|dc.description.abstract||The development of biomarkers for the identification of infertile heifers has the potential to improve the efficiency of cow-calf production. In this study, we utilized metabolomic profiling to look for biomarkers in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Angus and Angus-cross heifers, undergoing a 7 - day estrous synchronization protocol, were utilized for analysis. The heifers were housed at three separate Research and Extension Centers (Black Belt, Gulf Coast, and Wiregrass) located across the state of Alabama, U.S.A. and compared across two breeding seasons (2015 – 2016 and 2016 – 2017).
We compiled data on a total of 166 heifers (N = 166) from three locations across two breeding seasons for phenotypic parameters including body condition score (BCS), weight at time of weaning, reproductive tract score (RTS), and age at AI to determine the utility of using “traditional” heifer assessment metrics to predict reproductive outcome. These phenotypic parameters proved to not be significantly different (p > 0.05 for BCS, RTS, Weight at Weaning, and Age at AI) in heifers undergoing fixed-time artificial insemination that became pregnant by AI or those remaining open. These parameters also proved to not be significant (except for BCS across locations) when compared across two separate breeding seasons, three separate locations, and based on pregnancy outcome.
Heifers were compared to determine if there were metabolomic profile differences between heifers housed at two separate Research and Extension Centers [Black Belt (N = 8) and Wiregrass (N = 8)] across two breeding seasons (2015 – 2016 and 2016 – 2017). Analysis revealed six metabolites present at differential levels (T-test; p < 0.05; fold change > 2, FDR = 0.05) between Black Belt heifers and Wiregrass heifers. Tocopherol-alpha, Ornithine, Myristic Acid, P-tolyl Glucuronide, Sulfuric Acid, and Alpha-ketoglutarate were all found to be differentially expressed by at least 2-fold in Black Belt heifer’s blood plasma compared to Wiregrass heifer’s blood plasma.
In a second study, a total of N = 20 heifers were analyzed for phenotypic heifer assessment, as well as metabolomic profiling, to identify metabolite differences in infertile (open) heifer blood plasma compared to fertile (AI) heifer blood plasma. Heifers were deemed fertile (AI) if they conceived from the artificial insemination following estrous synchronization. Heifers were deemed infertile if they did not maintain a conceptus following estrous synchronization, fixed-time AI, and three consecutive estrus cycles in the presence of a fertile bull. Phenotypic parameters were determined to not be significantly different (p > 0.05 for BCS, RTS, Weight at Weaning, and Age at AI) between categorized fertile and infertile heifers.
Metabolomics profiles of N = 20 heifers revealed seven metabolites present at different levels (T-test; p < 0.05; fold change > 2; FDR = 0.05) between infertile and fertile heifers. Tryptophan, Cystine, Histidine, Ornithine, Asparagine, Glutamine, and Lysine were all found to be at least 2-fold less in the infertile heifer’s blood plasma compared to the fertile heifer’s blood plasma. We further characterized the utility of using the levels of these metabolites in the blood plasma to discriminate between fertile and infertile heifers. In order to identify the predictive ability of the significant metabolites discovered (P < 0.05; >2-fold change), we calculated the Receiver Operating Characteristic (ROC) area under the curve (AUC) value. We tested the models on the blood plasma metabolomes of the 20 selected heifers to determine their ability to predict pregnancy outcomes. Glutamine and Histidine alone, and in combination, predicted the correct pregnancy outcome in 90% of the animals. They did not incorrectly categorize a fertile heifer as infertile.
Finally, we investigated the potential role that inflammation might play by comparing the expression of inflammatory cytokines in the white blood cells of infertile heifers to that of fertile heifers. We found significantly higher expression (p < 0.05) of the proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα), C-X-C Motif Chemokine Ligand 5 (CXCL-5), and Interleukin 6 (IL-6) in infertile heifers when compared to fertile heifers. The study presented offers potentially valuable information regarding the identification of fertility problems in heifers undergoing AI.||en_US