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A review of nitrogen utilisation efficiency in the dairy cow, and what milk producers can do now to improve it

J.M. Moorby and H.R. McConochie

September 2002 Table of Contents

SUMMARY...... 3

INTRODUCTION...... 4

OBJECTIVE...... 6

FORAGE EFFECTS ON NITROGEN UTILISATION EFFICIENCY...... 6

EFFECTS OF FERTILISER NITROGEN APPLICATION ON NITROGEN UTILISATION EFFICIENCY ...... 6 IMPROVED RUMEN NUTRIENT BALANCE AT GRAZING...... 7 EFFECTS OF ENSILING GRASS ON NITROGEN UTILISATION EFFICIENCY...... 9 EFFECTS OF FEED SUPPLEMENTS ON NITROGEN UTILISATION EFFICIENCY...... 10

ACTION OF SUPPLEMENTS...... 10 EFFECTIVE SUPPLEMENTS FOR IMPROVING NITROGEN UTILISATION EFFICIENCY...... 13 MILK PROTEIN OUTPUT AND NITROGEN UTILISATION EFFICIENCY...... 16

MODE OF ACTION OF SUPPLEMENTARY AMINO ACIDS...... 18 Energy balance...... 19 Amino acid profile...... 19 EFFECTS OF DAIRY COW MANAGEMENT ON NITROGEN UTILISATION EFFICIENCY....20

MILKING FREQUENCY AND BST...... 20 MILK UREA NITROGEN...... 21 EFFECTS OF FORAGE CONSERVATION ON NITROGEN UTILISATION EFFICIENCY...... 22

GRASS SILAGE...... 22 Crop maturity...... 23 Wilting...... 24 Silage additives...... 24 LEGUME SILAGES...... 26 Nitrogen utilisation efficiency of ruminants fed forage and grain legumes...... 27 Tannin-containing legumes...... 29 Non-tannin containing legumes...... 30 Harvest management of legumes and nitrogen utilisation efficiency...... 33 RECOMMENDATIONS...... 34

FORAGE...... 34 Grazed forages...... 34 Conserved forages...... 35 RUMEN ENVIRONMENT...... 36 PERIPHERAL AND MAMMARY TISSUE METABOLISM...... 36 DAIRY COW MANAGEMENT...... 37 APPENDIX 1...... 38

REFERENCES...... 41

2 Summary Nitrogen utilisation efficiency (NUE) of dairy cattle on UK dairy farms is typically 20 – 30%. This means that 70 – 80% of dietary nitrogen is excreted, costing the dairy farmers in lost revenue, and contributing to environmental pollution. There are a number of things that the dairy farmer can do immediately to reduce N excretion from his cows, most of which are related to feeding the animal. The greatest loss of dietary N results from an imbalance between the supply of N and fermentable energy sources in the rumen, leading to the absorption of ammonia by the cow, a large proportion of which is excreted in urine. Crop management, cow management, silage making processes, and accurate diet formulation can all contribute to improving dairy cow NUE.

Post rumen inefficiencies in the utilisation of dietary N also occur, the most important of which is the use of amino acids for gluconeogenesis instead of for protein synthesis. Another cause of post-rumen inefficiency is an imbalanced supply of amino acids in relation to requirements for milk protein synthesis. The impact of both of these processes can be reduced through accurate diet formulation, ensuring that appropriate supplements are provided to the animals.

Specific recommendations for actions by dairy farmers which would lead to improvements in nitrogen utilisation efficiency by dairy cows include:  Reduce levels of N fertiliser application to grazing swards  Use grass varieties in new swards that contain high concentrations of water soluble carbohydrates  Move cows to fresh pastures after afternoon milking  Rapid wilt silage crops and use an effective bacterial inoculant to maximise residual water soluble carbohydrate concentrations and reduce proteolysis  Supply appropriate feed supplements (containing starches or sugars) to allow rumen microbes to make use of rapidly available N fractions of the diet  Consider the use of supplements containing rumen-protected methionine and lysine with maize-based diets  Use total-mixed rations to allow more accurate diet formulations  Use milk urea concentrations as an indicator of inefficient use of feed nitrogen.

3 Introduction In comparison with other ruminant and non-ruminant food production systems in the United Kingdom, the dairy industry is the most important contributor to nitrogen pollution (Castillo et al., 2000b). Nitrogen losses in agriculture are responsible for both atmospheric pollution, by volatilisation and denitrification of ammonia and nitrous oxide, and hydropheric pollution, by the leaching of nitrates into water. Intensification of the agricultural industry throughout Europe, characterised by the use of higher levels of nitrogen fertiliser and feed protein, corresponds to an increase in atmospheric pollution. Atmospheric ammonia inputs have increased 50% since 1950, and the agricultural sector is responsible for about 40% of anthropogenic nitrous oxide emissions (Morard, 1999). Hydropheric nitrate pollution is the subject of European directives that stipulate that the nitrate concentration of water must not exceed 50 mg nitrate/litre. However, this has been found to be exceeded in many waterways in the UK and is likely to be a consequence of leaching of surplus agricultural nitrogen which can amount to 40% of the nitrogen applied to agricultural land (Jarvis, 1994; Ledgard et al., 1998).

The 1991 Nitrate Directive, which is in the process of being fully implemented in England through the designation of Nitrate Vulnerable Zones (NVZ), concern over emissions of greenhouse gases, and current government interest in environmental protection, are all likely to result in greater regulation regarding the use of nitrogen and the production and disposal of nitrogenous by-products. Such regulation is likely to incur additional costs of production on the dairy industry. Implementation of the 1991 Nitrates Directive alone is forecast to cost the agricultural industry £26-36 million per year (DEFRA, 2001). Future regulation regarding nitrate pollution may follow that of the Netherlands, which involves a tax on nitrogen balance surpluses. The prospect of such a situation occurring in the UK increases the importance of improving nitrogen utilisation efficiency (NUE) on dairy farms. However, one must not ignore the potential financial benefits of improving NUE in the milk production system as improvements would be associated with either higher returns or reduced feed costs. Throughout this review NUE has been calculated as follows: NUE (%) = [N used for productive purposes (milk, growth) / N intake] x 100. In general, NUE on the dairy farm is very low (20 to 30 %) and in very intensive situations it can be even lower. Korevaar (1992) reported that in the Netherlands whole farm NUE levels were as low as 14 % in intensive systems. Bussink and Oenema (1998) calculated that whole farm NUE of up to 46 % is possible and whole farm experiments conducted in the United States confirm this: using an integrated approach to nitrogen utilisation on mixed crop and dairy farms, NUE of 42 and 47% have been realised (Dou et al., 1996).

There are two main areas of production that affect NUE. Firstly, in recent years there has been a shift in European farming practices from mixed to specialised intensive milk production operations. According to Weissbach and Ernst (1994), the loss of integration between livestock and crop production has led to a situation in which grassland alone cannot utilise the nitrogen excreted by livestock, and this in turn has led to increased losses of N and the importation of additional nitrogen onto the farm in the form of feed and fertiliser. Secondly, ruminants are inherently inefficient converters of nitrogen into protein. An analysis of nitrogen balance experiments conducted on 91 diets and 580 dairy cows in different countries (Castillo et al., 2000b) revealed that the average efficiency of utilisation of N intake for milk production is 28%. Analysis of additional N balance data (100 dairy cow diets and 27 sheep diets; Appendix 1) agrees with this, with a mean NUE of 26%. The greatest proportion of nitrogen loss occurs in the rumen in which ammonia produced from the microbial catabolism of proteins and amino acids and which is not incorporated into microbial protein, is absorbed across the rumen wall and either excreted as urea in urine or recycled back to the gut in saliva or across the intestinal mucosa. This inefficiency, through losses in urine, is the predominant reason for the poor NUE of the dairy farm.

We estimate that improvements in NUE from 26% to 35% would be worth in excess of £100 million per year to the UK dairy industry and could cut surplus nitrogen production by 49 thousand tonnes.

5 Objective The purpose of this review is to highlight the areas of the whole milk production system including nutrition and forage conservation, where the inefficiencies of nitrogen utilisation exist. The effect of inefficiencies on milk production and milk quality will also be examined. The review will conclude with a number of suggestions for improving NUE on dairy farm in the short term.

Forage effects on nitrogen utilisation efficiency In the UK, fresh and conserved grass constitutes a considerable proportion of the dairy cow’s diet. The quality of the forage protein, in terms of the proportions of true protein to non-protein nitrogen (NPN), can have a major bearing on the utilisation efficiency of the nitrogenous fractions of grass and grass silage. In fresh herbage true protein constitutes between 75% to 90% of the nitrogen fraction, however during ensilage 45% to 60% of this may be degraded to NPN (Woolford, 1984). Pasture management practices such as N application and maturity of herbage at harvest have been shown to affect the ratio of true protein to NPN, its degradability, and the concentrations of other important fractions such as water soluble carbohydrates (WSC; Peyraud et al., 1997). Although much of the protein entering the rumen is degraded into NPN and incorporated into microbial protein, this process tends to be the point at which inefficiencies occur, leading to poor NUE in the dairy cow. Van Straalen et al. (1993) found that 70% of the crude protein (CP) in grass silage is degraded in the rumen compared to around 58% of the CP in fresh ryegrass and clover. Dietary supplementation can influence the fate of N released in the rumen, and NUE has been found to increase from 17% to 30% in dairy cows offered herbage with starchy or fibrous concentrate supplements (Valk et al., 1990).

Effects of fertiliser nitrogen application on nitrogen utilisation efficiency Intensification within the dairy industry has led to an increase in the rate of fertiliser N being applied to grassland in order to realise higher stocking rates. Nitrogen balance data from a number of experiments which explored increased rates of N fertiliser application on grassland (Astigarraga et al., 1993; Delagarde et al., 1997; Peyraud et al., 1997), showed that NUE decreased with increasing N application rate. This is probably due to an increase in the rumen degradability of the forage nitrogen as a result of increased N

6 application and a reduction in the concentration of WSC (Peyraud and Astigarraga, 1998). Van Vuuren et al. (1992) showed that rumen ammonia concentrations in dairy cows offered herbage receiving 500 kg N/ha/year was higher than that of animals offered herbage receiving 275 kg N/ha/year. Likewise Astigarraga et al. (1993) showed that higher rates of fertiliser N application increased rumen ammonia concentrations and as a result increased blood urea concentrations. Although some of this N is utilised through the recycling of urea to the rumen, much N is lost in urine. The inability of rumen microbes to convert rumen available N into protein is the main reason for the poor NUE associated with heavily fertilised herbage. This is not simply a response to increased CP intake and the ratio between true protein and NPN in the CP. Of equal or greater importance is the dietary supply of fermentable carbohydrates (FC) and the balance of these with CP in the herbage. There is an inverse correlation between FC and CP in fresh grass, which is directly related to the rate of N fertiliser application (Peyraud and Astigarraga, 1998). As higher rates of N fertiliser are applied, grass CP concentration increases and water soluble carbohydrate (WSC) concentrations decrease, which can result in a reduction in NUE (Figure 1).

) 40 % (

y

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f 30 f e

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n e

g 20 o r t i 15 N 0 50 100 150 200 250 300 N application, kg/ha of pasture

Figure 1. Relationship between N fertiliser application to grazing pastures and nitrogen use efficiency in the dairy cow (data from Keady and Murphy, 1998, Keady et al., 1998 and Peyraud et al., 1997).

Improved rumen nutrient balance at grazing Beever and Siddons (1986) suggested that N losses arising from ammonia absorption from the rumen are a consequence of the imbalance that exists between the degradation

7 rates of ammonia-producing herbage N, and energy yielding organic matter (the so-called asynchrony in supply of energy and nitrogen). The effect of improved balance between dietary N and FC has been characterised in experiments studying the effect of offering cows perennial ryegrass bred for increased WSC concentrations on milk and milk protein production (Miller et al., 2001b; Moorby and Miller, unpublished data). In these experiments NUE was increased by up to 7% above a control variety of ryegrass, and overall efficiency of milk protein production was as high as 37%, apparently in response to the additional WSC present in the herbage. Work carried in vivo (Lee et al., 2002b) and in vitro (Miller et al., 2001b; Lee et al., 2002a) has demonstrated that incubation of grass with a high WSC concentration produced a lower concentration of ammonia and increased the production of propionate in rumen liquor compared with control grass.

Diurnal changes in grass WSC concentrations (Figure 2) mean that concentrations are higher in the afternoon than in the morning, when the plant has had time to photosynthesis. Orr et al. (2001) showed positive milk yield benefits from allocating fresh grass after afternoon milking, rather than after morning milking, using a strip grazing system. Dry matter intake was not different between animals offered fresh grass morning or afternoon, but milk yield was 5% higher from animals offered fresh grass after afternoon milking, probably as a result of increased DM and WSC concentrations at this time (Orr et al., 2001). The authors did not report milk composition, so it is not possible to calculate NUE from their results, although increased milk yields from similar rates of DM intake indicate increased efficiencies.

8

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c r n e t o a c 250 W

200

150

Figure 2.100 Diurnal changes in grass WSC concentration (from Miller et al., 2001b).

Effects of 50ensiling grass on nitrogen utilisation efficiency As a result of proteolysis and deamination reactions that occur during ensiling, the proportion 0of true protein in forage is reduced and the concentration of other nitrogenous 06.00 08.00 10.00 12.00 14.00 16.00 18.00 20.00 components, predominantly ammonia and free amino acids, is increased. The increased Time of day (h) ratio of non protein nitrogen (NPN) constituents increases the rumen degradability of grass silage nitrogen compared to the fresh material (Keady and Murphy, 1998; Van Straalen et al., 1993). However, this does not necessarily lead to differences in NUE, particularly in non-lactating animals with low N intakes (Keady and Murphy, 1998) which increases the importance of urea recycling (Van Soest, 1994). In the same study, NUE declined from 21% for grazed herbage to 18% for ensiled herbage in lactating animals (Keady and Murphy, 1998) while supplemental FC, in the form of sucrose added to the grass silage diet, appeared to increase NUE and milk protein concentration in the lactating cows. The authors concluded that this was a result of an improved microbial protein supply to the small intestine. The need for accurate and appropriate supplementation of grass silage diets is therefore important if high levels of NUE are to

9 be achieved, predominantly because of the increased protein breakdown products in the forage.

Effects of feed supplements on nitrogen utilisation efficiency Dairy cows are fed a variety of feeds to supply protein and energy, with the objective of providing the best diet to supply nutrients for good productivity. Increased supplies of protein in the diet often result in increased milk protein production, although this frequently occurs at the expense of NUE. In experiments in which grazing dairy cows received a supplement of 2 kg per day of soya (Astigarraga et al., 1993; Delagarde et al., 1997) NUE fell by up to 6 %, and this reduction in efficiency was predominantly a result of increased urinary N excretion. Similarly, supplementation of grass silage-based diets with different levels of protein, and protein of varying degradability, resulted in a decrease in NUE as crude protein concentration and degradability increased, although diet had no effect on milk or milk protein production despite an increase in the flow of rumen undegradable protein (RUP) to the small intestine (Castillo et al., 2000a). A recent review of the literature on the effect of RUP on dairy cow performance concluded that increased concentrations of dietary RUP had little effect on performance, especially when it results in a shortage of rumen degradable protein (RDP; Santos et al., 1998). Improvements in the efficiency of microbial protein synthesis are integral to improving NUE, and the microbial protein synthesised, in terms of its essential amino acid content compared to milk protein, is as good as most supplements containing high concentrations of RUP (Santos et al., 1998). On grass-based diets which are abundant in N, provision of additional carbohydrates to the diet have been shown to improve NUE while maintaining animal performance (Keady et al., 1998; Reynolds et al., 2001; Valk, 1994).

Action of supplements The efficient capture of dietary nitrogen and the synthesis of microbial protein by the rumen microbial population both require a balanced supply of both nitrogen and energy. When supply becomes unbalanced, losses of nitrogen as ammonia occur across the rumen wall and this is subsequently lost as urea in urine. This is primarily due to a shortage of ATP obtained from the fermentation of carbohydrates, which is necessary for protein synthesis from amino acids and ammonia. Limited supplies of carbohydrate cause rumen

10 bacteria to degrade amino acids for the production of ATP, which produces additional ammonia (Nocek and Russell, 1988).

Although the need for a balance between energy and nitrogen is well recognised (Dewhurst et al., 2000), some authors advocate the requirement for strict synchronicity in the supply of both fractions (Sinclair et al., 1993; Sinclair et al., 1995). Although it has been shown that strict synchronicity results in lower urea plasma concentrations, which indicates a more efficient use of dietary nitrogen (Witt et al., 2000), in vivo and in vitro experiments have shown that strict synchrony is not necessary for maximum microbial protein synthesis (Newbold and Rust, 1992), or production (Casper et al., 1999; Chamberlain and Choung, 1995; Newbold and Rust, 1992). Theoretically, a high degree of synchrony should result in improvements in NUE by reducing the amount of ammonia escaping the rumen. However, nitrogen balance experiments in which synchronous and asynchronous diets based on grazed herbage have been compared, showed little difference in NUE or milk production, despite the fact that blood urea concentrations were elevated in cows offered the asynchronous diet (Kolver et al., 1998). Experiments carried out by Sinclair et al. (1993; 1995) showed an increase in the production of microbial protein by sheep offered synchronous diets, although this did not appear to extend to benefits in terms of increased milk production (Witt et al., 2000). Newbold and Rust (1992) demonstrated in vitro that microbial protein production was not altered by a synchronous supply of energy and nitrogen compared to an asynchronous supply. This was probably due to the ability of rumen microbes to respond to transient periods of energy depravation by storing polysaccharides (Van Kessel and Russel, 1997), and then drawing on these reserves for protein synthesis when ammonia concentrations are elevated. However, the situation in vivo differs to that occurring in the artificial in vitro environment in two ways. Firstly, the rumen microbes do not remain in the rumen, but are continually passing out of the rumen and therefore taking their stored polysaccharides with them. Although it could be argued that any stored polysaccharide would be metabolised in futile non-protein producing, self maintaining cycles, these would cease in the acidic environment found post ruminally. Secondly, ammonia not captured by the rumen microbes crosses the rumen epithelium, and although can return as urea via saliva or transfer back across the rumen wall, losses inevitably occur.

11 The questions are therefore, if the efficiency of capture of rumen ammonia is reduced on asynchronous diets then why do animals perform as well as those receiving synchronous diets and why are no differences in nitrogen balance observed between the two regimes? The recycling of N back to the rumen via plasma urea as an explanation seems inadequate, since the animal’s urea pool is under physiological control. Van Soest (1994) suggested that at the homeostatic level, urea recycling is very efficient, and this is demonstrated by the NUE results obtained by Keady et al. (1998) with dry cows consuming small quantities of N. However, as rumen absorption of ammonia increases as a response to increased dietary N, the efficiency of urea N recycling is reduced and losses in urine increase proportionally. This would be an important factor on low protein diets, although it appears therefore that some other factor is responsible for reducing urinary N loss and maintaining animal performance on asynchronous diets.

One possible mode of action is that microbial storage polysaccharides synthesised in the rumen are degraded to glucose and absorbed in the lower gut. This supply of glucose above that produced endogenously from propionate in the liver has the potential to be metabolised to -ketoglutarate via the tricarboxylic acid cycle. Alpha-ketoglutarate may then be involved in amination reactions with ammonia leading to the production of glutamate, which itself is the precursor of numerous amino acids. This pathway may serve as an alternative for detoxification of absorbed ammonia, and unlike the ornithine cycle, theoretically does not consume amino acids or result in an increase in output of urea in urine. This hypothesis is supported by the findings of Obitsu et al. (2000) who demonstrated that glucose supplied post-ruminally resulted in lower plasma urea concentrations. Similarly, Reynolds et al. (2001) found that post-rumen glucose infusions reduced urinary nitrogen excretion and partitioned the retained N into tissue, rather than increasing milk protein output, which may have been an effect of insulin secretion in response to blood glucose concentrations. Absorption of glucose from the gut may also improve NUE by reducing the deamination of glucogenic amino acids for the production of energy, a degradative pathway that produces urea as a by-product. In this way, increased glucose supplies allow milk yields to be maintained or improved on diets of

12 lower CP content. In a similar but different area, supplementation of diets with feeds that supply otherwise limiting amino acids can help improve animal performance and NUE.

Although high concentrations of ammonia in the rumen can lead to losses of N in urine, a number of in vivo and in vitro studies (Annison, 1970; Hume et al., 1970; Mehrez et al., 1977; Mercer and Annison, 1976; Rooke et al., 1987; Satter and Slyter, 1974) have suggested that if the rumen ammonia concentration falls below a concentration of about 8 mM (470 mg/litre), microbial protein synthesis can be limited. Kanjanapruthipong and Leng (1998) found that the efficiency of microbial protein synthesis increased by up to 47 % when ammonia concentration was greater than 200 mg of N/litre. Although minimum rumen ammonia concentration is important because microbial protein production contributes a highly significant proportion of total protein supply to the animal, defining a concentration that is optimal in terms of efficiency is difficult. This is because of the close relationship between N and energy availability within the rumen, which governs the ability of the microbial population to convert N into microbial protein.

The evidence suggests, therefore, that NUE in the dairy cow can be improved by correct supplementation of grazed or ensiled forage with either a source of starch with a low degradability in the rumen, which acts post-ruminally, or FCs that closely mirror the degradative pattern of the nitrogenous components in the forage. Alternatively increasing the WSC content of the forage through grass breeding or management manipulation can result in improvements in NUE.

Effective supplements for improving nitrogen utilisation efficiency There is considerable experimental evidence to suggest that there are two major means of improving the NUE in dairy cows offered forage based diets: by improving the synchrony and/or balance in supply of energy and nitrogen in the rumen, or by increasing the post ruminal supply of glucose.

Synchrony may be improved by providing additional rumen FC to diets containing high levels of soluble N or by reducing the N content or N solubility of the forage. The latter two may be achieved by reducing the level of fertiliser N application, and the first may

13 be achieved by treating conserved forages with formic acid or an effective bacterial inoculant.

A multiple regression analysis of published data (Astigarraga et al., 1993; Valk and Hobbelink, 1992; Valk et al., 1990; Van Vuuren, 1993; Valk, 1994; Petit and Tremblay, 1995; Delagarde et al., 1997; Peyraud et al., 1997; Keady et al., 1998; Keady and Murphy, 1998; Moorby and Theobald, 1999; Castillo et al., 2000b; Reynolds et al., 2001; Castillo et al., 2000a; Aston et al., 1998; Kolver et al., 1998; Kebreab et al., 2000; Miller et al., 2001a; Miller et al., 2001b; Kebreab et al., 2001; Moorby et al., 2001) explored the relationship between NUE, N intake and N excretion in urine. Even though it may be assumed that NUE and N intake would be closely correlated (Figure 3) because N intake is used in the calculation of NUE, the most significant relationship is between NUE and N excretion in urine (Figure 4). The regression equation was found to be NUE% = 37.1 + 0.00048  N intake [g/d, P = 0.95] – 0.058  urine N excretion [g/d, P < 0.001]; s.e. obs. = 3.98; r2 = 0.59. The intercept figure of approximately 40% indicates the mean theoretical maximum NUE for the animals used in the calculation, the remaining 60% representing obligate losses through protein turnover and other maintenance requirements. The effect of N intake on NUE was found to be non- significant while the effect of the coefficient for urine N excretion was highly significant. This suggests that the major effects on NUE are those occurring within the animal which lead to losses of N in urine essentially regardless of the level of N intake. Many of these effects act at the rumen level, and appropriate nutritional manipulation of the animal can help to alleviate this problem.

14 45 %

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g 10 o r t i 5 N 0 100 200 300 400 500 600 700 800 N intake, g/d

Figure 3. Relationship between N intake and nitrogen utilisation efficiency in the dairy cow. References to data sources are given in the text.

45 %

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g 10 o r t i 5 N 0 0 100 200 300 400 500 Urine N excretion, g/d

Figure 4. Relationship between excretion of N in urine and nitrogen utilisation efficiency in the dairy cow. References to data sources are given in the text.

15 Experimentally, supplying glucose post ruminally has been achieved by intra gastric infusions of glucogenic precursors or glucose. In practice, however, this appears to be best achieved by increasing the supply of starch or by providing low rumen degradable starch. The supply of starch to the small intestine is affected by a number of factors, including source, maturity of forage and extent of processing of the grain supplement. Resistance to rumen degradation of starch present in maize silage has been shown to decrease with plant maturity, which was shown to alter N partitioning with an increase in milk N and a decrease in urinary N (Cammell et al., 2000a). Cammell et al. (2000b) showed non-ammnonia N flow to the duodenum was unaffected, and that the response found was possibly mediated through a sparing of amino acids as discussed previously. Hameelers (1998) found no effect on NUE or milk protein production of dairy cows offered whole crop maize or wheat starch, but noted that neither crop was in the optimal condition, the maize was low in starch and the wheat was too mature. Sutton et al. (1998) and Abdalla et al. (1999) found that feeding whole crop wheat resulted in an increase in supply of starch to the duodenum, but insufficient processing during ensilage meant that this was largely due to the presence of whole grains in the digesta. In this situation the effect of starch by-passing the rumen would be limited because it is not digested, and although increases in protein concentration were obtained there was no reduction in urinary N excretion (Abdalla et al., 1999; Sutton et al., 1998), which is in contrast to results obtained by supplying glucose in a highly absorbable form via intra gastric infusions (Obitsu et al., 2000; Reynolds et al., 2001). It can be concluded that if imbalance or asynchrony in the rumen leads to a large uptake of ammonia by the animal, a supply of glucose from the gut can help to reduce further urea production by sparing amino acids or even utilising some of the ammonia as a source of N for transamination processes. If sufficient glucose is not available, the ammonia-N will mostly be lost in urine.

Milk protein output and nitrogen utilisation efficiency The process of milk protein synthesis in the mammary gland is another source of inefficiency associated with nitrogen utilisation. For this reason an understanding of the processes involved, and the possibilities of manipulating them are central to improving NUE in the dairy cow. In order to put into context the importance of improving the

16 efficiency of mammary protein synthesis on whole animal NUE, a summary of reported literature on the use of encapsulated ruminally-protected methionine and lysine showed that supplementation had a consistent positive effect on N excretion in milk and on NUE (Robinson et al., 1995). Lysine and methionine tend to be the two first limiting amino acids on maize-based diets. Inefficiencies are due to the fact that the ratio between mammary protein synthesis and protein excreted in the milk is in the region of 1.5-2.6 : 1. This results in a continual cycling of proteins and amino acids through degradative and synthetic pathways, that not only results in the loss of non-recyclable nitrogenous products, but also incurs a high energetic cost in terms of ATP requirements (Bequette et al., 1998). The cellular mechanisms regulating amino acid uptake and protein synthesis by the mammary gland are complex, and results from experiments are often confounded which makes it difficult to derive specific conclusions. This stems from the fact that factors influencing the regulation of amino acid uptake are numerous, and include stage of lactation, hormonal status, source of amino acids, and the profile of amino acids absorbed by the mammary gland. However, discussion on all these interactions are outside the scope of this review and the reader is referred to Bequette et al. (1998) for further information.

Despite the complexity of interactions between numerous factors regulating milk protein production, a number conclusions can be drawn and are important in terms of improving the NUE of the dairy cow. One of the main benefits of improving the pattern of amino acid supply to mammary gland, in terms of its homology with that of milk protein, is the ability to reduce the total nitrogen intake of the dairy cow. Bach et al. (2000) showed that by provision of an improved amino acid supply, dairy cows could perform as well on a diet containing 15% CP as they did on a diet containing 18% CP, and as a result NUE was increased from 35 to 45%. Bach et al. (2000) also showed that as the CP content of the diet decreased, the amino acid profile of the diet became more important in order to maintain production. Post ruminal infusions of methionine elicited a linear response in milk casein production when a sufficient supply of lysine (7.3% of the total amino acids absorbed in the small intestine) was provided (Pisulewski et al., 1996). A concomitant decrease in plasma urea concentration was also observed, as were small increases in NUE, which suggests that improvements in amino acid supply have the potential to

17 improve NUE. Robinson et al. (1995) showed a 2% improvement in NUE (30.6% to 32.6%) by addition of rumen protected methionine and lysine to the diet. A lack of response to infusions of methionine alone has also been reported (Polan et al., 1991; Younge et al., 2001), indicating the importance of a number of amino acids and the basal diet offered to the animals.

On grass silage-based diets the data is more confused, with a number of experiments showing that amino acids other than lysine and methionine, predominantly histidine, are the most important. Vanhatalo et al. (1999) fed grass silage-based diets supplemented with abomasal infusions of either histidine alone or in combination with methionine and lysine. They found that milk yield and protein production was increased by supplementary infusions of histidine, and further increases associated with the addition of the other amino acids were additive to the histidine response. On the other hand, Varvikko et al. (1999) demonstrated that supplementary methionine and lysine had no effect on milk or milk protein production in cows fed grass silage diets, although it did result in increases in fat production. However, Younge et al. (2001) showed that methionine and lysine supplementation, either in a rumen-protected form or supplied by raw materials containing high concentrations of these amino acids, was effective at increasing milk protein concentration and milk protein yield. Utilising rumen protected amino acids however, resulted in better utilisation efficiency of dietary nitrogen, than when lysine was derived from raw materials, the latter increased the urea concentration of both plasma and milk, which predisposes to losses of N in urine. Iwanska et al. (1999) also reported that methionine was the limiting amino acid on grass silage-based diets, as supplementation with it improved all lactation parameters. Similarly, for diets based on whole-crop wheat, arginine (Ragland-Gray et al., 1997), methionine and lysine (Baker et al., 1996) have been found to be first limiting.

Mode of action of supplementary amino acids From the literature reviewed it appears that energy balance, protein supply, amino acid supplement, amino acid profile (both absorbed from the small intestine and supplied to the mammary gland), and stage of lactation can all affect NUE in response to supplementary amino acids.

18 Energy balance Increases in milk fat are more common in early lactation or cows in negative energy balance receiving supplementary rumen-protected methionine than those in positive energy balance or in mid to late lactation (Iwanska et al., 1999; Lindquist et al., 1983; Robinson et al., 1995; Rulquin and Delaby, 1997). Lysine can also have an effect on milk fat and protein concentrations (Guinard and Rulquin, 1994). When the cow is in negative energy balance, amino acids are important in two major ways. The first is as constituents of casein and other proteins, and the second is as precursors of other molecules involved in the energy metabolism of the animal. Methionine is involved in the synthesis of very low density lipoproteins (VLDL) in the liver from circulating non- esterified fatty acids (NEFA) and when the animal is in negative energy balance plasma NEFA concentrations increase together with the hepatic demand for methionine. Synthesised VLDL are taken up by the mammary gland and converted into milk fat. Increased plasma concentrations of NEFA also increases the demand for lysine, which is involved in hepatic metabolism of NEFA as a component of the transport molecule carnitine. Therefore, at times of negative energy balance, particularly in animals consuming diets based on maize, competition for lysine and methionine between milk proteins and other sinks for these amino acids can reduce the NUE of the animal.

Amino acid profile Over supply of protein and consequently improper profile of the amino acids supplied to the mammary gland are probably the main reason for a lack of response to supplementary rumen protected amino acids. This may be a result of competition for amino acid transport binding sites in the mammary gland epithelium (Piepenbrink et al., 1996); for example arginine competes for the same binding site as lysine, and this can result in loss of nitrogen due to catabolism of excess amino acids. In the majority of experiments supplementation has led to increased arterial concentrations of amino acids, although this does not necessarily lead to an increase in milk protein output because mammary blood flow can be decreased leading to the same rate of delivery of amino acids (Guinard and Rulquin, 1994). The importance of the biological value of a mixture of amino acids, compared to overall quantity, has been demonstrated a number of times. Nitrogen utilisation efficiency is increased by abomasal infusions of casein or a mixture of amino

19 acids similar to that of casein (Bach et al., 2000; Cant et al., 1991; Cant et al., 1989; Choung and Chamberlain, 1993a; Choung and Chamberlain, 1995; Choung and Chamberlain, 1993b) and although this is not a practical method of increasing milk protein output for a specific protein intake, it demonstrates that the biological value of the mixture amino acids leaving the rumen needs to be considered.

Effects of dairy cow management on nitrogen utilisation efficiency Milking frequency and bST Work done at the University of Maryland demonstrated the potential effect of using bovine somatotropin (bST), three times a day milking (3 milking) and long day photo period simulation (16 hours light and 8 hours dark; LDPP) on NUE (Dunlap et al., 2000). From N balance experiments conducted during the study it was predicted that the application of these technologies had the potential to reduce environmental N loading by up to 16%. The effects of the individual technologies on NUE efficiency are shown in Table 1.

Table 1. Mean effects (per cow) of adopting bST, 3 times a day milking (3), long day photoperiod (LDPP) alone, in combination or all three on nitrogen utilisation efficiency (from Dunlap et al., 2000). Treatment1 Control bST 3 LDPP BST BST 3 BST +3 +LDPP +LDPP +3 +LDPP Milk yield, kg/d 29.1 33.3 32.3 30.8 36.2 34.7 34.0 37.9 N intake, g/d 470 515 500 490 543 534 519 562 N excretion, g/d 313 337 329 324 352 347 339 362 N use efficiency2 33.4 34.6 34.2 33.9 35.3 35.0 34.7 35.7 1Treatments were no treatment (Control), administration of bovine somatotropin (bST), three times a day milking (3), or long daily photo period (LDPP). Treatment combinations are indicated by +. 2 % = ((N intake – N excretion) / N intake) x 100

Similar results were found in a recent survey of 454 American dairy farms (Jonker et al., 2002), in which average NUE for milk production was found to be 28.4%. Interestingly, farmers fed on average 6.6% more protein than recommended by published guidelines

20 (NRC, 1989), leading to a 16% increase in urinary N excretion (Jonker et al., 2002), and although increased frequency of ration formulation was associated with an increase in milk production it was not associated with increased NUE.

Although the use of bST is not relevant in Europe because it is not licensed for use, other management tools such as 3 milking and LDPP have potential benefits. Three times a day milking increases milk yield, and increases NUE through a dilution of maintenance requirements (Dunlap et al., 2000). Increasing milking frequency can also increase the efficiency of utilisation of amino acids for milk production by reducing the turnover of milk and constitutive proteins in the mammary gland (Bequette et al., 1998).

Milk urea nitrogen Milk urea concentration has been advocated as a useful management tool which can be used to monitor the efficiency of nitrogen utilisation in commercial herds (Godden et al., 2001; Jonker et al., 1998). Milk urea concentration is correlated with rumen ammonia concentrations and blood urea concentrations, which are in turn affected by dietary concentrations of CP and the diet protein-to-energy ratio (Blauwiekel and Kincaid, 1986; DePeters and Ferguson, 1992; Oltner et al., 1985; Oltner and Wiktorsson, 1983; Roseler et al., 1993). However, the interpretation of milk urea concentrations in relation to nutritional management is not easy, because some studies have shown a positive relationship between milk urea concentrations and milk production (Oltner et al., 1985) while others have demonstrated a negative relationship or no relationship (Baker et al., 1995; Carroll et al., 1988). Despite this, Frank and Swensson (2002) found that cows offered diets which resulted in the most efficient use of dietary N (up to about 43%) had the lowest concentrations of milk urea.

Despite the difficulties in interpreting milk urea N concentrations, Jonker et al. (1999) suggested producers should aim for a concentration somewhere between 10 to 16 mg/dl (100 to 160 mg/l). Bulk tank values above about 160 mg/l indicate that either dietary protein concentrations are too high, rumen fermentable energy supplies are too low, or a combination of the two. Bulk tank values below about 100 mg/l indicate that crude protein concentrations are too low, the balance of rumen degradable protein and rumen

21 undegradable protein is not correct, or rumen fermentable energy supplies are too high. High milk urea N concentrations (above about 160 mg urea/l milk) therefore indicate inefficient use of dietary protein, while low concentrations (below about 10 mg/l) can indicate restricted milk and milk protein yields.

Effects of forage conservation on nitrogen utilisation efficiency Grass silage The protein fraction in grass silage is very vulnerable to breakdown during ensilage. Extensive proteolysis and deamination by bacteria present in the ensiled forage can result in a large proportion of the N in grass silage being present as NPN (mainly free amino acids and ammonia). In poor quality silage the NPN component can constitute 80% of the N fraction, 25% of which may be present as ammonia. In contrast, in well made silage in which a pH of less than 4.0 has been rapidly established the NPN component may only account for 50% of the total N, with ammonia only accounting for 10% of that (Davies et al., 1998; Merry et al., 1995; Winters et al., 2000; Winters et al., 2001; Winters et al., 2002). Although there is a difference in the proportion of NPN in the total N betweeen differently quality silages, there tends to be an overall imbalance between the availability of N and energy required for microbial protein synthesis (quickly degradable N and slowly fermentable plant cell-walls). The deficient supply of energy from grass silage organic matter is further accentuated by the fact that the organic matter fraction can consist of up to 17% lactic acid and 26% of other fermentation acids (Harrison et al., 1994). Siddons et al. (1985) found that in sheep fed grass silage, the yield of ATP from the fermentation of organic acids and degradable nitrogenous compounds was insufficient to optimise microbial protein synthesis. Despite these limitations, NUE of ruminants offered grass silage only is not necessarily low. Nitrogen balance experiments at IGER using wether lambs have found NUE of up to 44% on a range of silages (Table 2). Management of the crop prior to ensiling, and methods used during ensiling, can affect the efficiency of utilisation of the nitrogenous components, as discussed below.

Table 2. N balance data (g N/d) from experiments investigating the effect of inoculation of high DM grass silages on NUE in wether lambs (MD Fraser, unpublished results). Control and inoculated silages 1 had a mean DM content of 370 g/kg, and control and inoculated silages 2 had a mean DM content of 514 g/kg.

22 Treatment N intake Faeces N Urine N N balance NUE %1 Control 1 23.5 9.2 7.0 7.3 30.4 Inoculated 1 25.9 9.0 5.5 11.5 44.4 Significance NS NS * *** *** Control 2 20.3 6.2 5.5 8.54 41.1 Inoculated 2 22.1 6.6 5.9 9.57 43.1 Significance NS NS NS NS NS 1 NUE = (N retained/N intake) × 100. *, P < 0.05; ***, P < 0.001.

Crop maturity McAllan et al. (1994) proposed that the efficiency of microbial protein synthesis is reduced as crop maturity increases because lignification of plant fibres reduces fermentability of plant tissues. This hypothesis was not confirmed by the study of Rinne et al. (1997) who found that the efficiency of microbial protein synthesis in cattle offered grass silages was unaffected by increasing grass maturity. However, higher rumen losses are associated young highly digestible herbage, due mainly to the increased solubility of the protein fractions of the grass caused by weaker associations with the fibre fraction of the plant. Comparisons of parent herbage and silage produced, showed that nitrogen present as the slowly degradable fractions were reduced as a result of herbage maturation, and that the nitrogen was converted into the highly soluble fraction (Rinne et al., 1997).

However, NUE of cows offered grass silage is not heavily dependent on the efficiency of microbial protein synthesis in the rumen expressed as a ratio of microbial protein produced to the amount of organic matter apparently digested in the rumen (OMADR). This is because it takes no account of the N losses that occur in the rumen, only the amount of N captured by microbes per unit of OMADR. Therefore the results of Rinne et al. (1997) do not indicate that grass should be harvested at a later stage of maturity, but emphasises the necessity to provide effective supplementation, or to manage the ensilage process to retain higher concentrations of FC, when feeding silage made from young forage.

Wilting It is generally accepted that wilting of fresh grass prior to ensilage restricts the ensiling fermentation, resulting in a higher pH value, lower total VFA production and higher

23 residual WSC concentrations. In terms of protein quality the speed and extent of wilting is very important, as it has been shown that proteolysis and deamination within the cut grass increases as the wilting period is extended, resulting in an increase in silage ammonia content (Gordon, 1981; Yan et al., 1996). A reduction in the wilting time taken to achieve the desired forage dry matter (DM) content reduces the extent of proteolysis and deamination (Yan et al., 1996). For this reason, spreading the cut crop during wilting, rather than leaving the crop in a swath can help control protein degradation (Wilkinson et al., 1999): compared with material left in the swath for 48 hours, material spread out for the same length of time had lower ammonia and higher amino N content. Despite the benefits of wilting in terms of increased intake (Dawson et al., 1999; Gordon and Peoples, 1986), there seems to be little benefit in terms of NUE in ruminants offered wilted rather than unwilted silage (Gordon, 1981; Kebreab et al., 2000; Kebreab et al., 2001). Indeed, Gordon and Peoples (1986) found cows ate significantly more wilted silage, but gave significantly less milk from it compared to unwilted silage, leading to a significant reduction in NUE of milk production (25.3% for unwilted and 22.5% for wilted silage). This may be due to losses of FCs during wilting, through the action of microbial organisms on the cut crop, or through an increase in the instantly available fraction of feed N (Gordon and Peoples, 1986). For this reason, it would appear that NUE was higher in cows offered more mature herbage, probably due to the presence of an improved balance between soluble N and FC, although this would not necessarily lead to best levels of milk production.

Silage additives Silage additives can be categorised into two main groups: those that either stimulate fermentation, such as bacterial inoculants, molasses and enzymes, or those that inhibit fermentation, such as formic or sulphuric acids. The effects of these two groups of additives on the performance of ruminants fed silage treated with them has been the focus of many experiments. In general terms the two types of additive produce silages with different properties. Compared with untreated silage, bacterial inoculants tend to increase lactic acid concentrations and tend to decrease water-soluble carbohydrates. Inoculants containing freshly cultured bacteria (e.g. Lactobacillus plantarum) tend to lead to a faster reduction in silage pH and therefore reduce N degradation, while freeze dried inoculants

24 tend to act more slowly and result insilages with a higher concentration of protein breakdown products (Merry et al., 1995). Silage additives which inhibit fermentation prevent or reduce the extent of lactic acid production, therefore increasing the amount of residual sugars and reducing the concentration of lactic acid and protein breakdown products.

In terms of animal production performance there is little difference between formic acid and freeze dried bacterial inoculants as silage additives. However, a number of experiments have shown that despite inferior NUE and DM intakes by animals consuming inoculated silage, their performance is equal to or superior to that of animals consuming silage treated with formic acid or no additive (Mayne, 1993). It has been proposed that the reason for this is metabolisable energy utilisation is improved with the use of a bacterial inoculant (Chamberlain et al., 1992). Similarly Mayne (1990) showed that despite the lower NUE of cows offered inoculated silage, milk production and milk energy output was significantly higher than from cows offered control and formic acid treated silage.

Silages made using freeze-dried inoculants tend to have lower concentrations of residual WSC and greater quantities of protein breakdown products than formic acid treated silages. This leads to the freeze-dried inoculant treated silages having lower NUE potential than formic acid treated silages, which have a better ratio of highly degradable N to WSC. In contrast to formic acid treated silage, untreated and inoculated silage contains high levels of lactic acid, which can be metabolised by rumen bacteria via the acrylate pathway to form propionate (Martin et al., 1990) which has the potential to improve glucose supply, and spares glucogenic amino acids from catabolism. The acrylate pathway in the rumen also consumes hydrogen, a by-product of acetate and butyrate production, which can help to reduce methane emissions and result in an improvement in energy utilisation.

Although acid treated silage may have the potential of leading to better NUE than fermented silage, many farmers and contractors are reluctant to use acid because of the health and safety issues, and increased wear on farm equipment. Recent experiments

25 using freshly cultured lactic acid bacteria have also shown that they are more effective than their freeze-dried counterparts and formic acid at reducing protein degradation and preserving WSC (Merry et al., 1995). Combined with the improved energy utilisation efficiencies associated with higher lactic acid content this may result in greater improvements in animal performance. Despite this, Winters et al. (2001) found no difference between the growth rates or feed conversion efficiency of steers offered grass silage treated with formic acid or a bacterial inoculant. In some situations, however, inoculation of grass silage has shown to be beneficial in terms of NUE (Table 2; MD Fraser unpublished data). A comparison of the effect of inoculation on N balance in wether lambs offered high DM grass silage showed that treatment improved N retention and NUE. Significant improvements in NUE were associated with significantly lower N excretion in urine, suggesting that the improvements in NUE were associated with improved rumen efficiency.

Legume silages Legumes are important livestock forages because of their ability to fix atmospheric nitrogen through their symbiotic relationship with rhizobacteria. In this manner, legumes offer a mechanism to immediately improve the overall farm nitrogen utilisation efficiency because of the reduced requirement for synthetic nitrogen fertilisers. Red clover (Trifolium pratense) and lucerne (Medicago sativa) are able to produce yields of up to 12 tonnes of forage DM annually (Fychan et al., 2002). A sward of hybrid ryegrass, which is comparable to the legumes in terms of longevity, can yield nearly 16 tonnes of DM per year, but this requires the application of 400 kg N/ha (NIAB, 2001). From 2004 the amount of nitrogen that farmers will be able to apply in areas designated as NVZ will be 170 kg/ha, which means that grass yields will be significantly reduced. In countries such as New Zealand where legumes constitute a significant proportion of the sown species, annual N fixation has been estimated to be in the region of one million tonnes and only 10k – 20k tonnes of fertiliser N are used. In comparison, the UK has a similar farmed area but uses 750k tonnes of fertiliser N, and as a result the estimated annual N fixation is only 80k tonnes (Frame et al., 1998). Legumes therefore have a tremendous potential to reduce the synthetic fertiliser N inputs and due to their high protein content, also have the ability to reduce supplementary feed N inputs, and as a result have cost saving benefits.

26 However, it must not be forgotten that N fixation is still a route for N input into the production system, and therefore the pollution potential still exists if utilisation efficiency is sub optimal.

Nitrogen utilisation efficiency of ruminants fed forage and grain legumes Comparative N balance data for a range of forage and grain legumes have been compiled at IGER (Table 3). This dataset is of particular relevance in our attempts to improve the NUE of dairy cows, as it provides us with data highlighting those forages that require energy supplementation. The partitioning of N outputs between urine and faeces are indicative of the nature of the losses. High urinary losses of N are associated with ruminal loss of ammonia, whereas faecal N loss is associated with the proportion of indigestible N in the forage or feed.

In the experiment of Fraser et al. (2000; Table 3, Experiment 1), for example, the data shows that the nitrogen fraction in sainfoin (Onobrychis viciifolia) silage was extremely indigestible, and a high proportion of N in the diet was excreted in the animals’ faeces. In contrast, the N fraction in lucerne silage was apparently extremely degradable in the rumen, leading to a high proportion of dietary N being excreted in urine as ammonia was absorbed from the rumen rather than being captured as microbial protein. Fraser et al. (2000) concluded that lotus (Lotus corniculatus) had the greatest potential for conservation as a high protein forage. However, recent lotus variety evaluation experiments at IGER (R. Fychan, personal communication) have demonstrated that establishment and persistency under conservation regimens are unreliable, and this will be a crucial factor in determining its adoption by livestock producers. This aside, tannin- containing legumes have the ability to reduce rumen N losses, and therefore have a potential contribution to make to improving the NUE of the dairy cow, and in this context are worth reviewing here.

Table 3. Nitrogen partitioning data (g N/d) compiled at IGER for a range of diets based on forage legumes and grain legumes fed with grass silage. All N figures rounded to the nearest whole number. Diet N intake Faeces N Urine N N balance NUE2

27 Experiment 1 Early cut red clover 51 16 25 10 19.6 Late cut red clover 51 17 24 11 21.6 Early cut lucerne 38 12 28 -2 -5.3 Late cut lucerne 39 12 24 3 7.9 Lotus 55 15 24 16 29.1 Sainfoin 24 20 7 -3 -12.5 Experiment 2 Lucerne, baled, control 41 13 16 11 26.8 Lucerne, baled, inoculated 41 12 19 10 24.4 Luc, clamped 45 13 17 15 33.3 Red clover, baled, control 44 19 13 11 25.0 Red clover, baled, inoculated 42 18 13 11 26.2 Red clover, clamped 40 19 13 8 20.0 Experiment 3 Barley/pea, control 15 6 6 2 13.3 Barley/pea, inoculated 14 6 6 3 21.4 Experiment 4 Field bean silage, control 30 9 16 5 16.7 Field bean silage, inoculated 26 10 15 1 3.9 Forage pea silage, control 28 8 13 7 25.0 Forage pea silage, inoculated 26 7 14 5 19.2 Lupin whole-crop silage1 24 6 11 6 25.0 Experiment 5 Zero-grazed grass 33 7 11 16 48.5 Zero-grazed lotus 46 13 17 16 34.8 Zero-grazed lucerne 50 10 21 20 40.0 Zero-grazed red clover 68 13 29 26 38.2 Zero-grazed white clover 69 12 29 27 39.1 1R Fychan, unpublished data. Experiment 1: Fraser et al., 2000; Experiment 2: R Fychan, unpublished data; Experiment 3: MD Fraser, unpublished data; Experiment 4: Fraser et al., 2001; Experiment 5: MD Fraser, unpublished data. †NUE = (N balance/N intake) × 100

Tannin-containing legumes The effects of condensed tannins (CT) on protein nutrition were reviewed by Waghorn et al. (1990). In vitro studies have shown that CT precipitate dietary protein in neutral to weakly acidic conditions similar to those found in the rumen. This precipitation protects protein from microbial degradation and increases the supply of intact protein to the small intestine. In the more acidic conditions found in the small intestine, the tannin-protein complexes dissociate and the protein is then available for digestion as normal. Using an

28 in vitro limited substrate inhibitor procedure, Broderick and Albrecht (1997) found that differences in the rate of protein degradation and rumen escape of protein of nine forage legume species were proportional to tannin concentration. In vivo effects of CT in lotus on the performance of ewes nursing twin lambs was investigated in two groups of ewes, one receiving lotus herbage (control) and the other receiving lotus herbage supplemented with polyethylene glycol, which inactivates CTs (Wang et al., 1996). They found that the control ewes had lower rumen ammonia concentrations and increased rates of milk yield and of lactose and protein secretion. It was concluded that CT improved the efficiency of milk production, as the improvement in performance was obtained from statistically identical nutrient intakes. Apart from reducing rumen ammonia and increasing the flow of NAN to the small intestine, CT have also been shown to have positive affects on the flow of essential amino acids (EAA) to the small intestine. Waghorn et al. (1990) reported experiments with abomasally cannulated sheep fed either active CT diets or inactivated CT diets, in which the flow of EAA was 50% higher in sheep offered the CT active herbage. However, high levels of CT intake can be deleterious to animal performance, as extensive protein precipitation in the rumen can result in reduced microbial growth and a reduction in plant fibre degradation. Waghorn et al. (1990) demonstrated this with sheep, whose performance was decreased when fed Lotus pendunculatus containing 5.5 % CT in the DM, although performance was not decreased in sheep for lotus containing 2.2 % CT in the DM. Barry and Manley (1984) proposed that CT concentration should not exceed 4 % of plant DM, and that 2-3 % is sufficient to reduce excess proteolysis in the rumen. The effect of high levels of CT may have been a reason for the poor NUE of sheep fed sainfoin in the experiment of Fraser et al. (2000); analysis of CT concentration in nine forage legume species (Broderick and Albrecht, 1997) showed that the average conentration of CT in sainfoin was more than four times that of lotus.

A tannin-containing forage would be especially valuable in a mixed forage diet if its protein protecting properties could be conveyed to forages such as grass, lucerne or white clover silage, which contain proteins that are highly degradable in the rumen. There is mixed evidence for this effect in the literature. Beever and Siddons (1986) reported that mixing sainfoin with red clover had no interactive effects on duodenal amino acid supply

29 compared to red clover alone. In contrast, the review by Waghorn et al. (1990) of 10 diets consisting of combinations of low and CT-free legumes with CT-containing legumes showed a large response in N-retention and EAA absorption from the small intestine.

Non-tannin containing legumes As already mentioned, agronomic factors such as low annual DM yield, slow establishment and inferior competitiveness with weeds, make lotus and sainfoin of limited value in the United Kingdom, although breeding programmes would help to address these problems. As a consequence, red clover seems to have the best potential as a high-protein legume crop for use by dairy cattle in the UK (Sheldrick et al., 1995; Wilkins and Jones, 2000). Results of the recent EU-funded LEGSIL project (Wilkins, 2001) showed that forage legumes can increase profitability in milk production systems, and that red clover and lucerne were the most suitable species for preserving as silage. White clover silage offers good potential for milk production, although the NUE for milk protein production of animals fed on this tends to be less than that of animals fed on other forages, including ryegrass silage (Bertilsson and Murphy, in press).

The performance of lactating dairy cows offered red clover silage (RCS), lucerne silage (LS) and white clover silage (WCS) has been the focus of numerous studies. Broderick (1995) reviewed the desirable characteristics of forage legumes, concentrating mainly on LS. He summarised experiments that demonstrated the poor efficiency of N utilisation associated with feeding LS supplemented with RUP, which although improved animal performance in terms of milk production, resulted in reductions in NUE. The addition of protein to high forage diets is often used to correct sub-optimal performance on such diets, and although it is often successful it tends to result in a reduction in NUE. Poor NUE in animals offered LS originates from the high levels of NPN present in the forage, resulting in high rumen ammonia concentrations and losses of N in urine (Broderick, 1995). In a complete lactation study, Dhiman and Satter (1994) showed that by increasing the supply of rumen FC, by replacing a third of the LS with maize silage, total lactation yields were increased by 577 kg per cow from a diet that was lower in CP concentration. Addition of FC reduced rumen ammonia concentrations, which combined with a lower CP concentration in the diet and improved performance (Dhiman and Satter, 1994)

30 suggests that this improved NUE. A similar response to the addition of FC on NUE and the performance of dairy cows offered legume silages was shown by Castle et al. (1984). They supplemented white clover silage with barley grain or soyabean meal, or a combination of both and found that barley, compared to no supplement, improved milk and milk protein production, as well as improving NUE from 18 % to 23 %. Castle et al. (1983) reported similar results from the addition of barley grain to a white clover silage diet. In contrast, the addition of soyabean meal alone to WCS led to a reduction in NUE, but an increase in animal performance, which is consistent with the effect of adding protein to legume based diets found by Broderick (1995). Supplementing WCS with both barley and soyabean meal led to an increase in both animal performance and NUE (Castle et al., 1984).

Unlike LS and WCS, RCS is recognised as being less susceptible to proteolysis both in the clamp (Winters et al., 1999) and in the rumen (Broderick and Albrecht, 1997). Broderick and Albrecht (1997) concluded that red clover displayed the characteristics of legumes containing low levels of tannins, but does not contain tannins. Hatfield and Muck (1999) proposed that this was due to the presence of polyphenol oxidases and polyphenols that have the ability to inactivate hydrolytic enzymes responsible for proteolysis and deamination during ensilage. This would account for the lower levels of NPN found in RCS compared to LS, and lower rumen ammonia and milk urea concentrations in animals offered RCS compared to LS (Broderick et al., 2001; Broderick et al., 2000). However, further analysis of data comparing animal performance on RCS and LS in the lactating dairy cow (Broderick et al., 2001; Broderick et al., 2000; Hoffman et al., 1997) shows no difference in the correlation between N intake and NUE (Figure 5). These data suggest that the lower levels of protein breakdown products in RCS have little effect on improving NUE.

31 35% Lucerne

y 33% c Red clover n

e 31% i c i f

f 29% e

n 27% o i t a

s 25% i l i t 23% u

n

e 21% g o r

t 19% i

N 17% 15% 400 450 500 550 600 650 700 750 Nitrogen intake, g/day

Figure 5. Relationship between nitrogen intake from silages made from red clover or lucerne and nitrogen utilisation efficiency ([milk N output/N intake] × 100) in dairy cows. There was no difference between forages. Data from Broderick et al. (2000), Broderick et al. (2001) and Hoffman et al. (1997).

The effect however, may have been masked by the concentrates fed with the forages, as data from N balance experiments conducted at IGER showed that wether lambs offered RCS as the sole component of the forage achieved a significantly higher level of NUE than those offered LS (means of 20.6% and 1.3% for RCS and LS respectively; Experiment 1, Table 3), although in a subsequent experiment no differences were observed (grand mean of 25.6 % NUE; Experiment 2, Table 3). The use of high levels of concentrates in the diets evaluated during the LEGSIL project may also have been responsible for the poor NUE obtained by dairy cows offered ensiled legumes (Bertilsson and Murphy, in press; Wilkins, 2001). Reis and Combs (2000) showed that by supplying a source of rumen FC to dairy cows grazing mixed red clover, lucerne and grass swards, milk yield (21.38 to 30.4 kg/d), milk protein production (620 to 930 g/d), NUE (22 to 30%) and DM intake were increased, despite a reduction in the CP concentration of the diet (195 to 155 g/kg). Reductions in rumen ammonia concentration in cows offered a FC supplement highlight the importance of providing the appropriate supplement to diets

32 based on forage legumes (Reis and Combs, 2000), and indicates that the potential exists to reduce dietary CP content without detrimental effects on performance.

Harvest management of legumes and nitrogen utilisation efficiency Drying legumes for preservation as hay can result in high field losses due to the fragility of the plants, which tend to shed leaves at higher DMs. For this reason legumes are best conserved as silage, although their chemical composition needs to be taken into account to enable effecting silage production. Low concentrations of WSC in the forage makes them difficult to ensile, as there is limited substrate available for bacterial fermentation and the production of lactic acid. When WSC availability is limited, the extent of protein breakdown is increased, producing ammonia that buffers the drop in pH. Extending the wilting period prior to ensiling RCS and LS can result in a decrease in silage ammonia-N concentration (Fychan et al., 2002). The decline in the ammonia-N content of RCS following wilting was not statistically significant, and is evidence for the role of polyphenol oxidases in the inhibition of protein breakdown in RC (Hatfield and Muck, 1999; Jones et al., 1995). Waldo (1984) recognised the importance of correct preservation technique to NUE. He showed that ensilage of low DM forage resulted in extensive protein breakdown, lower intakes, reduced animal performance and low or negative N retention.

The extent of protein degradation in legumes during conservation is of major concern due to its effect on NUE. Vagnoni et al. (1997) showed that compared to no additive, the application of formic acid or sulphuric acid increased the B protein fraction (ammonia and free AA) of LS by 60 %, whereas application of trichloroacetic acid increased the B fraction by 98%. This was thought to be due to the ability of acid additives to induce acidic conditions below the optimal for the activity of plant proteases. Nagel and Broderick (1992) also showed that compared to no additive, applying formic acid reduced both the ammonia-N and free amino acid-N content of LS. Reducing protein degradation in the silo resulted in an increase in milk yield, protein content, protein yield, milk urea and a reduction in plasma urea concentrations.

33 Table 4. Proportion of NPN in the total N content of a range of alternative forages, fresh and ensiled with (inoculated) or without (control) the use a bacterial inoculant. Figures in % NPN in total N content. Crop Fresh herbage Control silage Inoculated silage Pea/barley bi-crop 20 49 45 Red clover, early maturity 9 29 26 Red clover, late maturity 10 29 25 Kale silage 16 39 36 First cut lotus silage 12 33 31 Second cut lotus silage 6 30 29 Lucerne/red clover, early maturity 13 33 31 Lucerne silage, early maturity 17 38 42 Lucerne silage, late maturity 15 42 42 Pea silage 7 38 35 Mean NPN, % of total N 13 36 34

Analysis of alternative forage silage quality data (in the DEFRA-funded Alternative Forages programme, LS0803) showed that compared to control untreated silages, the amount of N present as NPN (free amino acids and ammonia) was not statistically different to those treated with a freshly cultured bacterial inoculant, although in most cases NPN values for inoculated silages were numerically lower (Table 4). Recommendations From the literature review it is possible to draw certain conclusions and produce recommendations which can be applied on farm to help improve NUE.

Forage Grazed forages In order to improve the NUE of the grazing dairy cow we must have a clear perspective of the factors predisposing to poor NUE. The review has already highlighted the high solubility of forage N, as a result of increased fertiliser N application and the imbalance that exists between forage N and FC as being the main causes. An obvious recommendation would be to reduce the level of N application. However, levels of fertiliser application are related to the output expected per hectare, and the stock that the land must carry. Therefore, although reducing N application rate may improve NUE, many farmers may be reluctant to do so because of the resultant loss in grass yield. However, some farmers may have to reduce fertiliser application rates if their land falls within an NVZ, which should therefore lead to improvements in the NUE of their dairy

34 cattle. An alternative view would be to recognise the increased requirement for FC associated with higher levels of N application, and for dairy cows grazing pastures with a high white clover content. This will allow the rumen microbes to better utilise the available N. Provision of FC may be achieved by providing supplements containing sugars, starches or digestible fibre. Alternative options include turning cows out to fresh grass in the afternoon when WSC is higher, and when renewing pastures, selecting grass varieties that have been bred for higher WSC concentrations. Sources of low-degradable starch can be a valuable supplement for grazing dairy cows, and supplementation of animals with sources of DUP, which may improve performance of grazing dairy cows, masks inefficiencies in rumen N utilisation.

Conserved forages The problem of a high soluble N content is further exaggerated when forages are ensiled, and therefore the recommendations of increasing the FC content of the diet also applies in the case of ensiled forages. Special attention should be paid to the soluble N content of legume forages such as white clover and lucerne, grass silage and poorly preserved forages that may contain high levels of soluble N. The objective is to minimise the extent of protein breakdown by promoting rapid acidification of the crop during ensilage. Main recommendations include:  Maximising the WSC concentration of the forage through rapid wilting or selection of forage species high in WSC, as these are essential for a homofermentative lactic acid fermentation.  Use of an effective bacterial inoculant that stimulates a rapid lactic acid fermentation, or a fermentation inhibitor such as formic acid that acidifies the forage thus preventing protein degradation by bacterial proteases.

At present no measure of the soluble N content of forage is widely available in the UK. As a guide, forage with a high soluble N content often arises from material that has been harvested too soon after fertiliser application, material harvested after long periods of wet weather, and legumes harvested below the optimal DM content (less than 25% DM).

35 Rumen environment The rumen is the main source of N utilisation inefficiency, and therefore the use of dietary components which are high in DUP tends to increase NUE. However, soluble protein and non-protein N are the most easily accessible source of N for the rumen microbes and a shortage can lead to a reduction in microbial activity, leading to a reduction in VFA and microbial protein production. Improving the capture of dietary N in the rumen therefore improves NUE, and improves the protein nutrition of the animal. This requires accurate balancing of N and FC supply to the rumen, although this is made difficult by current descriptions of the N and carbohydrate fractions in the diet.

Peripheral and mammary tissue metabolism Provision of sufficient energy to the peripheral and productive tissues of the animals is of the highest importance. Limited uptake of energy-yielding nutrients results in the inefficient utilisation of amino acids as sources of energy, which produces ammonia as a by-product. Reducing the length and severity of periods of negative energy balance by providing diets of optimal energy density may have positive effects on NUE. Feeding rumen-protected amino acids has the potential of reducing the CP content of the diet without having a detrimental effect on production, while improving NUE. Results obtained from use of such supplements, however, are variable due to the many interacting factors that effect amino acid metabolism discussed in the review. However, a number of conclusions can be drawn:  Improving the profile of absorbed amino acids with regards to the amino acid profile of milk protein would allow the overall diet CP concentration to be reduced.  As dietary CP level is reduced, protein quality in terms of amino acid profile becomes more important in order to maintain performance.  The demand for lysine and methionine is increased when animals are mobilising body fat depots.  Simply increasing plasma concentrations of amino acids is not effective at increasing milk protein yield and NUE as it results in reduced mammary uptake and greater catabolism of amino acids.  Lysine and methionine tend to be the first limiting amino acids in animals offered maize-based diets.

36  Responses to methionine are dependent on the availability of dietary lysine.

However, there are currently no methods available to accurately determine the whole animal amino acid requirements or the microbial amino acid supply to the small intestine in a farming situation. This means that diet formulation remains the only way to calculate and supply approximate requirements.

Dairy cow management Feeding and milking management can also affect NUE. Positive effects of artificially maintaining a constant photoperiod of 16 hours of light in every 24 hours, and the use of three times a day milking have been found. However, not all dairy farmers are able or willing to milk their cows more than twice a day, in which case milking at 12 hour intervals would probably lead to the most efficient compromise.

Feeding a total mixed ration (TMR) offers the opportunity of accurately balancing dietary N and energy sources, provided that the diet has been formulated with that aim, and that the components of the diet are present in the TMR in their correct proportions. Accurate feeding of individual dietary components is more difficult when animals are offered separate rations of forage and concentrate feeds, making it more difficult to balance protein and energy components of the total diet. Monitoring milk urea concentrations may help with this. Many of the limitations of the UK’s ME and MP systems used for diet formulation (AFRC, 1993) were addressed by the Feed into Milk project (Offer et al., 2002), the results of which are available to dairy farmers as a ration formulation system both as a computer program for use on farm and through the use of feed compounders who were part of the Sustainable Livestock Production LINK consortium which funded the work.

37 Appendix 1 This table summarises some of the data used in the review. Data were taken from measurements of nitrogen partitioning in dairy cows, and nitrogen utilisation efficiency (NUE) was calculated as the proportion of dietary N that was used for productive purposes (growth and milk production).

N N in N in N in N NUE§ Source† Diet* intake faeces urine milk retained 1 GS + high CP, low deg. concentrate 465 150 162 114 40 33 1 GS + low CP concentrate 422 134 151 110 26 32 1 GS + high CP, medium deg. concentrate 469 143 184 118 26 31 1 GS + high CP, high deg. concentrate 472 131 219 116 5.20 26 1 GS + high CP concentrate 516 148 225 122 21 28

2 GS + concentrate + NH3 infusion 523 178 260 106 -20 16 2 GS + concentrate + acetate infusion 470 174 220 107 -31 16 3 TMR + starch infusion 532 174 175 125 58 34 3 TMR + water infusion 531 159 226 127 19 27 4 GS + high NDF concentrate 354 112 96 98 48 41 4 GS + soluble sugar concentrate 365 140 97 95 34 35 4 GS + low degradability starch 341 137 87 102 15 34 4 GS + high degradability starch 369 125 134 99 10 30 5 GS 569 143 241 139 46 33 5 GS + barley straw + concentrate 548 150 232 137 29 30 5 GS + concentrate 485 123 217 118 27 30 5 GS + prairie meal + concentrate 553 147 245 143 19 29 5 GS + barley straw + concentrate 485 133 227 116 10 26 5 GS + prairie meal + concentrate 490 132 241 111 5 24 5 GS + prairie meal 314 86 178 50 16 5 GS 261 76 151 34 13 5 GS + barley straw 174 64 101 9 5 6 GS + low CP concentrate 391 112 180 88 11 25 6 GS + medium CP concentrate 492 133 245 106 3 22 6 GS + high CP concentrate 555 134 299 104 16 22 7 GS + 44% maize silage 417 173 110 134 32 7 GS + 24% maize silage 469 167 166 136 29 7 GS + 45% maize silage 534 174 216 144 27 7 GS + 43% maize silage 510 150 225 124 24 7 GS 726 157 437 132 18 8 GG + high fibre concentrate 386 162 112 112 29 8 GS + high starch concentrate 424 152 148 123 29 8 GS + fibre and starch concentrate 460 179 156 129 28 8 GS + high starch concentrate 455 159 173 123 27 8 GS + high fibre concentrate 485 194 160 131 27 8 GS + maize silage 467 140 205 121 26 8 GS + sugar beet pulp 476 138 233 107 22 8 GS + high moisture ear corn silage 468 140 229 98 21 8 GS + high moisture ear corn silage 477 138 239 100 21 8 GS + ensiled sugar beet pulp 468 126 248 94 20

38 N N in N in N in N NUE§ Source† Diet* intake faeces urine milk retained 9 Energy and nitrogen synchronous diets 491 114 149 121 25 9 Energy and nitrogen asynchronous diets 510 125 150 125 25 10 GG (high sugar PRG) + concentrate 358 133 64 107 30 10 GG (low sugar PRG) + concentrate 453 132 121 111 25 11 GG (high sugar PRG) + concentrate 280 110 71 83 30 11 GG (low sugar PRG) + concentrate 290 121 100 68 23 12 GG (high sugar PRG) + concentrate 362 137 75 136 38 12 GG (low sugar PRG) + concentrate 320 123 87 119 37 13 GS + maize grain 555 191 211 112 20 13 GS + sugar beet + fish meal 565 201 234 112 20 13 GS + barley + oat grain 543 179 216 107 20 13 GS + sugar beet + soya 559 190 187 109 19 13 GS 502 177 216 93 19 14 GS (organic acids) + supplement 482 167 145 146 30 14 GS + maize grain 462 164 140 137 30 14 GS + barley grain 476 161 154 138 29

14 GS (NH4 tetraformate) + supplement 467 157 153 135 29 14 GS (bacterial inoculant) + supplement 459 163 144 130 28 15 GS + starch 378 149 94 129 34 15 GS – low fertiliser + starch & fibre 345 133 87 115 33 15 GS – medium fertiliser + starch & fibre 409 157 105 131 32 15 GS – high fertiliser + starch & fibre 395 143 118 122 31 15 GS + fibre 387 140 113 116 30 16 ZG + high starch concentrate 318 165 38 122 38 16 ZG + Maize silage + concentrate 372 166 51 139 37 16 ZG + sugar beet concentrate 376 150 98 135 36 16 GG + maize silage + concentrate 418 177 106 139 33 16 ZG + maize silage + concentrate 540 161 238 141 26 16 ZG + high starch concentrate 481 111 330 123 26 16 ZG + sugar beet concentrate 491 87 285 124 25 16 GG + maize silage 501 151 223 124 25 16 GG + maize silage + concentrate 537 140 214 120 22 16 GG + concentrate 726 157 437 132 18 16 GG + concentrate 604 149 347 109 18 16 ZG + corn cob mix 535 162 261 93 17 16 ZG + corn cob mix 562 130 271 95 17 16 ZG + ground ear maize 562 130 271 95 17 16 ZG + wet beet pulp 562 139 281 95 17 16 ZG + wet beet pulp 554 149 294 93 17 16 GG + concentrate 629 164 363 102 16 16 ZG + ground ear maize 547 157 272 87 16 16 ZG + dry beet pulp 554 162 272 83 15 16 ZG + dry beet pulp 592 127 291 88 15 17 GG – 0 kg N/ha 315 102 115 98 31 17 GG – 0 kg N/ha + 2 kg soya 466 119 220 125 27 17 GG – 250 kg N/ha + concentrate 585 118 348 119 20 18 GG – 60 kg N 423 110 204 109 26 18 GG – 300 kg N/ha 503 113 281 109 22 18 GG – 60 kg N/ha + 2 kg soya 594 132 350 123 21

39 N N in N in N in N NUE§ Source† Diet* intake faeces urine milk retained 19 ZG - 0 kg N/ha 257 91 76 91 35 19 ZG - 250 kg N/ha 365 91 181 93 25 20 GS + 209g starch 521 157 243 112 21 20 GS + 384g starch 543 152 269 111 20 20 GS + 50g starch 528 159 242 106 20 21 GS + sucrose 203 65 108 66 33 21 ZG 266 93 104 78 29 21 GS + sucrose + fishmeal 284 83 148 79 28 21 GS 218 63 100 54 25 22 GS + sugar beet pulp 466 139 30 22 GS + starch concentrate 524 153.92 30 22 GS + sugar beet & starch 576 159.09 28 22 GS + sugar beet pulp 590 160.03 27 22 GS + starch 596 161 27 * GS, grass silage; GG, grazed grass; ZG, zero-grazed grass; NDF = neutral detergent fibre; deg., degradable; CP, crude protein.

† Source references: 1. Castillo et al., 2000b; 2. Moorby and Theobald, 1999; 3. Reynolds et al., 2001; 4. Castillo et al., 2000a; 5. Moorby et al., 2000; 6. Aston et al., 1998; 7. Valk, 1994; 8. Van Vuuren, 1993; 9. Kolver et al., 1998; 10. Miller et al., 2001a; 11. Miller et al., 2001b; 12. Moorby et al., 2001; 13. Petit and Tremblay, 1995; 14. Kebreab et al., 2000; 15. Kebreab et al., 2001; 16. Valk and Hobbelink, 1992; 17. Delagarde et al., 1997; 18. Astigarraga et al., 1993; 19. Peyraud et al., 1997; 20. Keady et al., 1998; 21. Keady and Murphy, 1998; 22. Valk et al., 1990.

§NUE = [(N retained + N in milk)/N intake]*100

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