Whole-body and mammary gland metabolism in dairy cattle: impact of postabsorptive energetic substrates and amino acid profiles

Ruminants play a key role in converting human-inedible feedstuffs into high-quality human edible food. In dairy cattle, the type of energy and protein delivered through dietary ingredients are important factors influencing nutrient transfer from feed into milk components. In particular, the capture of dietary nitrogen (N) into milk N (milk N efficiency) is important to the profitability of dairy farms and impacts the level of N emissions to the environment.

At low and high dietary protein levels, increased supply of glucogenic nutrients can improve the postabsorptive transfer of amino acids (AA) into milk protein. Lipogenic nutrients can increase milk energy output through the direct transfer of dietary fatty acids (FA) into milk, but the impact of lipogenic supplements and their interaction with protein supply on milk N efficiency and N partitioning at the whole-body and mammary gland level represented a significant knowledge gap. Furthermore, knowledge was lacking on whole-body energy and N metabolism of cows receiving similar metabolizable protein (MP) levels that differ in AA profile. Therefore, a key objective of this thesis was to investigate effects of postruminal absorption of lipogenic, glucogenic, and aminogenic energy sources at the whole-body and mammary gland level with respect to their application for improving milk N efficiency in dairy cattle.

In the first experiment, isoenergetic levels of rumen-protected protein (xylose-treated soybean meal and rapeseed meal) and rumen-inert hydrogenated palm FA (C16:0 and C18:0) were tested in a 2 × 2 factorial arrangement using 56 Holstein-Friesian cows in a randomized complete block design. This study demonstrated independent and additive stimulation of milk yield when protein and fat were supplemented at isoenergetic levels. Furthermore, rumen inert hydrogenated palm FA was an effective energy source to improve N utilization by lactating cows at high and low MP levels, although the effect on milk N efficiency was more pronounced at the low MP level.

This study also demonstrated the metabolic flexibility of the mammary gland in its use of aminogenic versus lipogenic substrates for milk synthesis. Mammary plasma flow was not affected by energy from protein or fat. Energy from fat had no effect on mammary net uptake of any AA group, but increased uptake of triacylglycerol (TAG) and long-chain fatty acids (LCFA). Mammary net uptake of total EAA and group 2 AA was increased in response to energy from protein, regardless of dietary fat level. Greater milk protein synthesis in response to energy from protein was supported by increased intramammary metabolism of group 2 AA, evidenced by an increase in their mammary uptake to milk protein output ratio (U:O). Notably, neither energy from protein nor energy from fat affected mammary glucose balance, and mammary glucose uptake was in excess of estimated requirements for the observed lactose and fat synthesis. These results show that postruminal fat supplementation has little effect on mammary gland AA utilization, and suggests that factors other than mammary glucose supply regulate lactose yield when extra energy is supplemented from protein and fat.

In the same study, the effects of energy from protein and fat on expression of genes associated with mammary gland cellular pathways contributing to energy generation and secretory capacity were studied using RNA isolated from milk fat. mRNA expression of enzymes regulating branched-chain AA catabolism and of mitochondrial malic enzyme suggested that energy from protein affected cellular energy-yielding pathways differently in the presence or absence of energy from fat, and may suggest a link between regulation of branched-chain AA catabolism and anaplerotic flux through the tricarboxylic acid cycle. Energy from protein may increase mammary secretory capacity through endoplasmic reticulum biogenesis and secretory cell differentiation, suggested by increased expression of the active form of X-box binding protein 1. Stimulation of protein synthetic activity when energy from protein and fat are supplemented together may have been supported through increased expression of protein phosphatase 1 regulatory subunit 15A. These results show that mammary cells use aminogenic and lipogenic precursors differently in support of milk component production when AA and FA supply is altered by dietary intervention. They also suggest that mammary cells respond to increased AA supply through mechanisms of increased secretory capacity and secretory cell differentiation, dependent on the presence of extra energy from fat supplementation.

A second experiment was conducted to test the effects of energy from abomasally infused glucogenic (glucose) or lipogenic (palm olein; mainly C16:0 and C18:1) substrates at low and high MP levels (infused EAA mixture in the profile of casein at a constant 844 g/d). Six Holstein-Friesian dairy cows were housed in climate respiration chambers and were used in a 6 × 6 Latin square design where each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion. Postruminal glucose and palm olein did not affect total milk, protein, or lactose yields, and did not affect milk production differently at the high MP level than at the low MP level. Similarly, alterations in whole-body energy and N partitioning observed in response to glucose or palm olein infusion were largely independent of MP level. Infusing EAA (the high MP level) increased milk protein, fat, and lactose production without negatively affecting energy balance, but decreased milk N efficiency. Important differences found between glucogenic and lipogenic energy were that regardless of MP level, glucose supplementation promoted energy retention and improved milk N efficiency, whereas palm olein supplementation partitioned extra energy intake into milk and had no effect on milk N efficiency.

Increased absorptive supply of glucose and palm olein differently affected mammary gland metabolite utilization, irrespective of MP level. In response to glucose infusion, arterial plasma concentration of group 2 AA decreased and mammary plasma flow increased, both regardless of MP level. The observed reduction of intramammary catabolism of group 2 AA (lower mammary U:O) at the low MP level was attributed to the anabolic effects of insulin on extra-mammary peripheral tissues. Regardless of MP level, palm olein did not affect arterial AA concentrations or mammary AA utilization. Infusion of EAA (the high MP level) increased arterial EAA concentrations to 2.5-times that of the low MP level, and mammary net uptake of EAA increased. Intramammary catabolism of group 2 AA increased at the high MP level, and that of non-EAA decreased, suggesting group 2 AA supported the increase in milk protein yield during EAA infusion. Similar to the findings of the first experiment, these results illustrate flexibility of mammary metabolite utilization when absorptive supply of glucogenic, lipogenic, and aminogenic substrates are increased.

A third experiment examined the effects of EAA profile within MP supply by removing different groups of EAA from a complete EAA profile, but compensating with the supply of the other EAA such that the total supplemented MP level remained constant. Abomasal infusion was used to deliver the following EAA profiles: 1) a complete EAA mixture, 2) Ile, Leu, and Val (BCAA), 3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and 4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). Within each infusion, EAA were infused in amounts relative to their content in casein, and the total infused EAA level was constant at 562 g/d. Five Holstein-Friesian dairy cows were housed in climate respiration chambers and treatments were applied according to a 5 × 5 Latin square design. Each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion.

An important result of this study was that compared with a complete EAA profile, the same level of total milk, protein, fat, and lactose yield can be achieved whether Arg, Lys, and Thr, or Ile, Leu, and Val are absent, if the other 7 EAA are present to compensate the MP supply. Supplementing only Ile, Leu, and Val reduced feed intake, was inhibitory to efficient milk protein synthesis, increased the proportion of N intake excreted in urine, and resulted in negative energy balance. Most notably, extra MP from a complete EAA profile resulted in 25% greater milk protein yield with the same milk N efficiency as the saline control, extra MP from the GR1+ILV profile resulted in the same milk N efficiency and N excretion in manure as the complete EAA profile, and extra MP from the GR1+ALT profile reduced milk N efficiency to below control levels and resulted in higher manure N excretion.

When Arg, Lys, and Thr, or Ile, Leu, and Val were absent from the infusions, intramammary catabolism of the present group 2 AA compensated for the lower mammary uptake of the absent group 2 AA, evidenced by greater U:O of the absent group. Notably, mammary uptake of Lys may have been inhibited by high levels of arterial branched-chain AA during BCAA and GR1+ILV infusion, and in both treatments the intramammary catabolism of branched-chain AA could have compensated for the lower intramammary Lys level. When Ile, Leu, and Val were supplemented alone, the U:O of these EAA increased to approximately double the level as on the other infusions, suggesting the mammary gland maintained a high capacity for their uptake at this infusion level, and increased their catabolism to stimulate milk protein synthesis and likely milk fat and lactose synthesis as well. These results illustrate flexibility in mammary uptake and intramammary catabolism of AA within the group 2 category to support milk protein synthesis when the supplemented MP level is maintained but the EAA profile is incomplete with respect to casein.

Altogether, the experiments described in this thesis contribute to the gaps in knowledge around effects of postruminal energy type, particularly lipogenic energy compared with glucogenic energy, at low and high MP levels, and around effects of AA profile of MP. Results show that postruminal fat increases the transfer of AA into milk protein at low MP levels, and may improve this transfer at high MP levels. Further, postruminal fat has the potential to reduce AA catabolism by increasing utilization of non-EAA for milk protein synthesis, particularly at low MP levels. Aside from this, in contrast to increased levels of circulating glucose, increased levels of circulating FA largely do not affect mammary gland AA metabolism. With regard to AA profile of MP, similar responses in milk production and milk N efficiency to a complete EAA profile can be achieved by supplying group 1 AA plus Ile, Leu, and Val. Postruminal supplementation of EAA can result in increased milk protein yields at milk N efficiencies (approximately 36%) that are the same or greater than that achieved at a low dietary MP level.