317 Maternal citrulline supplementation to nutrient-restricted ewes during gestation programs glycine metabolism in the postnatal lamb.

Abstract Glycine plays important roles in protein synthesis, reducing oxidative stress, one-carbon metabolism, creatine synthesis, and is a conditionally essential amino acid in animal nutrition. Maternal arginine supplementation from gestational days (GD) 75 to 125 (term = ~147) has been shown to increase circulating glycine levels in the neonatal lamb. However, the need for protection against ruminal degradation decreases the efficacy of this supplemental approach. The present study aimed to determine if maternal citrulline supplementation (the precursor for arginine synthesis) improves intrauterine and postnatal growth as well as program glycine metabolism in lambs. Rambouillet donor ewes were superovulated and laparoscopically inseminated using semen from a single Hampshire sire. On day 6, embryos were collected and a single grade 1 blastocyst was transferred into Hampshire recipients. Recipient individual housing began on GD 21 and ewes received 100% National Research Council (NRC) requirements for a one-week acclimation period. Following pregnancy determination on GD28, ewes were nutritionally restricted to receive 50% NRC requirements and randomly assigned to receive either L-alanine (isonitrogenous control) at 0.61% of the diet (ALA, n=17) or L-citrulline at 0.40% of the diet (CIT, n=17) fed twice daily as a top-dress. The dietary and supplementation treatments continued until GD140 at which time supplementation ceased and ewes were returned to a 100% NRC dietary intake. Upon lambing, birth weights were recorded, and a jugular blood sample collected from each lamb before suckling. On postnatal day (PND) 60, lambs were euthanized, organ weights recorded, and samples were snap-frozen in liquid nitrogen for future analyses. Birth weight, PND60 body weight, and PND60 liver weights were heavier (P< 0.05) in lambs born from CIT-treated mothers. High-performance liquid chromatography (HPLC) was performed to determine circulating amino acid (AA) concentrations of lambs at birth and PND60. Glycine levels on PND60 were greater (P< 0.05) in lambs from CIT mothers than lambs from control ewes. Due to their roles in AA metabolism, liver and kidney tissues were subjected to qPCR to determine mRNA expression of enzymes involved in glycine metabolism. In these tissues, there were tendencies (P< 0.10) and differences (P< 0.05) in expression of SHMT1, SHMT2, GATM, AMT, GCSH, DLD, DAO, ALAS1, and GCAT enzymes in lambs born to citrulline-treated ewes (P< 0.05). Because both enzymes for converting glycine into creatine were elevated, creatine levels were measured. However, there was no difference (P >0.10) in plasma creatine levels on PND60 between treatment groups. The differences in expression of enzymes involved in glycine metabolism appear to favor its net synthesis and explain the increased levels of circulating glycine in lambs born to citrulline-treated mothers. These findings warrant further investigation to determine the mechanism by which glycine metabolism is programmed in the offspring. (Supported by a USDA/NIFA grant #2022-67015-37272)