Some amino acids are limiting for optimal milk production as evidenced by an increase in milk and protein yields, and percentage of protein in milk after supplementation with specific, rumen-protected amino acids. Many amino acids can have positive effects on physiological processes that are independent of their effects on synthesis of proteins. This has been termed “functional effects” of amino acids and methionine and arginine effects are the best studied “functional amino acids” that have been linked to reproduction.

Most amino acids are more concentrated in the oviduct and uterus than in the blood. In addition to the mechanisms that concentrate amino acids in the uterus in non-pregnant ruminants, there are additional mechanisms that result in further increases in concentrations of amino acids in the uterine lumen in pregnant ruminants near the time of embryo elongation (Day 14 to 18 of development). Of particular interest for dairy cattle, the three amino acids that are considered rate-limiting for milk production, Met, His, and Lys, are the amino acids with the greatest increase in concentrations in the uterine lumen during embryo elongation (>10-fold increase on average. Arginine is another amino acid that has been studied extensively in relation to reproduction and it is also highly concentrated in the pregnant uterus.


How does supplementation of rumen protected amino acids effect reproduction?

Numerous studies have evaluated the effects of rumen-protected amino acids, particularly methionine, on milk production. Unfortunately, we have been unable to find studies in the scientific literature which were specifically designed and adequately powered to evaluate the effects of specific amino acids on reproductive efficiency of lactating dairy cows. The largest study combined results from 259 cows at 6 Universities evaluating rumen-protected methionine and lysine supplementation. They detected no significant effect on days to first service, services per conception, or calving interval, although no details were provided on reproductive measures in each specific treatment group.


Are there any studies that might give us insight?

One particularly interesting study used serum from lactating dairy cows in the media to grow 9.5 day old rat embryos. Complete development of these embryos requires serum and development is normal in rat serum. When embryos were grown in serum from dairy cows embryonic development was abnormal. Supplementation of bovine serum with amino acids and vitamins produced normal development. Amino acid supplementation alone but not vitamin supplementation produced normal development. Supplementation of methionine alone was sufficient to produce normal development of the rat embryos in cow serum. In a separate experiment, use of serum from cows that were supplemented with rumen-protected methionine also produced normal embryo development. Thus, bovine serum has such low methionine concentrations that normal development of rat embryos is retarded.

The requirements for complete development of bovine embryos have not yet been determined. Conditions have not been developed that allow elongation of embryos in vitro, and definitely do not allow culture of bovine embryos to the stage that was analyzed in the rat embryo experiments.


What about studies with bovine embryos?

We recently evaluated the effect of supplementation with rumen-protected Met on early embryo development in superstimulated cows. We used superstimulated cows so that we would have sufficient statistical power by evaluating numerous embryos in order to validly test the in vivo effects of methionine supplementation on early embryo development in lactating dairy cows. In this experiment, cows randomly assigned to two treatments differing in level of dietary Met supplementation: 1) Met; diet supplemented with a blood meal-based product (ProVAAl Ultra; Perdue Agribusiness) with the rumen protected Met Smartamine (Adisseo), formulated to deliver 2,875 g of metabolizable protein (MP) with 6.8 Lys as % of MP and 2.43 Met as % of MP; 2) Control; cows fed the same basal diet but replacing ProVAAl Ultra by ProVAAl Advantage, which contains no added rumen protected Met, formulated to deliver 2,875 gr MP with 6.8 Lys as % of MP and 1.89 Met a % of MP. There was an increase in both kg of milk protein produced and percentage of protein in the milkg.

Thus, from a protein production standpoint, Met appeared to be rate-limiting. We measured plasma Met concentrations in this study and found a large effect of feeding rumen-protected Met on circulating Met concentrations.

Our primary interest was the effect of supplemental Met on embryo quality. We evaluated a total of 570 embryos in this experiment and found no differences in fertilization or embryo quality. Thus, Met supplementation did not alter early embryo development, at least from a gross morphological standpoint.


Does methionine have other effects on the embryos?

Even though Met supplementation during the later stages of follicle development and early embryo development may not have produced morphological changes in the

early embryo, it is well known that Met during this time can have dramatic effects on the gene expression of the embryo. This means that the genes can be changed in such a way that they are not expressed in the same way due to addition of groups, generally methyl groups to the DNA of the cells. For example, a previous study in sheep restricted methyl donors by restricting Met, vitamin B12, and folate before and for the first 6 days after breeding. They then transferred normally-appearing embryos into control sheep and then evaluated the lambs after parturition. The embryos that were produced in low methionine produced lambs that had substantial differences in blood pressure and immune function. To test this idea in cattle, we evaluated whether the embryos that were recovered from cows that had been supplemented or not supplemented with Met had differences in gene expression.

The objective of this part of the study was to evaluate the effect of maternal Met supplementation on the transcriptome of bovine preimplantation embryos. Only high quality embryos from individual cows were pooled and then analyzed by a powerful technique that allows evaluation of all genes that are expressed in these embryos, called RNA sequencing (RNAseq). Remarkably, the small difference produced in circulating methionine produced a substantial difference in expression of genes in the embryo. A total of 10,662 genes were significantly expressed in the bovine embryos. A total of 276 genes were expressed differently, statistically, in embryos from cows supplemented or not supplemented with methionine. Thus Met supplementation seemed to change gene expression in a way that may lead to improved pregnancy outcomes and improved physiology of the offspring. Many of the genes are involved in immune function and later stages of embryo development that may be critical for pregnancy progression and normal immune function after birth.


What is the conclusion?

Thus, supplementation of rate-limiting amino acids can have substantial effects on milk protein content and yield; however, effects on reproduction have not yet been adequately evaluated. The dramatic induction of the rate-limiting amino acids, Met, His, and Lys, in the uterine fluid of pregnant cows near the time of embryo elongation suggests that elevated amounts of these amino acids may be critical for this important stage of embryo development. Supplementation of cows with methionine during the final stages of follicular development and early embryo development, until Day 7 after breeding, did not lead to gross morphological changes in the embryos but did result in dramatic differences in gene expression in the embryo. Further studies are needed to evaluate whether supplementation with these essential amino acids to lactating cows would have a beneficial impact on embryo survival and if these changes in the early embryo translate into changes in pregnancy outcomes or physiology of the resulting calf.


Author: Milo C. Wiltbank, University of Wisconsin-Madison

Adapted from Florida Ruminant Nutrition Symposium Proceedings