Responses of the bovine mammary glands to absorptive supply of single amino acids

Responses of the bovine mammary glands to absorptive supply of single amino acids J. P. Cant 1, R. Berthiaume 2, H. Lapierre 2, P. H. Luimes 2,3, B. W. McBride 1, and D. Pacheco 2 1 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1; 2 Agriculture and Agri-Food Canada, Dairy & Swine Research and Development Centre, Lennoxville, Quebec, Canada J1M 1Z3; 3 Department of Animal Science, McGill University, Ste. Anne-de-Bellevue, Quebec, Canada H9X 3V9. Received 10 September 2002, accepted 24 March 2003. Cant, J. P., Berthiaume, R., Lapierre, H., Luimes, P. H., McBride, B. W. and Pacheco, D. 2003. Responses of the bovine mammary glands to absorptive supply of single amino acids. Can. J. Anim. Sci. 83: 341 355. In this review, we discuss the mechanisms of responses of various tissues of the lactating dairy cow, particularly the mammary glands, to perturbations in supply of single amino acids that result in observed milk protein yields. Additions of methionine, lysine, histidine or leucine to the absorptive supply cause arterial concentrations of these amino acids to increase, mammary extractions to drop and mammary blood flow to decrease. Single subtractions of essential amino acids have the opposite effect. Changes in mammary blood flow that have been recorded can be explained as attempts by the mammary glands to restore intracellular ATP balance in the face of altered concentrations of energy metabolites in the general circulation. In a quantitative sense, milk protein yield is relatively insensitive to fluctuations in arterial amino acid concentrations but can be stimulated by any one of a number of amino acids. In this context, it is suggested that the designation of a limiting amino acid is not appropriate to the purpose of predicting milk protein yield. Rather, milk protein synthesis appears to operate at a predetermined rate set by external communications of milk withdrawal rate, physiological state and overall nutritional status. Utilization of amino acids (AA) by splanchnic and peripheral tissues, in coordination with the mammary setpoint, offsets imperfections in the dietary AA supply. How strongly an individual AA influences the mammary setpoint, arterial concentrations of energy metabolites, and mammary AA transport capacity will determine the magnitude of the milk protein yield response when its absorptive supply is changed. Key words: Dairy cows, milk protein, amino acid Cant, J. P., Berthiaume, R., Lapierre, H., Luimes, P. H., McBride, B. W. et Pacheco, D. 2003. Réaction de la glande mammaire des bovins à l absorption des acides aminés. Can. J. Anim. Sci. 83: 341 355. Dans cet article, les auteurs examinent comment certains tissus de la vache laitière en lactation, principalement la glande mammaire, réagissent à une modification de l apport de certains acides aminés entraînant un changement du rendement du lait en protéines. L addition de méthionine, de lysine, d histidine ou de leucine aux acides aminés absorbés augmente leur concentration dans le sang artériel et la diminue dans les sécrétions et le sang de la glande mammaire. La soustraction d un de ces acides aminés a l effet contraire. On explique les variations observées au niveau de la circulation sanguine dans la glande mammaire par les efforts que cette dernière déploie pour rétablir l équilibre de l ATP après modification de la quantité de métabolites porteurs d énergie dans la circulation générale. Sur le plan quantitatif, le rendement du lait en protéines est relativement insensible aux fluctuations de la concentration d acides aminés dans le sang artériel, mais il est stimulé par plusieurs acides aminés. On estime qu il est impossible de prévoir le rendement du lait en protéines à partir d un acide aminé limitant. En effet, les protéines laitières semblent être synthétisées à un taux préétabli par les rapports externes entre la quantité de lait prélevée, la condition physique de l animal et son bilan nutritionnel. L usage des acides aminés par les viscères et les tissus périphériques compense les variations de l apport d acides aminés par les aliments, de concert avec le taux de synthèse prédéterminé des glandes mammaires. La mesure dans laquelle un acide aminé donné influe sur le taux de synthèse de la glande, la concentration des métabolites porteurs d énergie dans le sang artériel et la capacité de transport des acides aminés par la glande mammaire détermineront comment le rendement du lait en protéines réagira quand il y a modification de la quantité d acides aminés absorbée. The purpose of this review is to discuss features of AA metabolism in the lactating dairy cow with an eye to develop systems to predict milk protein yield. Specifically, we will address the question: for a given change in the absorptive supply of a single amino acid, how much will milk protein yield change? To build a predictive model from descriptions of underlying functions, the elements that need to be included must first be identified, keeping in mind the Presented at the session Les acides aminés: du lait de la viande et plus! Amino Acids: Meat, Milk, and More during the CSAS/ADSA/ASAS 2002 Joint Meeting, Québec City, QC, 20 25 July 2002. Mots clés: Vache laitière, lait, protéine, acides aminés 341 advice of Baldwin (1995) that preconceived notions regarding the importance of regulation are not introduced a priori and only when warranted by model failure. This review is an attempt, then, to identify the important underlying mechanisms of the response in milk protein yield to changes in supply of single amino acids. The reader is referred to the review of Hanigan et al. (2001) for a masterful exposition of the elements of mammary AA metabolism Abbreviations: AA, amino acid; EAA, essential amino acid; IGF-I, insulin-like growth factor-i; MBF, mammary blood flow; 2C, two-carbon

342 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 1. Leucine, Lys and Phe flux from small intestinal disappearance to milk protein, presented as a proportion of available flux (mesenteric appearance minus endogenous loss) and as a proportion (mol 100 mol 1 ) of the sum of essential AA fluxes [adapted from MacRae et al. (1997), Berthiaume et al. (2001) and unpublished data, Blouin et al. (2002), and Ouellet et al. (2002)]. that must be considered in a predictive model of milk protein yield, and options for their mathematical representation. Several of the points raised therein will be reiterated here. Balancing dietary AA supplies against requirements and supplementing diets with single AA have been used for years in monogastric species and only recently in the dairy industry (National Research Council 2001). The main theory for describing effects of AA nutrition on milk protein yield is that of the limiting AA in which the ratio of input of each AA to its obligatory utilization for milk protein output is considered. It is universally accepted that the activities of the rumen so transform the profile of AA consumed in feed that it is irrelevant to evaluate AA adequacy of a diet through diet AA analysis alone. Most current approaches base their evaluations on duodenal AA flows (Bateman et al. 2001). But the story does not end at the entrance to the small intestine. Tissues between the site of absorption of AA and the milk into which they are secreted as protein have a profound influence on the profile of AA (Fig. 1) and, therefore, on the declaration of which single AA apparently limits milk protein yield. The mesenteric-drained viscera remove essential AA for secretion into the GI tract and oxidation (Lobley 2002). The remainder of the portal-drained viscera takes a further amount (also for oxidation and non-reabsorbed endogenous secretions), while hepatic contributions may be positive or negative depending on the AA in question. In the experiments used to generate Fig. 1, post-hepatic Leu flux was increased over the net portal flux so that the proportion of Leu, which was lower in the absorbed profile than in milk protein, became higher than in milk protein. Based on small intestinal disappearance, one might classify Leu as deficient but posthepatic flows do not lead to the same conclusion. In contrast, Phe was absorbed at a greater proportion of essential AA than in milk protein but net uptake by the liver reduced that proportion to less than that in milk. Lysine flows and proportions were changed little by hepatic activities. The mammary glands themselves remove more Lys, Thr, Arg, Ile, Leu and Val than are secreted in milk protein, catabolizing the excess to produce non-essential AA and ATP. From which profile is it appropriate to designate essential AA as potentially limiting? In this review, we present the remedy that the designation of limiting is inappropriate for predicting effects of absorbed AA supply on milk protein yield. Part of the reasoning rests on the relationship between splanchnic and mammary utilization of individual AA. The splanchnic bed uses up to 50% of the absorbed

CANT ET AL. MAMMARY RESPONSES TO SINGLE AMINO ACIDS 343 essential AA and 50 to 100% of the remainder is secreted into milk (Berthiaume et al. 1999, 2002; Blouin et al. 2002). However, the proportions are not static (Lapierre et al. 2000). When essential AA are infused intravenously into lactating cows, milk protein yield increases (Metcalf et al. 1996; Cant et al. 2001; Berthiaume et al. 2002) but Berthiaume et al. (2002) found that the increase in hepatic utilization of essential AA was greater than the exogenous infusion rate. Similarly, when net portal flux of α-amino N was increased in steers by 38 mmol h 1 through abomasal casein infusion, hepatic uptake of AA increased by the same amount such that no additional AA passed into peripheral circulation, and yet AA concentrations in the peripheral circulation increased (Guerino et al. 1991). These observations suggest that the relationship between splanchnic and peripheral utilization of AA is not a simple matter of the splanchnic tissues taking a proportion of incoming AA with the remainder used for milk or muscle protein synthesis. Nor is it immediately apparent how multi-organ utilization of AA results in the emergent phenomenon of a limiting AA, in which intestinal supply of one AA can limit incorporation of all others into milk protein. DEVELOPMENT OF LIMITING AMINO ACID THEORY The concept of a limiting amino acid is an application of the Law of the Minimum proposed by the German chemist, von Liebig, to explain growth responses of field crops to mineral fertilizers: Where, in any given case, the crops of any plant are limited by a minimum of phosphoric acid in the field, these crops will increase by augmenting the quantity of phosphoric acid up to the point at which the additional phosphoric acid bears a proper proportion to the next minimum constituent in the field. (von Liebig 1863) It is a testament to the strength of von Liebig s contributions that the ideas he developed for one area of agricultural science would be universally applied 150 yr later in the separate discipline of protein nutrition that he was to pioneer through the study of products of protein degradation. On the discovery that some of these degradation products were essential in the diet for construction of tissue, and that tissue proteins had specific AA structures, Osborne and Mendel (1915) logically concluded that an essential amino acid (EAA) could be the first-limiting factor of the nutritive value of dietary protein. von Liebig s Law of the Minimum is often illustrated as a barrel constructed of staves of different lengths representing the available nutrient supplies. Even if several EAA are in deficient supply, as in the corn protein depicted in Fig. 2, only supplementation with the most deficient EAA will improve protein gain inside the barrel. This concept is the popular basis for interpretation of most AA and protein supplementation experiments, especially those that failed to elicit a response to the supplemental AA or protein. There is much evidence to support the concept of a single limiting AA such as supplementation of corn (Fig. 2) with Fig. 2. Barrel made of staves of different lengths to represent the essential amino acid content of corn protein relative to an ideal (from Mitchell 1964). Trp, which had no effect on rat growth until Lys was also supplemented (Mendel 1923). And it is obvious that the limit on the number of protein molecules that can be produced, on a net basis, is determined as in Fig. 2. But the mechanism behind having only one AA stimulate protein accretion is not simple to delineate. Polypeptide chain elongation can be considered a ping-pong reaction mechanism, where the ribosome complex binds one amino-acylated trna at a time and catalyzes formation of the peptide bond before moving on. The velocity equation for such a mechanism (Segel 1975) shows that elongation rate (v) should be affected by each of the AA i trna i complexes separately: V v = n K max AA AA trna n K AA 1 + 1 + 2 1 1 2 AA trna2 1 2 KAA + + n 20 20 AA trna20 20 where n i is the number of i AA residues coded for in the mrna template. The point is actually moot because it has long been recognized that an AA deficiency must be severe to have any effect on intracellular concentrations of aminoacylated trna for that particular AA (Rogers 1976; Flaim et al. 1982). It has now been established that AA stimulate protein synthesis, not through mass action as substrates, but through a signaling cascade that affects activity of translation initiation factors by phosphorylation (Davis et al. 2000; Shah et al. 2000; Anthony et al. 2002). Thus, despite illustrations that invoke coded AA sequences of proteins, it is important to clarify that a single AA limitation to growth is a phenomenon that emerges at the whole animal level as a consequence of regulation of protein synthesis, protein degradation and AA catabolism in multiple organs of the growing animal.

344 CANADIAN JOURNAL OF ANIMAL SCIENCE Not surprisingly, given that the AA limitation is an emergent phenomenon, there are instances throughout the history of AA nutrition research in which the Law of the Minimum has been violated. Valine and Lys each individually stimulated growth of weanling rats fed whole-egg protein at 9% of dry matter (DM) intake (Mitchell 1959). Kapoor and Gupta (1975) found that supplemental Met and Trp each individually improved the protein efficiency ratio on 25% soybean meal diets, but that these two AA together caused a further improvement. Rats force-fed diets entirely lacking in His were able to maintain a positive N balance for some days, which was attributed to a redistribution of His stores within the body (Forbes and Vaughan 1954). In other words, redistribution of AA between various body pools, although a simple mechanism to understand, can cause a violation of the Law of the Minimum. In the lactating animal, redistribution of body protein stores to milk is expected (Pine et al. 1994; Bell et al. 2000). Arguably, the more appropriate notion to arise out of attempts to correct AA deficiencies of dietary proteins was that of AA balance. Rosenberg (1959) pointed out that the first limiting amino acid was often supplemented to the level of its requirement without consideration for the second limiting amino acid, thereby creating an unintentional AA imbalance and reducing growth rate. From Rose s (1937) essential AA requirements and Mitchell and Block s (1946) chemical score to the ideal AA profiles of the Agricultural Research Council (1981) and the National Research Council (1998), there has been an awareness that absorption of too much or too little of an individual EAA relative to others necessitates a shift in metabolism that has an adverse impact on protein deposition rate. A depression in DMI is an important part of the response (Harper et al. 1970). The drop in intake is mediated by parts of the brain that are thought to respond to very low intracellular concentrations of the dietary deficient AA (Gietzen et al. 1998). Additionally, high circulating concentrations of deficient AA stimulate hepatic catabolism of all AA, including those in deficiency, further exacerbating the circulating deficiency (Keene and Austic 2001). SINGLE AMINO ACID RESPONSES IN LACTAT- ING COWS Having established that the limiting AA phenomenon is not universal and arises from the interplay between anabolic and catabolic processes in multiple tissues, we now turn our attention to the dairy cow. In one of the seminal experiments to identify methionine and lysine as colimiting AA for milk protein synthesis, Schwab et al. (1976) showed that abomasal infusion of Lys could account for 8% of the increase in milk protein yield obtained with abomasal casein infusion and Met plus Lys accounted for 32%. However, Arg plus His in the same experiment caused 19% of the abomasal casein effect. If Met and Lys limit milk protein production according to a barrel stave analogy, how can Arg plus His increase it? Even at the mammary secretory cell level, Clark et al. (1978) found that Trp, Met, Cys and Thr each individually stimulated milk protein synthesis in vitro. An interesting paradox from the limiting AA view is provided by Kim et al. (2000) who found that i.v. infusion of His alone into cows accounted for 56% of the response in milk protein yield to infusion of all EAA simultaneously and His was therefore first-limiting. In a follow-up experiment, omission of Met from an infusate of four EAA, including His, completely abolished the stimulatory effect of the infusate on milk protein yield, so Met was, therefore, first-limiting. The diets fed in the two experiments were based on two different batches of grass silage, so the apparent paradox might be explained as due to differences in absorptive AA supply. Basal plasma His was at the very low concentration of 7 µm in the first experiment when His had an effect, but was at 22 µm in the second when Met had an effect, effectively suggesting a lower supply of His in the first study. Basal Met was 14 µm in both experiments. There are very few other experiments in which different AA mixtures have been infused postruminally to confirm or refute the possibility that no one single AA limits milk protein production. Responses to infusions of mixtures of AA have never been attributable to just one AA within the mix (Schwab et al. 1976; Kim et al. 2000). From a review of postruminal infusion results in young ruminants, Merchen and Titgemeyer (1992) concluded that Met, Lys, His, Thr, Val, Ile and Leu could all be colimiting in rumen microbial protein for tissue protein accretion in growth. The nonessential AA, either singly or as a group, have not been shown to stimulate milk protein production (Mepham and Linzell 1974; Alumot et al. 1983; Teller et al. 1988; Fraser et al. 1991; Plaizier et al. 2001). Fraser et al. (1991) found that simultaneous removal of approximately 45% of the Arg, Ile, Leu, Val and Trp from an intragastric infusate had no effect on milk protein output or N retention in the body of cows; only Lys and His removal affected milk synthesis. On the other hand, Hanigan et al. (2000) were able to explain a greater proportion of the variance in milk protein yield from 21 published experiments with a mathematical model of mammary metabolism that considered each essential AA to have a unique mass action effect on protein secretion than with protein secretion rate determined by the one AA in lowest relative supply. Before considering mechanisms of how different essential AA might uniquely stimulate milk synthesis, we will briefly summarize the milk composition responses to single AA that have been documented. Addition Experiments Since Schwab et al. (1976), there have been at least 180 publications describing milk production responses to postruminal Met and Lys supplementation (CABDirect 2002). However, these experiments have not, in general, had as their objective the testing of the hypothesis that Met and Lys limit milk protein production but, rather, as Robinson et al. (2000) pointed out,... of documenting a positive production response of dairy cows to rumen-protected amino acids in situations where those amino acids would be expected to limit productive performance. Milk composition responses to Met and Lys have been extensively reviewed (Rulquin et al. 1993; Robinson 1996; Schwab 1995). Feeding approximately 12 g d 1 of rumen-protected methionine and 23 g d 1 of protected lysine in seven dif-

CANT ET AL. MAMMARY RESPONSES TO SINGLE AMINO ACIDS 345 Table 1. Dose responses of bovine mammary glands to postruminal infusion of single amino acids Dose Contrast 1 2 3 4 5 Linear Quadratic Guinard and Rulquin (1995) DL-Met infusion rate (g d 1 ) 0 8 16 32 plasma [Met] (µm) 22 41 48 85 0.003 0.776 mammary Met extraction, (%) z 39.4 24.6 15.3 11.1 0.002 0.038 mammary blood flow (L h 1 ) 634 586 546 582 0.122 0.039 mammary Met uptake (g d 1 ) 13.9 14.4 10.1 14.4 0.926 0.429 milk protein yield (g d 1 ) 674 660 707 689 0.433 0.618 milk fat yield (g d 1 ) 984 969 1003 1021 0.410 0.854 Varvikko et al. (1999) DL-Met infusion rate (g d 1 ) 0 10 20 30 40 plasma [Met] (µm) 17 30 49 72 155 <0.001 NS mammary Met extraction (%) 54.8 38.3 26.5 18.2 10.5 <0.001 NS mammary plasma flow (L h 1 ) y 654 548 635 679 651 n/d n/d mammary Met uptake (g d 1 ) 21.9 22.6 30.0 31.9 38.0 n/d n/d milk protein yield (g d 1 ) 788 777 810 815 804 NS NS milk fat yield (g d 1 ) 992 999 1037 1038 1064 <0.001 NS Guinard and Rulquin (1994) Lys-HCl infusion rate (g d 1 ) 0 9 27 63 plasma {Lys] (µm) 77 88 124 131 0.003 0.025 mammary Lys extraction, (%) 61.1 62.9 56.7 51.2 0.013 0.887 mammary plasma flow (L h 1 ) 411 365 375 438 NS NS mammary Lys uptake (g d 1 ) 63 71 92 100 0.024 0.195 milk protein yield (g d 1 ) 670 702 698 683 0.966 0.369 milk fat yield (g d 1 ) 949 972 908 993 0.371 0.169 Varvikko et al. (1999) L-Lys infusion rate (g d 1 ) 0 15 30 45 60 plasma [Lys] (µm) 90 97 119 138 185 <0.001 <0.010 mammary Lys extraction (%) 52.2 50.4 48.4 39.1 30.1 <0.001 <0.100 mammary plasma flow (L h 1y ) 469 465 466 508 619 n/d n/d mammary Lys uptake (g d 1 ) 79 82 96 98 124 n/d n/d milk protein yield (g d 1 ) 702 710 709 704 694 NS NS milk fat yield (g d 1 ) 919 951 954 912 918 NS <0.100 Korhonen et al. (2000) His infusion rate (g d 1 ) 0 2 4 6 plasma [His] (µm) 23 28 51 64 <0.001 NS mammary His extraction (%) 55.7 52.4 22.6 27.6 <0.001 NS mammary plasma flow (L h 1 ) 622 574 630 493 NS NS mammary His uptake (g d 1 ) 26 28 28 28 NS NS milk protein yield (g d 1 ) 861 877 907 919 <0.010 NS milk fat yield (g d 1 ) 1240 1167 1296 1177 NS NS Rulquin and Pisulewski (2000b) L-His infusion rate (g d 1 ) 0 27 41 55 plasma [His] (µm) 11 82 81 97 <0.050 mammary His extraction (%) 55 16 15 15 <0.050 mammary plasma flow (L h 1 ) 862 792 562 572 <0.050 mammary His uptake (g d 1 ) 16 34 34 40 <0.050 milk protein yield 610 675 694 720 <0.050 Rulquin and Pisulewski (2000a) Leu infusion rate (g d 1 ) 0 40 80 120 plasma [Leu] (µm) 42 107 143 162 <0.050 mammary Leu extraction (%) 72 47 44 36 <0.050 mammary plasma flow (L h 1 ) 415 354 389 368 NS mammary Leu uptake (g d 1 ) 39 56 77 67 <0.050 milk protein yield (g d 1 ) 570 652 648 624 <0.050 z Recalculated from mean arterial concentrations and arteriovenous differences published. ferent experiments increased milk protein yields by 50 g d 1, on average, and fat yields were often elevated, up to 100 g d 1 (Robinson 1996). Varvikko et al. (1999) found that Met infused up to 40 g d 1 into the abomasum of cows had no effect on either milk protein yield or percentage, but fat percentage increased linearly with Met dose (Table 1). These results indicate the mechanism of the increase in milk fat is distinct from the mechanism of the increase in milk protein.

346 CANADIAN JOURNAL OF ANIMAL SCIENCE Histidine supplementation has received renewed interest in recent years as a result of the demonstration by Vanhatalo et al. (1999) that His infused into the abomasum at 6.5 g d 1 increased outputs of His and protein in milk by 0.7 and 26 g d 1, respectively. Plasma His concentrations increased from 18 to 53 µm. On the same grass silage-based diet, milk protein was not affected by abomasal infusions of Met that increased plasma Met from 17 to 72 µm, nor by Lys infusions that increased plasma Lys from 90 to 138 µm (Varvikko et al. 1999). Instead, milk fat yield and percentage increased in a linear relationship to Met dose. In contrast, graded His infusions up to 6 g d 1 resulted in a dose-dependent decline in milk fat yield and percentage (Korhonen et al. 2000). Infusing Met, Lys, Met plus Lys, or Leu into cows receiving His infusions had no further effect on milk composition besides the typical increase in fat content with Met (Vanhatalo et al. 1999; Huhtanen et al. 2002). Rulquin and Pisulewski (2000b) infused His at higher doses, and to higher concentrations in plasma (up to 97 µm), and observed continued increases in milk protein yields. Infusing glucose at 250 g d 1 into His-supplemented cows resulted in an additional 33 g d 1 milk protein production (Huhtanen et al. 2002). None of the plasma AA increased in concentration and branched-chain AA concentrations actually declined with glucose infusion. Duodenal flows of branched-chain AA are often calculated to be in lowest supply relative to a desired output of milk protein (Fig. 1b; Robinson et al. 1999). Isoleucine infusion at 30 g d 1 into the abomasum of cows did not affect yields or contents of milk protein or fat, but lactose secretion, lactose percentage and overall milk yield were increased (Robinson et al. 1999). In these same cows, feeding of rumen-protected Met plus Lys increased milk protein percentage. Paradoxically, the combination of Ile, Met and Lys did not affect any milk components compared to the control. Rulquin and Pisulewski (2000a) observed an 82 g d 1 increase in milk protein yield when plasma Leu concentrations rose from 42 to 107 µm as a result of duodenal leucine infusion at 40 g d 1. At 80 and 120 g d 1 Leu, plasma Leu concentrations continued to rise, but protein yields were not affected (Table 1). Others have found no effect on milk composition of Leu by itself at 12 g d 1 (Huhtanen et al. 2002) or all three branched-chain AA at 150 g d 1 (Mackle et al. 1999a). However, plasma Leu in control cows were higher than 42 µm, being 77 and 164 µm in the two experiments, respectively. A brief list of results obtained on infusing graded doses of Met, Lys, His or Leu into the abomasum or duodenum of lactating cows is presented in Table 1. Keeping in mind that the basal diets were different for each experiment, there are a few common features to the dose responses. In all cases, whether milk protein yield was increased statistically significantly or not, plasma concentrations of the infused AA increased and mammary extractions of the infused AA out of plasma decreased. In the Met experiment of Guinard and Rulquin (1995) and the His experiment of Rulquin and Pisulewski (2000b), rates of mammary blood flow (MBF) also decreased with increasing doses of single AA. Despite two- to fourfold increases in plasma concentrations of Met, Lys and His, uptakes of these AA by the mammary glands did not increase. In the experiments of Rulquin and Pisulewski (2000a,b) where plasma His and Leu concentrations increased eight- and fourfold, respectively, mammary uptakes of these AA approximately doubled. Subtraction Experiments With an addition experiment, it is difficult to know whether the milk composition response is due to an improvement of the absorbed AA balance or its deterioration. Often, researchers have attempted to induce the former result by formulating a basal diet that is predicted to undersupply the AA in question. However, depending on parameters and assumptions of the mathematical model used to make the predictions of AA balance, different AA may appear to be undersupplied. For example, in an experiment in which Lys, Met and Ile were supplemented postruminally, Robinson et al. (1999) found that CNCPS version 2.13A (O Connor et al. 1993) predicted those three AA were in deficient supply in the basal diet but Shield (Robinson 1998) predicted deficiencies of Arg, Ile and Lys instead. An experimental approach, which guarantees that milk composition responses will be due to improvement of AA balance, is to administer all AA postruminally as a positive control and then subtract from the mix considerable quantities of the single AA in question. The first subtraction experiment on lactating cows was carried out by Fraser et al. (1991) using three cows provided with all nutrients by intragastric infusion. Casein was infused at a basal rate of approximately 1 kg d 1 and, from a supplement of the EAA plus Tyr and Cys, AA were subtracted in various pairs and groupings over 15 consecutive periods. The subtractions removed approximately 45% of the gastric supply of each AA. From multiple regression analysis of the results, it was determined that Lys removal caused the greatest depression in milk protein yield followed by His and Met. Nitrogen retention in the body was also decreased by these removals and by Phe subtraction. The results can also be interpreted to suggest that correction of a deficiency in any one of Lys, Met, His or Phe improved N balance and milk protein yield. Correction of a His deficiency has repeatedly been shown in subtraction experiments to increase milk and milk protein yields and decrease milk fat yield (Fraser et al. 1991; Kim et al. 1999; Bequette et al. 2000; Weekes and Cant 2000; Cant et al. 2001). In early-lactation cows fed a 9%-protein diet plus 1.2 kg d 1 abomasal AA, Weekes and Cant (2000) found that improvements in each of Met, Lys and His supplies caused protein yields to increase, while fat yields dropped, but the effect was most pronounced for His. Subtraction of 30 g d 1 His caused a decrease of 186 g d 1 in protein yield and an increase of 181 g d 1 in fat yield. Bequette et al. (2000) induced very low circulating concentrations of His in lactating goats by subtracting approximately 40% of the His from the total duodenal supply. Mammary blood flow rate was 36% higher at 8 µm plasma His compared to 73 µm. Rulquin and Pisulewski (2000b) observed a 51% increase in MBF between 97 and 11 µm plasma His (Table 1). In short-term, 10-h intraarterial infu-

CANT ET AL. MAMMARY RESPONSES TO SINGLE AMINO ACIDS 347 sions of AA mixtures at 30 g h 1 into cows, subtraction of His had no effect on iliac arterial blood flow (Cant et al. 2001), but this artery supplies two mammary glands and a hind limb, tissues which may respond differently to AA perturbations (Bequette et al. 2001). Bequette et al. (2000) observed a 40-fold increase in unidirectional His clearance by the mammary glands so that, in conjunction with the hyperemia, net uptake of His and milk protein yields were only reduced by 15% at 8 µm His. A Leu imbalance, created by infusion of a mix of 18 AA lacking Leu, had no effect on milk protein yield in goats but caused MBF to increase by 17% (Bequette et al. 1996). Subtraction of all three branched-chain AA from an abomasal infusate of 1.2 kg d 1 into cows fed a low-protein diet had no effect on milk or milk component yields after 5 d of infusion (Weekes and Cant 2000). Likewise, subtraction of 45% of the gastric Arg, Thr and branched-chain AA supply had no effect on milk production variables or N retention in the experiment of Fraser et al. (1991). Instead, N excretion in urine was increased when this group of AA was infused. The common features of essential AA imbalance are a low protein and high fat output in milk. Responses to added Met and Lys, which may include an increase in milk fat yield (Robinson 1996; Varvikko et al. 1999), are not always consistent with the correction of an AA deficiency. Mammary Blood Flow The single AA subtraction experiments conducted to date support the following conclusions obtained from the addition experiments: MBF and net extraction of deficient AA are elevated during a single AA deficiency. Mammary blood flow is presumed to be under some degree of control by vasodilators released from epithelial and endothelial cells in the gland proper (reviewed by Prosser et al. 1996). Most of what is known about local vasodilatory mechanisms has been worked out in exercising muscle tissue, although the picture is still far from complete because of the number of vasodilatory compounds that interact and overlap in function. One working hypothesis is that as O 2 consumption in muscle tissue increases, the muscle fibers themselves release adenosine into the extracellular fluid and endothelial cells of the arteriole release NO (Laughlin and Korzick 2001). Adenosine stimulates relaxation of smooth muscle cells surrounding arterioles through activation of a Ca 2+ pump on the sarcoplasmic reticulum and an ATP-sensitive K + channel on the plasma membrane. Nitric oxide stimulates relaxation through a cgmp cascade. In this scenario, blockage of any one vasodilatory pathway can be compensated by the others until O 2 supply matches consumption (Ishibashi et al. 1998). A variation on this theme is that adenosine acts to relax the smooth muscle cell exclusively through stimulation of the ATP-sensitive K + channel and, furthermore, is responsible for NO release from the endothelium (Hein and Kuo 1999; Murrant and Sarelius 2000). A third suggestion is that adenosine, K + ATP channels and NO have no role in exercise hyperemia, although there remains a linear relationship between O 2 consumption and blood flow rate (Tune et al. 2001). Adenosine and NO donors infused into the arterial supply of the bovine mammary glands have elicited hyperemia (Oguro et al. 1982; Nielsen et al. 1995; Lacasse et al. 1996) and blockers of NO synthase and K + ATP channels have reduced MBF (Lacasse et al. 1996; Madsen et al. 2001; Madsen, T.G., S. Cieslar, M.O. Nielsen, and J.P. Cant, unpublished data). It is tempting to conclude from the AA addition and subtraction experiments that AA participate in local vasodilatory control mechanisms in mammary tissue. In fact, Arg is the substrate for NO synthesis and i.v. Arg infusion increases blood flow to skeletal muscle (Schellong et al. 1997; Meneilly et al. 2001). However, Arg infused into the mammary arterial supply of cows did not affect MBF (Luimes, P.H. and D. Petitclerc, unpublished data) and plasma Arg was elevated more by complete AA infusion where MBF did not change (Bequette et al. 2001) than by His-deficient AA infusion where MBF increased 36% (Bequette et al. 2000). Histamine, a metabolite of His, also does not appear to be vasoactive in mammary circulation (Madsen et al. 2002). To the authors knowledge, there are no reports of circulating AA profile influencing blood flow to other organs, so before proposing a novel element of control in the mammary glands, the established role of metabolic rate should be considered. Cant and McBride (1995) adapted the blood flow prediction equations of Granger and Shepherd (1973) for a mathematical model of bovine mammary metabolism. The central hypothesis of the model, without identifying any particular vasodilators, was that mammary blood flow is locally regulated to match intracellular production of ATP with utilization. The other hypotheses were that net uptake of milk precursors from blood followed distributed-in-capillary extraction kinetics and milk component synthesis was a function of precursor uptake. In a test of the model s ability to predict effects of an infusion of glucose into the arterial supply of the bovine mammary glands (Cant et al. 2002), the first two hypotheses were upheld: MBF decreased and glucose, acetate, β-hydroxybutyrate and long-chain fatty acid uptakes were affected accordingly. However, lactose, fat and protein secretion into milk were insensitive to changes in precursor uptakes, so the third hypothesis was rejected. There is considerable evidence corroborating the insensitivity of milk component secretion rate to precursor concentration. Table 1 shows that two- to ninefold increases in arterial concentrations of single AA caused, at most, an 18% increase in milk protein yield. Tripling essential AA concentrations in the arterial supply of the mammary glands only increased milk protein yields by 13% (Cant et al. 2001). Infusing insulin i.v., with glucose to maintain glycemia, increased milk protein yields despite a drop in circulating AA concentrations (McGuire et al. 1995; Mackle et al. 1999b; Bequette et al. 2001). Milk lactose yield was unchanged with a 1.8-fold increase in arterial glucose concentration (Cant et al. 2002). Thus, milk component yields exhibit a low sensitivity to precursor concentrations. Presumably, MBF is changing when the glands are faced with an excessive or deficient supply of milk precursors such as His or glucose in order to alter extracellular and intracellular concentrations of those precursors. If the con-

348 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 3. Flow of nutrients in the mathematical model of mammary metabolism [revised from Cant and McBride (1995)]. Boxes represent state variables described by differential equations, arrows represent flux rates. Intracellular catabolic and anabolic fluxes were simulated with Michaelis-Menten equations. All simulations used Michaelis constants of K Gl, Gl Lc = 0.172 mm, K Gl, Gl Cd = 3.0 mm, K Ac, Ac Cd = 1.0 mm, K Ac, Ac Fa = 0.2 mm, J Fa, Ac Fa = 0.02 mm, K Fa, Fa Tg = 1.0 mm, K Aa, Aa Cd = 1.0 mm, and K Aa, Aa Pt = 0.05 mm. centrations do not greatly influence milk component synthesis, the alternative is that the precursor concentrations influence rates of their catabolism. Thus, mammary blood flow is regulated to provide substrates for ATP production (catabolism) while ATP utilization (biosynthesis) remains inelastic. The model of Cant and McBride (1995) has been adjusted to reflect this last proposition (Fig. 3). Hemodynamic and uptake mechanisms remain as previously described but utilization of glucose, 2-carbon (2C) units (acetate + 2 β-hydroxybutyrate), fatty acids and AA for biosynthesis and catabolism are represented by Michaelis-Menten-type functions of intracellular precursor concentrations. The Michaelis constants for biosynthesis were set one to two orders of magnitude lower than those for catabolism to fit the observed responses to close arterial glucose infusion (Cant et al. 2002). Sensitivity to concentration is directly related to the value of the Michaelis constant. The four treatments of Guinard and Rulquin (1995), in which MBF decreased as duodenal Met infusion rate increased (Table 1), were simulated from inputs to the revised model of the observed arterial concentrations of glucose, 2C, fatty acids and AA (Table 2) after setting transport parameters and V max s to match prediction with observation on the control treatment. The model predicted a very similar pattern of MBF decline with Met dose to that observed (Fig. 4a). Since there is no provision for any direct effect of AA on MBF in the model, the simulated response to duodenal Met was entirely due to the increases in circulating 2C unit concentrations that were observed (Table 2). In other words, a local energy effect, not an AA effect, can explain the mammary hemodynamic response. Two different groups have reported that euglycemic insulin infusion into lactating ruminants increases both MBF and milk protein yield (Mackle et al. 2000a; Bequette et al. 2001), the former ostensibly to provide AA for the latter. Mackle et al. (2000a) infused cows abomasally with water or casein, each with or without euglycemic insulin infusion for 4 d. Neither acetate nor β-hydroxybutyrate concentrations in plasma were reported but a decline in both would be expected. Increasing plasma insulin concentration sixfold in sheep resulted in a 60% drop in 2C concentration (Brockman and Laarveld 1985). Eisemann and Huntington (1994) observed a 25% drop in arterial acetate in hyperinsulinemic, euglycemic beef steers. For the hyperinsulinemia simulations, assuming a 42% decrease in 2C concentration from a normal value of 4.0 mm (Table 2) resulted in increases in predicted MBF similar to those observed (Fig. 4b). The implication that restoration of ATP balance was again responsible for the hemodynamic response is supported by the observation that abomasal casein infusion during hyperinsulinemia did not influence MBF (Mackle et al. 2000a). Were the hyperemia an attempt to provide AA for protein synthesis, then the casein infusion should have lessened the demand. The insulin-induced increase in milk protein yield was not simulated by the model (Fig. 4b) but neither was the increased extraction of essential AA from plasma (Mackle et al. 2000a; Bequette et al. 2001) which would have increased predicted protein yields, ATP utilization and MBF. The mechanism by which insulin stimulates milk protein secretion is unknown (Mackle et al. 2000b) and so it is premature to model putative effects on AA transport for this discussion. However, one particularly dramatic example of an increase in AA transport was observed in the His subtraction experiment with goats of Bequette et al. (2000; Fig. 4C). Unidirectional His clearance, a measure of the collective activity of all His transporters in the mammary

CANT ET AL. MAMMARY RESPONSES TO SINGLE AMINO ACIDS 349 Table 2. Inputs used in the simulations of mammary metabolism shown in Fig. 4. 2C = acetate + 2 β-hydroxybutyrate arterial concentration (mm) fatty amino glucose 2C acids acids Guinard and Rulquin (1995) Control, 0 g d 1 Met 3.50 5.21 0.253 z 1.88 8 g d 1 Met 3.48 5.40 0.253 1.79 16 g d 1 Met 3.47 5.96 0.253 1.85 32 g d 1 Met 3.41 5.57 0.253 1.77 Mackle et al. (2000a) Control, water 2.89 4.00 y 1.207 x 1.86 casein 2.89 4.00 1.207 2.08 water + insulin 2.89 2.30 1.231 1.62 casein + insulin 2.89 2.30 1.144 1.73 Bequette et al. (2000) Control, with His 2.15 w 3.93 w 0.699 w 2.19 without His 2.88 2.03 0.725 2.15 z Triacylglycerol from Rigout et al. (2002) and NEFA from Guinard and Rulquin (1994). y Set arbitrarily. x Triacylglycerol from Griinari et al. (1997). w From Weekes and Cant (2000). glands, increased 40-fold when His was removed from the abomasal infusate and MBF also increased 36%. As energy metabolism was not the focus of the experiment, glucose, 2C and fatty acid concentrations in plasma were not measured. To create a data set to evaluate the response of the mathematical model to a His deficiency (Table 2), the milk composition and energy metabolite concentrations observed when cows were infused abomasally with all AA except His (Weekes and Cant 2000) were combined with AA concentrations and MBF:milk yield ratios observed in goats (Bequette et al. 2000). The deficiency of His in blood was represented by assigning the arterial AA pool an idealness factor of 1.0 and 0.36 for the control and His-subtracted treatments, respectively (based on the essential AA profile observed in arterial plasma relative to the profile of milk protein, the maximum milk protein yield possible without His was 0.36 of that with His). In the model, protein synthesis was calculated from the ideal fraction of AA concentrations in the cell, while catabolism remained a function of total AA concentrations. Increasing the transport k Ia for the ideal AA group from 629 h 1 to 1176 h 1 on the minus His Fig. 4. Comparison of predicted (black bars) mammary plasma flows (upper panel) and milk protein yields (lower panel) with observed (white bars). Model parameters were set to match predictions with observations on the control treatment for each experiment (a = Guinard and Rulquin (1995), b = Mackle et al. (2000a), c = Bequette et al. (2000), Weekes and Cant (2000)]. Using these same parameter values, predictions were obtained of the response to observed concentrations of the milk precursors on the other treatments (Table 2).

350 CANADIAN JOURNAL OF ANIMAL SCIENCE treatment fit the milk protein yield observations (Fig. 4c). Predicted MBF increased by 15% because of the low 2C concentrations in plasma but the prediction was still less than the 36% hyperemia observed. On this treatment, AA catabolism was predicted to account for 42% of AA uptake, which is physiologically too high, and provided too much ATP in the simulation. Bequette et al. (2000) noted that clearances of many AA (but not His) by the mammary glands were decreased during the His deficiency. Reducing the transport k Aa for all AA from 629 h 1 to 535 h 1 on the minus His treatment while keeping the transport k Ia for ideal AA high at 1038 h 1 fit both the milk protein yield and MBF observations (data not shown). Modeling shows that if milk composition is relatively insensitive to precursor concentrations, and if the secretory cells can adjust AA transporter activity, the possibility cannot be ruled out that MBF responses to AA perturbations are an attempt to restore intracellular ATP balance. While AA are not directly involved in vasodilation, manipulation of AA supply or utilization in the dairy cow affects metabolism in non-mammary tissues to change the profile of energy metabolites in general circulation, which then induces the change in MBF. The underlying mechanisms behind these non-mammary adjustments remain to be elucidated and are beyond the scope of this review. Suffice it to say that an absorbed AA imbalance causes glucose, acetate and β-hydroxybutyrate concentrations in plasma to change (Guinard and Rulquin 1995; Weekes and Cant 2000), which, combined with the MBF effects, may have been responsible for the high milk fat yields observed during AA imbalance (Cant et al. 1999). The lack of a direct effect of AA on MBF explains why close arterial infusion of an AA solution lacking His for 10 h had no effect on iliac blood flow (Cant et al. 2001; Madsen et al. 2002), while long-term abomasal infusion for 6 d, which allowed systemic adaptations to come into effect, did elicit an MBF increase (Bequette et al. 2000). It also explains why long-term postruminal infusion of complete profiles of essential AA, which do not substantively affect circulating concentrations of energy metabolites (Cant et al. 1993b; Guinard et al. 1994; Griinari et al. 1997; Mackle et al. 2000a), have no effect on MBF (Cant et al. 1993b; Mackle et al. 2000a; Bequette et al. 2001). Of course, there may be other, equally valid explanations. To the authors knowledge, the definitive experiment has not yet been conducted to test the role of energy metabolites in single AA-induced hyperemia in the mammary glands. Amino Acid Extraction Ad hoc changes in AA transport efficiency allowed the simulation model to behave like actual mammary glands. A more complete description of mammary metabolism would have included the mechanism of such changes. Although the kinetic systems of AA transport, and some of the transport proteins themselves, have been identified in mammary tissue (Shennan and Peaker 2000), the pattern and mechanisms of their nutritional regulation have not been determined in any detail. Instead, we make inferences from observations of arteriovenous difference. First, Table 1 shows quite clearly that net extraction of an AA is inversely related to its concentration in arterial plasma. Second, stimulation of milk protein production with insulin is accompanied by an increase in AA extraction (Mackle et al. 2000a; Bequette et al. 2001). Additionally, when MBF was reduced in response to elevated fatty acid concentrations in plasma, AA extraction increased (Cant et al. 1993a). These observations are all consistent with the conclusion of Mackle et al. (2000a) that the mammary glands have the ability to modify AA extraction efficiencies to meet a pre-set need for AA utilization. The changes in net extraction may be a result of a rate of bidirectional transport across the plasma membrane which is much higher than a controlled rate of intracellular AA sequestration. An increase in sequestration rate (i.e., protein synthesis) would reduce intracellular AA available for cellular efflux, thereby increasing net extraction without any shift in transport parameters. Using the enrichment of milk casein with an intravenously infused tracer as a surrogate for intracellular AA enrichment, unidirectional influxes of individual essential AA were estimated to be 11 to 72% higher than net uptakes (Bequette et al. 2000; Guan et al. 2002). This recycling is sufficient to allow for modest changes in net extraction to occur without transporter modification but the fourfold increase in extraction of His and twofold decrease for other AA during His deficiency involved concordant up- and down-regulation of unidirectional transport efficiency, respectively (Bequette et al. 2000). Similarly, efficiency of unidirectional Lys transport into the mammary glands of lactating sows was increased threefold when Lys was fed at half its recommended intake (Guan et al. 2002). Thus, transporter regulation is a common mechanism to coordinate AA supply to the mammary glands with protein synthesis rate. In equations to predict net transport of individual AA, Hanigan et al. (2000) incorporated a term for inhibition by intracellular AA. In their model, stimulation of milk protein synthesis by insulin would reduce intracellular AA concentrations and relax the inhibition of transport so that unidirectional uptake would increase (Hanigan et al. 2001). Additionally, the equations would produce the correct pattern of transport changes during His deficiency through the low intracellular concentrations of His and high concentrations of the other AA that result. The mammary glands are a unique tissue in terms of AA utilization, in that milk protein synthesis rate is not at the mercy of fluctuations in extracellular AA concentrations. Rather, protein synthesis appears to operate at a predetermined rate set by external communications of milk withdrawal rate, physiological state and overall nutritional status. Thus, in the domestic dairy cow, stage of lactation and dietary energy intake have a much larger impact on milk protein yield than does the AA supply (Spörndly 1989; Hanigan et al. 1998; Cant 2002). The identities of the signals of nutritional and physiological status have not been identified, although components of the somatotropic axis are candidates. Alternatively, the signals may be as-yet undiscovered and akin to the feedback inhibitor of lactation which signals alveolar milk withdrawal (Wilde et al. 1995). The central directive to produce milk maintains what has