77 Essential amino acid supplementation on bovine trophectoderm: potential strategies to mitigate pregnancy loss in dairy cattle

Reproductive failure is the second most reported reason for female culling in dairy cattle operations. The majority of pregnancy loss occurs within the first month of pregnancy and presents a substantial financial burden for producers. During pregnancy, the uterine epithelium secretes and transports histotroph, a mixture of nutrients, hormones, growth factors, and so forth, to support conceptus development. Changes in amino acid (AA) composition and glucose concentrations in histotroph greatly influence conceptus elongation, affecting conceptus growth and attachment. It is hypothesized that modulation of essential AA (EAA) supplementation could directly affect trophectoderm (TE) proliferation, stimulating genes involved in nutrient supply, and pregnancy markers. This study evaluated the effects of dose-dependent EAA concentrations on TE growth in vitro. Hatched in vitro-derived blastocysts were cultured individually in 24-well gelatin-coated plates and incubated at 38.5°C and 5% CO₂ until Day 30 of culture. Primary TE cell lines were cultured in AA-free medium supplemented with standard DMEM concentrations of nonessential AA and increasing concentrations of total EAA ranging from 0, 0.25, 0.5, 1, and 1.5× DMEM. Pictures of individual cell lines were taken every other day. Cell lines were collected at Day 30 of culture for gene expression. A total of 64 TE cell lines were produced over four independent experiments. Growth measurements were performed using ImageJ, v1.53. Trophectoderm growth and gene expression data were analyzed by ANOVA using the GLM of SAS v9.4. Significant treatment differences were observed for EAA concentrations (P = 0.03) along with significant day effects (P < 0.0001). Treatment-by-day interactions were not observed (P = 0.98). Overall, TE cells cultured with 0.25× EAA medium had decreased growth compared with those cultured with to 0.5× (P = 0.006), 1.0× (P = 0.03), and 1.5× (P = 0.03). Gene expression among EAA concentrations did not differ for the mTOR pathway substrate eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1; P > 0.05). However, Unc-51-like autophagy activating kinase 1 (ULK1), an enzyme involved in autophagy in response to amino acid withdrawal, was significantly lower in cells cultured with 1.5× EAA than those cultured with 0.25× EAA (P = 0.03). There was no effect of EAA supplementation in the expression of pregnancy-associated glycoprotein 1 (PAG1) or AA and glucose transporters, solute carriers (SLC), SLC3A2, SLC7A1, and SLC7A5 (P > 0.05), except for the glutamate transporter SLC1A1, which was greater in TE cells cultured with 0.25× EAA compared with those cultured with 0.5× (P = 0.008), 1.0× (P = 0.05), or 1.5× (P = 0.03) EAA. These preliminary results demonstrate transcriptional differences in TE cell lines influenced by nutrient availability. Further research is necessary to evaluate the role of individual AA and nutrient signaling during early embryo development to identify if nutritional strategies can mitigate pregnancy loss.

This work was supported by the Wisconsin Dairy Innovation Hub and SciMed Graduate Research Scholars.