Assessment of Amino Acid Needs of Growing Cattle

Several factors make straightforward approaches to the study of amino acid utilization by growing ruminants unsatisfactory. For responses to a single amino acid to be measured, that amino acid must be the sole limiting factor. Thus, the amino acid of interest must be deficient, but other nutrients, including other amino acids, must be supplied in adequate or excess amounts. Due to the synthesis of microbial protein in the rumen, it is difficult to supply adequate amounts of energy to the animal without nearly meeting or exceeding the animal's amino acid needs. Also, because the amino acid profile of the ruminal microbial protein is relatively well balanced, no one amino acid is usually much more limiting than the others. Thus, a careful experimental approach is necessary to create a model with a single limiting amino acid.

Furthermore, due to ruminal degradation of proteins and amino acids, the supply of amino acids to a ruminant cannot be changed in a controlled manner simply by adding the amino acid to the diet. Amino acid supplementation must occur through feeding of rumen-protected amino acids or through postruminal (abomasal or duodenal) infusion of the amino acid. Currently, rumen-protected forms of amino acids are only commercially available for methionine and lysine.

When a single amino acid is supplemented, it is possible to determine that it is first-limiting for growth if the animal responds with an increase in protein deposition. However, animal performance will respond only be to the point where another amino acid (or other nutrient, such as energy) limits performance. Thus, research where only a single amino acid is supplemented is usually less than fruitful in identifying the magnitude of limitation.

Richardson and Hatfield (1978) evaluated the limiting amino acids in ruminal microbial protein for growing Holstein steers. They fed a semipurified diet that was low in true protein and supplemented the cattle with single amino acids. Methionine was determined to be the most limiting amino acid, and, in the presence of supplemental methionine, lysine also was found to be limiting. Although further additions indicated that threonine was third limiting, this conclusion was less than convincing.

Similar approaches have been used by a number of researchers to identify limiting amino acids for growing cattle. Steinacker et al. (1970) identified methionine as the first limiting amino acid for steers fed a timothy hay-based diet. For growing steers fed maize-based diets, lysine has been identified as the first limiting amino acid (Hill et al., 1980; Burris et al, 1976). It is perhaps not surprising that maize-based diets were most limiting in lysine, because the maize protein should supply enough methionine to complement this deficiency in microbial protein, but the maize provides little lysine, which also is deficient in microbial protein. This is supported by the work of Hill et al. (1980) where steers fed the maize-based diet did not respond to methionine supplementation, even in the presence of adequate supplemental lysine.

In the preruminant calf, ruminal fermentation and the difficulties that it imparts do not exist, so amino acids can be supplied by the diet in studies to assess amino acid requirements, van Weerden and Huisman (1985) conducted the most thorough evaluation of amino acid needs of calves (55-70 kg body weight) fed liquid diets. They fed diets containing 230 g kg-1 fat with 160 g kg-1 protein from skim milk powder plus additional essential amino acids in amounts equivalent to what would be provided by 90 g kg-1 protein from skim milk powder. The amino acid supplements supported N retentions as great as skim milk powder that supplied the same amounts of the essential amino acids. Performance of calves was generally good with gains of 0.85-1.0 kg day-1. Each essential amino acid was singly removed and changes in N balance measured; decreases in N balance indicated that the 160 g kg-1 protein diet was deficient in that amino acid. The greatest deficiencies were observed for total sulphur amino acids (methionine + cysteine) followed by lysine, isoleucine, threonine and leucine. Subsequently, they quantified the requirement for sulphur amino acids and lysine by feeding graded levels of those amino acids. Gains were optimized when 9.2 g day-1 total sulphur amino acids (6.8 g day-1 methionine) and 23 g day-1 lysine were fed, which corresponded to 7.4 and 18.1 g kg-1 of the diet, respectively. These estimates probably exceed the true values slightly because they correspond to maximal possible N balance from a quadratic model; breakpoint analysis would yield estimates 5-10% lower. Rough estimates of requirements for the other essential amino acids were also provided by the authors, although the published values for leucine are lower than would be supported by their data. The efficiency of methionine utilization for gain between intakes of 6.9 and 8.0 g day-1 of total sulphur amino acids was near 30%, and that for lysine utilization between intakes of 16.4 and 19.0 g day-1 was near 35%.

In a series of studies, Abe et al. (1997, 1998, 1999) evaluated amino acid deficiencies in young Holstein calves that were consuming solid feed. The calves were trained to suckle such that amino acid treatments could be fed as liquids and pass, via the reticular groove, directly to the abomasum. These workers did not attempt to quantify the animals' requirements for amino acids, but rather determined which amino acids were limiting. For smaller calves (75 kg body weight) fed maize-soybean meal diets (156 g kg-1 crude protein), methionine, lysine and tryptophan were marginally deficient (Abe et al., 1998). However, for similar calves fed maize-maize gluten meal diets, lysine was observed to be deficient (Abe et al., 1997). In contrast, larger calves (>150 kg body weight) did not appear to be limited by essential amino acid supply when fed either maize-soybean meal (Abe et al., 1999) or maize-maize gluten meal diets (Abe et al., 1997).

Another approach for studying amino acid utilization by ruminants is the use of intragastric nutrition, where the ruminal microbial population is removed and animals are maintained through ruminal infusions of VFA and abomasal infusions of protein and, in some cases, carbohydrates (0rskov et al., 1979; MacLeod et al., 1982). Because the microbial population has been removed, the issues surrounding the microbial contribution to metabolizable protein are eliminated. Similarly, the linkage between energy supply and protein supply has been removed. Because the protein (amino acid) supply is directly regulated by the abomasal infusions, it is possible to supply the animal with essentially any profile of amino acids and create a model where any essential amino acid is limiting. Another advantage of this system is that animals adapt very rapidly to postab-sorptive changes in nutrient supply, so very short experimental periods can be used. Nitrogen balance typically is used as a measure of protein deposition and can provide valid estimates over the short periods. The disadvantages of this system are that animal performance is usually less than industry-standards due to difficulties in infusing large amounts of nutrients while maintaining the health of the animal and its gastrointestinal tract. Also, animals maintained by intragastric nutrition may be physiologically different from normally fed animals. In particular, the intestine of intragastrically maintained animals appears somewhat atrophied, and this could impact amino acid metabolism. The economic costs associated with this technique (labour and infusates) also limit its widespread application, particularly to cattle. However, the approach has been used quite successfully to assess amino acid use in growing sheep (see below) and lactating dairy cows (Fraser et al., 1991).

One huge advantage of using intragastrically maintained animals for amino acid research is that the deletion approach can be utilized. This approach is discussed in some detail by Storm and 0rskov (1984). In essence, all amino acids (as well as other nutrients) are supplied in adequate or excess amounts and then the supply of a single amino acid is reduced. This allows the researcher to completely characterize the response to the single amino acid under conditions where the animal's response is not dictated by the supply of other limiting nutrients.

The deletion approach to evaluating amino acid utilization also has been applied to growing ruminants fed normal diets. Herein, cattle are fed a limited amount of a diet such that ruminal fermentation results in a less than optimal amount of amino acids reaching the small intestine. The cattle are then supplemented with energy (VFA infused ruminally and glucose infused abomasally) to ensure that the animal is limited by protein. Mixtures of amino acids are then supplied postruminally to exceed the animals' requirements for all of the amino acids except the one of interest. Graded amounts of the test amino acid are supplied, and N balance is used as an estimator of protein deposition. With this model, stark methionine deficiencies have been generated, and increases in N balance are easily measured when methionine is supplemented (Titgemeyer and Merchen, 1990; Campbell et ai, 1996, 1997; Froidmont et ai, 2000). Data can be used to calculate the efficiency of methionine utilization or to calculate the requirement for methionine at the point where animal performance is maximized. Similar models have been developed for the study of lysine and histidine (Greenwood and Titgemeyer, 2000) and of leucine and valine (Loest et ai, 2001), but have not yet been used in the study of those amino acids.

Methionine has been the most studied of the amino acids because it is first-limiting in microbial protein for growing cattle (Richardson and Hatfield, 1978) and because total sulphur amino acids also tend to be low in a number of protein sources fed to cattle (Titgemeyer et ai, 1989). The efficiency of protein utilization by animals is limited by the supply of the first-limiting amino acid as well as the efficiency of use of amino acids as a whole.

Using the deletion technique, the efficiency of methionine utilization in growing cattle (incremental increases in methionine deposition, based on N retention, divided by the incremental increase in methionine supply) has ranged from 14% to 66%. Figure 18.3 shows N balance in unsupplemented and methionine-supplemented steers using the deletion approach. The left-most point of each line in Fig. 18.3 represents the unsupplemented steers and the right-most point a value for steers receiving an amount of methionine less than or equal to that capable of yielding maximal N retention. Thus, each line represents a portion of the linear response surface for cattle demonstrating a methionine-dependent phase of growth. The slopes of the lines can be used to calculate efficiency of methionine use for gain.

A number of points should be made regarding Fig. 18.3. In all cases where more than two methionine levels were tested, N balance responses to methionine were linear. Retained methionine was calculated from nitrogen retention as: Met retained = N retention x 6.25 x 0.02, which accounts for the conversion of N into protein (6.25) and the methionine content of whole body protein (0.02). The line for y = x would correspond to 100% efficiency of methionine use. Points to the left of this line would indicate that the animal deposited more methionine than was absorbed, clearly an impossibility for an essential amino acid. This is probably accounted for by slight overestimation of protein deposition by N retention. Also, if the efficiency of methionine use is constant across all levels of gain, then these lines could be extrapolated to a methionine retention of zero, and the methionine intake at the point of zero methionine retention would correspond to the maintenance requirement. For most of the studies presented in Fig. 18.3, this would yield a negative maintenance requirement, which again is impossible. This may be a result of either the overestimation of protein deposition by N balance and(or) a higher efficiency of methionine use at intakes of methionine below those evaluated in these experiments.

These studies on methionine utilization based on the deletion technique (Fig. 18.3) represent a set with relatively standardized methods. With the exception of two studies (Titgemeyer and Merchen, 1990; Froidmont et ai, 2000), these experiments were conducted with lightweight (132-205 kg) Holstein steers. Froidmont et ai (2000) used larger (315 kg) double-muscled Belgian Blue bulls, and this accounts for the higher overall level of methionine retention. However, it is clear

Fig. 18.3. Responses of growing cattle to incremental increases in methionine supply. Methionine retention was calculated as N retention X 6.25 X 0.02, which accounts for the conversion of N into protein (6.25) and the methionine content of whole body protein (0.02). The /= xline corresponds to 100% efficiency in the utilization of methionine for protein deposition. The slopes of each individual line are the efficiencies of use of the supplemental methionine. References for the studies and the observed effic lencies [email protected] ♦, Titgemeyer and Merchen (1990), 41%; •, Campbell et al. (1996), 23%; ■, Campbell et al. (1997), 27% for both lines; A, Loest (1999), 43% and 58%; O, Froidmont et ai. (2000), 15%; □, Lambert (2001), 14%, 27% and 66%.

Methionine supply (g day-1)

Fig. 18.3. Responses of growing cattle to incremental increases in methionine supply. Methionine retention was calculated as N retention X 6.25 X 0.02, which accounts for the conversion of N into protein (6.25) and the methionine content of whole body protein (0.02). The /= xline corresponds to 100% efficiency in the utilization of methionine for protein deposition. The slopes of each individual line are the efficiencies of use of the supplemental methionine. References for the studies and the observed effic lencies [email protected] ♦, Titgemeyer and Merchen (1990), 41%; •, Campbell et al. (1996), 23%; ■, Campbell et al. (1997), 27% for both lines; A, Loest (1999), 43% and 58%; O, Froidmont et ai. (2000), 15%; □, Lambert (2001), 14%, 27% and 66%.

from Fig. 18.4 that factors beyond body weight are influencing the efficiency of methionine use for gain. Although the largest cattle (Froidmont et ai, 2000) had relatively low efficiencies (Fig. 18.4), there was not a strong relationship between body weight and efficiency. Taken as a whole, these studies would suggest that methionine is used less efficiently for gain than predicted by the Cornell Model (68-47% for cattle weighing 132-315 kg; Ainslie et al., 1993) and that maintenance requirements for methionine are lower than predicted by the Cornell Model. Whether these relationships would be true for other amino acids is unclear; there simply are not enough empirical measures available to make that determination.

Another factor to consider regarding the utilization of methionine is the transsulphura-tion pathway and the use of methionine as a source of cysteine (see Chapter 8). In mono-gastric animals, cysteine can be used to meet about half of the total sulphur amino acid requirement, and cysteine supplied in amounts less than the cysteine requirement can spare the animal's need for methionine. Thus, the total sulphur amino acid requirement is often considered as a sum of the needs for methionine plus cysteine where the cysteine supply is less than half of this total. However, in cattle, supplemental cysteine has not been found to effectively spare methionine (Campbell et al., 1997). Initial suggestions were that methionine's use in metabolic pathways other than protein synthesis (e.g. methyl donation) may be important in explaining this discrepancy, although studies with a similar model (Loest, 1999) did not observe any sparing of methionine by betaine or choline, methyl group sources. Regardless of the reason, the lack of sparing of methionine by cysteine in cattle suggests that the methionine requirement should be considered independent of the cysteine supply.

Cornell model

Body weight (kg)

Fig. 18.4. Plot of observed efficiencies of utilization of supplemental methionine as related to body weight. Key to symbols is as for Fig. 18.3. The line for the Cornell model provides estimates from Ainslie et al. (1993).

Growth assays with cattle fed diets supplying various amounts of metabolizable protein also have been used to assess amino acid utilization. As a general rule, growth is reflective of protein deposition because lean represents a large portion of the total weight gain by the animal. Fat deposition accounts for a large portion of energy deposited, but because it is energy dense and has little water associated with it, it does not alter weight gain as dramatically as protein. Wilkerson et al. (1993) summarized a number of growth assays in which cattle responded to supplementation with metabolizable protein by increasing their growth rate. The individual studies used basal diets containing little true protein, and the microbial protein supply was insufficient to meet the animal's needs (i.e. cattle fed only the basal diets were deficient in metabolizable protein). Across studies, weight gain was regressed on metabolizable protein supply. Maintenance requirements of 253 kg steers for metabolizable protein were estimated as 242 g day-1, and for each kg of gain the cattle required 305 g of additional metabolizable protein. Assuming that gain for cattle in these trials contained 150 g kg-1 protein, the effi ciency of use of metabolizable protein for gain was 49%. These authors also provided estimates of requirements for Individual amino acids, which were calculated as the lowest supply of the amino acid, across diets containing different protein sources, that yielded maximum gains. However, these can be considered as only maximal estimates because the supplies of all amino acids were altered by the supplementation of intact protein sources and it is impossible to know which amino acid(s) were supplied in excess and which ones were actually limiting. Additionally, in these experiments there was a strong correlation between energy supply and metabolizable protein supply because protein supply was heavily dependent on ruminal microbial protein synthesis. Thus, the relationship between metabolizable protein supply and gain may have been influenced by energy intake.

Using growth assays similar to those described above, Klemesrud et al. (2000a) evaluated the methionine and lysine requirements of growing steers. To evaluate methionine requirements, all steers were supplemented with meat and bone meal, which contains low amounts of methionine, to ensure that only methionine limited performance. Graded levels of rumen-protected methionine were then supplemented. Growth of 251 kg steers was maximal (0.39 kg day-1) when 2.9 g day-1 methionine was supplemented (total supply = 11.6 g day-1). Similarly, to evaluate lysine requirements, all steers were supplemented with maize gluten meal, which contains low amounts of lysine, to ensure that only lysine limited performance. Graded levels of rumen-protected lysine were then supplemented. Growth of 210 kg steers was maximal (0.56 kg day-1) when 0.9 g day-1 lysine was supplemented (total supply = 22.5 g day-1). The impact of growth rate on amino acid needs can be demonstrated by comparing the study by Klemesrud et al. (2000a) to that by Klemesrud et al. (2000b). In the latter study, steers (237 kg) were fed maize-based diets which allowed for much greater gains. During the initial 56 days of the growth assay, daily gains increased in response to supplemental rumen-protected lysine, with the maximal gain of 2.10 kg day-1 occurring when supplemental lysine supply was 2.6 g day-1 and total lysine supply was 40.5 g day-1. The much greater requirement for the more rapidly growing steers (40.5 vs. 22.5 g day-1) demonstrates the energetic limitation on gain in the study by Klemesrud et al. (2000a) such that maximal protein deposition and, subsequently, amino acid requirements were lower.

Concentrations of plasma amino acids in response to supplementation of graded levels of a supplemental amino acid also have been used to determine animal requirements. The concentration of an amino acid in blood should remain low and relatively constant when its supply is less than its requirement and then increase in concentration when the supply is above the animal's need (Bergen, 1979). Thus, the breakpoint can be used as an estimate of the animal's requirement. There are several disadvantages to this technique. It provides an estimate of the animal's requirement, but there is no clear measure of the performance level that corresponds to that requirement. More importantly, the procedure does not always yield estimates that match those based on measures of lean deposition (N balance, Campbell et al., 1997, Fig. 18.5; growth rate, Klemesrud et al., 2000b, Fig. 18.6).

Several additional points should be made with reference to the use of plasma amino acids for assessing animal requirements. The use of plasma amino acid profiles in response to supplementation of mixtures of amino acids is unwise, because many factors are altered simultaneously and it is never clear if the plasma responses relate to the supply of the amino acid being investigated or to changes in the supply of other amino acids (either due to changes in protein deposition or amino acid s? co

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Nitrogen balance

Nitrogen balance

Supplemental methionine (g day-"1)

Fig. 18.5. Nitrogen retention and plasma methionine as functions of supplemental methionine in calves (Campbell et al., 1997). The requirement was estimated to be 5.8 g day 1 supplemental methionine based on nitrogen balance, but only 2.0 g day-1 based on plasma methionine responses.

Plasma methionine

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Fig. 18.5. Nitrogen retention and plasma methionine as functions of supplemental methionine in calves (Campbell et al., 1997). The requirement was estimated to be 5.8 g day 1 supplemental methionine based on nitrogen balance, but only 2.0 g day-1 based on plasma methionine responses.

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Supplemental lysine (g day-1)

  1. 18.6. Daily gain and plasma lysine as functions of supplemental lysine in calves (Klemesrud et al., 2000b). Growth data are from the first 56 days of the trial, and blood samples were collected at the midpoint of this period. The requirement was estimated to be 2.6 g day"1 supplemental lysine based on gain, but plasma lysine concentrations did not demonstrate a pattern that could be used for prediction of animal requirements.
  2. Also, the isomeric form of the supplemental amino acid is important. Although the unnatural D-isomer of methionine can be used efficiently by growing calves for protein deposition (Campbell et al., 1996), it leads to higher concentrations of plasma methionine than does the natural L-methionine (Titgemeyer and Merchen, 1990; Campbell et al., 1996). Presumably, similar relationships would exist for isomers of other amino acids.
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