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Breeding and nutritional manipulation are the two major strategies to improve milk protein content. Breeding is possible, but the rate may be slow. So, dietary manipulations are more convenient and rapid way to improve the concentration and yield of milk protein. Nature of protein nutrition for animals is the AA supply. For available absorption and utilization of AA, ruminants must be provided with sufficient and well-balance AAs. Free AAs are the major form in which amino-N is taken up by lactating mammary tissues. Sometimes the uptake of certain EAA across the mammary gland appears to be insufficient for their output as milk protein in lactating dairy cows (Bickerstaffe et al., 1974; Metcalf et al., 1996). There is a need to activate their withdrawal from the peptide-bound amino acids (PBAA) pool. Identification of peptide transporters in the mammary gland, therefore, may provide new insights into protein metabolism and secretion by the gland. The present paper will focus on the major advances in biosynthesis of milk protein, and the strategies to improve its synthesis in dairy cattle by dietary manipulation.
THE BIOSYNTHESIS OF Milk Protein
The mammary gland has high metabolic activity, as protein synthesis by the mammary gland averaged 43% of whole body protein synthesis (Thivierge et al., 2002). Free AA, primarily extracted from blood are the precursors of milk protein, however it has been suggested that small peptides from blood may also be a source of protein (Backwell, et al., 1996). Milk protein synthesis and secretion within the mammary gland of the dairy cow is a complex biological process, containing several smaller integrated functions such as AA uptake, transcription of DNA to mRNA components, translation of mRNA to protein, and finally intracellular post-translational modification of the protein prior to secretion. All epithelial cells of the mammary gland are thought to contribute to this biosynthetic process, and each cell expressing all of the genes is thought to synthesize and secrete the casein and whey proteins.
The variability in milk protein content may be associated with differences in nutritional (AA and glucose availability), endocrine, and regulatory factors inside the gland downstream of transcription that influence protein metabolism in the mammary gland (Johnston et al., 2004; Bobe et al., 2009; Burgos et al., 2010). Recent studies have indicated that regulation of protein translation may be important in determining milk protein production by dairy cows. The translation initiation and elongation factors could be potential targets for the regulation of milk production and thus potentially milk protein synthesis (Hayashi et al., 2009). The mechanisms controlling milk protein synthesis must be more clearly understood before nutritional strategies can be devised.
The mammary gland has the ability to regulate its nutrient uptake in order to maintain milk synthesis. This regulation is exerted by adjusting mammary blood flow or by adjusting the removal of milk precursors from arterial supply, as it has been well demonstrated when an AA limits protein synthesis (Thivierge et al., 2002). The udder possesses intracellular controls that regulate milk synthesis, including a protein influencing milk output, the feedback inhibitor of lactation (FIL; Wilde et al., 1995). The regulatory effect of FIL on milk protein secretion is mediated through an increased degradation of newly synthesized casein and a down regulation of protein synthesis (Rennison et al., 1993).
The endocrine system, perhaps more than any other physiological system, plays a central role in all aspects of mammary development (mammogenesis), onset of lactation (lactogenesis), and maintenance of milk secretion (galactopoiesis) (Akers, 2006).
Lactogenic hormones (i.e., prolactin, hydrocortisone, and insulin) are required for functional differentiation of cultured mammary tissue/epithelial cells (Brennan et al., 2008; Lee et al., 2009). Induction of milk protein gene expression required the complement of prolactin, hydrocortisone and insulin. The polypeptide hormone, prolactin, plays a major role in driving the pregnancy and postpartum development of the mammary gland to produce successful lactation (Oakes et al., 2008). Prolactin stimulates the synthesis of milk protein, possibly at the transcriptional level (Nardacci et al., 1978). Accumulation of casein mRNAs induced by prolactin is amplified by hydrocortisone (Kabotyanski et al., 2009). Hydrocortisone belongs to glucocorticoid, and it induces casein gene expression through an indirect cellular mechanism. However, Puissant and Houdebine (1991) found that addition of cortisol directly stimulated rapid accumulation of the whey acid protein (WAP) mRNA. Insulin also plays an important role in milk protein synthesis (Nagaiah et al., 1981). Menzies et al. (2009) demonstrated that insulin stimulated milk protein synthesis at multiple levels (milk protein gene expression, casein synthesis and 14C-lysine uptake) within the mammary epithelial cells. Besides, combination of insulin and prolactin synergistically promoted the accumulation of casein mRNA in mammary tissue (Choi et al. 2004). These hormones may modulate mammary protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway (Yang et al., 2008).
Growth hormone (GH)
Growth hormone (GH) has a well-established galactopoietic effect on the bovine mammary gland (Molento et al., 2002); however, the molecular mechanisms mediating the effect of GH on protein synthesis remain largely unknown. Several studies have shown that the positive effects of GH on lactation were related to the increases in the proliferation and activity of mammary epithelial cells (Molento et al., 2002), as a result of either the direct effect of GH on mammary gland or an indirect effect via increased secretion of IGF-1 (Akers et al., 2000). The study of Hayashi et al. (2009) provided evidence that the effect of GH on milk production was mediated, at least in part, by up-regulating the initiation and elongation phases of protein translation and suggests that mRNA translation step is associated with milk protein synthesis in the lactating cow treated with GH. They also found that increased milk protein yield in the GH-treated cows was associated with increased phosphorylation of ribosomal protein S6 (rpS6), suggesting that the effects of GH on lactation may be mediated via the mTOR pathway (Hayashi et al., 2009).
It has been demonstrated that glucagon infusions, under various conditions, resulted in a rapid and short-term decrease in protein yield and concentration with minimal effects on milk yield or on other milk components in early lactation cows (Bobe et al., 2009). The most likely mechanism is that glucagon decreases AA availability and increases glucose availability to the mammary gland by increasing hepatic extraction of gluconeogenic AA for conversion to glucose, thereby decreasing arterial concentrations of gluconeogenic AA and increasing concentrations of glucose (Bobe et al., 2009).
Protein synthesis consumes AA and ATP, so an adequate supply of both is essential to meet the demands of lactation.
Many studies have examined responses in mammary gland protein metabolism to the altered AA supply pattern. Casein treatments increased milk protein concentration and yield of dairy cow (Raggio et al., 2006). Mammary protein synthesis was 50% higher with increased availability of AA in medium (Burgos et al., 2010). Increases in milk protein yield have been shown to be related to increases in uptake of mammary EAA, and some AAs have been identified to be potentially limiting for milk protein synthesis (Johnston et al., 2004). Besides working as the building blocks for milk protein synthesis, AAs also act as the signaling molecules. There is increasing recognition of the role of AA as signaling molecules in the regulation of protein synthesis (Kimball and Jefferson, 2002). Activity of mTOR signaling proteins in mammary cells increased with greater AA availability, and this response was enhanced by lactogenic hormones (Burgos et al., 2010). However, this molecular mechanism has not been fully understood in the mammary gland of lactating cows.
The lactating mammary gland also requires adequate energy supply. Hanigan et al. (2009) estimated that almost half of the ATP generated by the lactating mammary gland is used for synthesis of proteins. In general, increasing the intake of nonstructural carbohydrate increases milk and milk protein yield and N efficiency in lactating cows (Rius et al., 2010a; Raggio et al., 2006). Studies in animal and cell culture models have revealed that cellular energy availability play a role in regulating protein synthesis not only as substrates but also through direct signaling to the protein synthetic machinery (Proud, 2007). Rius et al. (2010b) also demonstrated that starch infusions increased phosphorylation of rpS6 and mTOR, consistent with the change model of milk protein yields.
DIETARY SUPPLY STRATEGIES TO IMPROVE Milk Protein
Dietary protein is an important nutrient for lactating dairy cow, especially during the early stage of lactation, because dairy cows at this stage have a high protein requirement at a relatively low dry matter intake. In ruminant nutrition, the degradation of dietary protein in rumen is an inefficient process, thus providing dairy cows with high-protein diet is not always an approving solution. Nature of protein nutrition for animals is the AA supply. For available absorption and utilization of AA, ruminants must be provided with sufficient and well-balanced AAs. With the development of biotechnology, mass-production of AA is now possible. Free AAs are the major form in which amino-N is taken up by lactating mammary tissues. Sometimes the uptake of certain EAA across the mammary gland appears to be insufficient for their output as milk protein in lactating dairy cows (Bickerstaffe et al., 1974; Metcalf et al., 1996). There is a need to activate their withdrawal from the PBAA pool. The ability to rapidly manipulate milk protein must occur through nutritional strategies.
Challenges to predicting AA requirement
Knowledge of both AA supply and requirement is required for prediction of milk protein yield. The NRC (2001) recommendations were made for two AAs, Lys and Met, based on works of Rulquin et al. (1993) and Schwab (1996). These AAs may be limiting in certain diets under intensive dairy systems. The predicted concentrations of Lys (7.2%) and Met (2.4%) in metabolizable protein are suggested to acquire maximum yield and content of milk protein. Thus, the optimum ratio of Lys: Met in metabolizable protein is 3.0:1. Results of Wang et al. (2010) also demonstrated that, based on meeting the requirement, an appropriate ratio (3:1) of Lys to Met was critical for maximizing the synthesis of milk protein, hence improving milk and milk protein yield and N utilization efficiency. However, in study of Socha et al. (2005), the evaluation of Lys: Met ratio in metabolizable protein was 3.8:1 or 3.9:1 for optimal milk protein content and yield. In all cases, it is concluded that Met is more limiting than Lys and that the cows have responded more to rumen-protected Met supplementation than rumen-protected Lys.
After absorption, individual AA flows first to the liver where substantial and differential net removal occurs, varying from zero for the BCAA to 50% of portal absorption for phenylalanine (Lapierre et al., 2006). This process alters the pattern of net supply to the mammary gland. Intermediary metabolism of AA between the duodenum and mammary gland results in the decreased efficiency of absorbed AA for milk protein synthesis. Therefore, variable factors for transfer efficiencies must be incorporated into the predictive models. Lapierre et al. (2006) summarized that the gut metabolism substantially and differentially alters the availability of AA supplied from microbial protein and rumen undegraded protein (RUP); and losses of AA through the routes of both endogenous proteins and gut oxidation will need to be incorporated into future predictive models to define the AA demands of the dairy cows.
Besides, accuracy of equations is an obvious goal to improve the predictability of factors influencing milk protein secretion. In a comprehensive meta-analysis of experiments studying substitution of various protein sources (Ipharraguerre and Clark, 2005), microbial protein flow to the duodenum was depressed on average by 7%, partially negating the benefit of the RUP source to increase overall supply of metabolizable protein. However, provision of the RUP source may increase the duodenal flow of either Lys or Met enough to compensate for the depressed metabolizable protein synthesis, even though it does not do so in all individual studies. With the increasing reliance on meta-analysis for either direct calibration of prediction equations or for evaluation of mechanistic predictions, researchers need to be aware of statistical pitfalls embedded with these approaches to ensure that quantitative estimates are accurate. Integrating knowledge on AA metabolism will help build predictive models that allow accurate definition of the dietary protein supply for milk protein synthesis.
AA supply and balance
Increasing AA supply to the mammary gland is the basis for most dietary manipulations to increase milk protein concentration or yield. The Met has been identified as the first limiting AA when the diet is poor in corn or rich in forage, or also when supplemental RUP is provided by soybean products, or both (NRC, 2001). Increasing Met supply through feeding rumen-protected Met (RPM) has the potential to augment milk protein in high-producing dairy cows, probably through increased protein synthesis (NRC, 2001). The RPM addition to diets can increase the true protein content in milk and production of true milk protein (Patton, 2010). Numerous derivatives and analogs of Met have been tested for their resistance to degradation, with the Met hydroxy analog, or D, L-2-hydroxy-4-(methylthio)-butanoic acid (HMB), being the most studied because it is currently used successfully for Met supply to monogastrics. Addition of HMB can increase milk yield, milk protein yield, and milk fat content (Wang et al., 2010). However, in study of St-Pierre and Sylvester (2005), they found HMB does not seem to be able to effectively meet the Met requirements of lactating cows, at least for milk protein synthesis. It has been also demonstrated that the HMBi (isopropyl ester of HMB) allows a significant Met supply to cows (Graulet et al., 2005) and an increase in their milk protein yield and content (Rulquin et al., 2006).
Lys is another limiting AA for dairy cow. Production responses of dairy cows to improved Lys and Met nutrition include variable increases in feed intake, milk production, and content and yield of milk protein (Socha et al., 2005). There are 4 observations regarding improvements in duodenal concentrations of Lys and Met (Socha et al., 2005). First, content of milk protein is more sensitive than milk yield (NRC, 2001). Second, results of several experiments indicate that milk casein is affected more than the whey and non-protein nitrogenous compounds fractions (Armentano et al., 1993). Third, increases in content of milk protein are greater than what would be expected by increasing dietary crud protein (NRC, 2001). Finally, increases in milk yield to supplemental Lys and Met generally are limited to cows in early lactation when the need for absorbable AA, relative to absorbable energy, is greatest (Rulquin et al., 1993).
The BCAA, namely, leucine, isoleucine, and valine possess several characteristics that make them unique among the EAA. The BCAAs play important roles in the regulation of AA and protein metabolism, which include insulin secretagogues, positive regulators of protein synthesis, donors for glutamine synthesis, and inter-organ signalers (Lal and Chugh, 1995). The BCAAs are also important in lactation. In addition to providing up to 20% of the total AA and up to 50% of total EAA in milk protein (Jenness, 1974b), BCAAs are also used to form intermediates of the glycolytic and tricarboxylic acid cycle pathways and contribute to the pool of non EAA required for milk protein synthesis (Mepham, 1982).
Imbalances created by excesses or deficiencies of dietary AA may limit milk protein synthesis and reduce lactation performance. In the previous studies, we found there was an optimal concentration of individual EAA (Met, Lys, Phe and Thr) for casein αs1 gene expression in cultured bovine mammary epithelial cells and excess or deficiency of any EAA depressed milk protein gene expression (Zhao et al., 2005; Zhou et al., 2008). Results of short-term experiments show that, in cows consuming a diet of grass silage and a cereal-based supplement containing feather meal as the sole protein supplement, milk yield is limited by a deficiency in the supply of specific AA such as His, Met, and Lys (Kim et al., 1999; 2000). Deficiencies in the dietary supply of specific AA for 6 wk, in both early/mid and mid/late lactation, markedly reduced milk yield (Yeo et al., 2003). Wang et al. (2008) evaluated the ratio of Lys to Met in metabolizable protein on lactation performance and AAs metabolism of Chinese Holstein dairy cows. Supplementation with Met or/and Lys improved the AA uptake of mammary gland. Milk yield was increased by supplementing Lys or Met, with no significant effects on milk compositions. To maximize milk protein and milk yield the optimal ratio of Lys to Met was at 3:1.
The advantage of improving the balance of absorbable AA is the increased efficiency of use of absorbed AA for milk protein production. It has been demonstrated that improved Lys and Met nutrition reduced the amount of dietary crude protein needed to achieve similar yields of milk protein (Rulquin et al., 1990). Dairy cows in early lactation are sensitive to changes in intestinal AA balance, and their lactation performance may be enhanced considerably by optimizing Lys and Met nutrition (Socha et al., 2005). These responses are typically interpreted according to a limiting AA theory in which there is but one AA under a given set of dietary and physiological conditions whose absorptive supply can influence milk protein yield (Weekes et al., 2006). In the previous study, we also demonstrated different ratios of Lys to Met and Thr to Phe in medium affected the expression of casein αs1 gene in mammary gland, with the optimal ratio at 3.5:1 (Liu et al., 2007) and 1.05:1 (Zhou et al., 2008), respectively. We also conducted a study to investigate the optimum ratios of EAA, including Lys, Met, Thr, and Phe on lactation performance and nitrogen utilization of Holstein dairy cows. The results indicated that, adding rumen protected EAA may balance the dietary AA and reduce protein level, and improve lactation performance and nitrogen efficiency (Yang, 2009). In conclusion, appropriate ratio of EAA can increase the synthesis of milk protein and improved AA balance is good for enhancing milk protein yield and dairy cow performance.
Role of AA in translational mechanisms governing milk protein synthesis
The underlying molecular mechanisms that control milk yield and milk protein yield in domestic animals are not completely understood. Hayashi et al. (2009) pointed out that the mTOR pathway might be a potential control point in the regulation of milk protein synthesis in the mammary gland. Besides as nutriment, AAs also act as the signaling molecules. There is increasing recognition of the role of AA as signaling molecules in the regulation of protein synthesis (Kimball and Jefferson, 2002). Dietary AA can regulate the initiation phase of mRNA translation in animals in vivo through the mTOR signal transduction pathway, which is a protein kinase that controls ribosome biogenesis (Jefferson and Kimball, 2003). The role of AA on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied by Moshel et al. (2006). They found that total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on β-lactoglobulin synthesis in bovine cells, and got the conclusion that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells (Moshel et al., 2006).
Free AAs are the major form in which amino-N is taken up by lactating mammary tissues. However, this concept has been challenged for a long time. In lactating dairy cows, the uptake of certain EAA across the mammary gland appears to be insufficient for their output as milk protein (Bickerstaffe et al., 1974; Metcalf et al., 1996). The AA which is short for milk protein synthesis requirement appears to activate their withdrawal from the PBAA pool.
In the study of Tagari et al. (2004), considerable amounts of PBAA were found in the portal-drained visceral flux of lactating cows fed steam-flaked or steam-rolled corn grain to densities of 360 or 490 g/L, respectively. Considerable amount of PBAA fractions involves in the portal-drained visceral flux, mammary uptake, liver flux, or splanchnic ¬‚uxes (Tagari et al., 2008). Results from in vivo experiments with lactating dairy goats using specific markers, such as 15N or 14C, indicate that many EAA are taken up by the mammary gland as PBAA from the circulation and utilized for protein synthesis (Bequette et al., 1999). Extraction of Lys from the free AA pool fell short of the needs for milk protein synthesis, and despite its apparently large reserve in blood cells (Hanigan et al., 1991), a substantial proportion was extracted from the peptide-bound Lys pool, thus once again indicating the PBAA pool as the first reserve source used. In our previous study, when the dipeptide containing Met was used as supplement of Met the expression of αs1 casein gene was increased in cultured mammary epithelial cell (Wu et al., 2007). Similarly, inclusion of Phe-containing dipeptides as source of Phe also promoted the casein αs1 gene expression in cultured mammary epithelial cells as compared with free AA (Zhou et al., 2011). These results are consistent with the finding that His-containing dipeptide enhanced milk protein production compared with free His (Backwell et al., 1996). Tagari et al. (2008) reported that total peptide-bound EAA in mammary uptake, as a proportion of total free EAA, varied between 3.7 and 4.8%, but the proportion of individual peptide-bound EAA varied from 2.5 to 23.8%.
The molecular bases for the uptake of intact oligopeptide into epithelial cells are the apically located H+/peptide cotransporters, peptide transporter 1 (PepT1) and peptide transporter 2 (PepT2). The high-capacity low-affinity transporter PepT1 is mainly expressed in small intestine (Adibi, 1997), and no hybridization is observed with PepT1 messenger RNA (mRNA) from the mammary gland of the dairy cows (Chen et al., 1999). The high-affinity low-capacity transporter PepT2 is mainly expressed in kidney tubule (Shen et al., 1999). By use of reverse transcriptase PCR (RT-PCR), PEPT2 mRNA was detected in rat mammary gland extracts, human milk epithelial cells (Groneberg et al., 2002), and bovine mammary gland explants (Zhou et al., 2009). The identification of peptide transporter in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland.
Lactating dairy cattle can supply plentiful milk protein for human nutrition and health. Milk protein synthesis in the mammary gland of the dairy cow is a complex biological process, which is strongly influenced by nutritional, endocrine, and other regulatory factors. Increasing AA supply to the mammary gland is the basis for most dietary manipulations to increase milk protein concentration or yield. It is well-documented that uptake of sufficient and well-balance AAs is all-important for milk protein synthesis and lactation performance in dairy cows. Integrating knowledge on AA metabolism and improved accuracy of estimate approach will help build predictive models that allow accurate definition of the dietary protein supply for milk protein synthesis. However, further researches are needed to ensure accurate prediction of lactating dairy cow AAs or peptides requirement for milk protein secretion. Also, there are still lots of problems need to solve, such as the identification of limiting AAs other than Met and Lys, effects of individual AA and peptide on milk protein synthesis, PBAA utilization by mammary gland, and effects of environment, nutrition, and physiological status on milk protein synthesis.