena leucocephala, Hibiscus rosa-sinensis and Ceiba pentadra on intake, digestion and rumen environment of growing goats. Livestock Research for Rural Development. Volume 10, Article #133.  http://www.lrrd.org/lrrd10/3/nhan1.htm

Pfister J A 1988 Nitrate intoxication of ruminant livestock. In: The Ecology and Economic Impact of Poisonous Plants on Livestock Production. James L F, Ralphs M H, and Nielsen D B (Editors). Westview Press, Boulder and London.

Preston T R and Leng R A 1978  Sugar cane as a cattle feed. Part 1: Nutritional constraints and perspectives. World Animal Review 27: 7-12

Preston T R and Leng R A 1987  Matching Livestock Systems to Available Resources in the Tropics and Subtropics. Penambul Books Australia

Preston T R 1972 Fattening beef cattle on molasses in the tropics. World Animal Review (FAO) 1: 24-29

Tillman A D, Sheriha G M and Sirny R J 1965  Nitrate reduction studies with sheep.  Journal of Animal Science 24: 1140-1146 http://jas.fass.org/cgi/reprint/24/4/1140

Received 19 November 2008; Accepted 4 December 2008; Published 1 January 2009

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