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Examining the ability of molecular methods to differentiate reproductive potentials in Bos taurus heifers




Mahoney, Erin

Type of Degree

Master's Thesis


Animal Sciences

Restriction Status


Restriction Type

Auburn University Users

Date Available



Informative selection of replacement heifers remains a challenge in cow-calf production. Traditional phenotypic methods are currently used to identify heifers with a high probability of conceiving early in their first breeding season. These parameters include body weight, age, body condition score (BCS) and reproductive tract scoring (RTS). Unfortunately, these metrics are unable to comprehensively detect all sub-fertile heifers. Reproductive technologies are advancing and increasing the success of beef production breeding seasons; however, a need remains for a more accurate selection tool for producers to use that would accurately predict the fertility status of a replacement heifer at an early timepoint in her life. The objective of this study was to identify molecular differences, at weaning, in heifers with different pregnancy outcomes following estrous synchronization (ES) and artificial insemination (AI). In this study, we compared age and weight, amino acid profiles, and microRNAomes between heifers with differing reproductive outcomes following the first service AI. At weaning, blood samples were collected and processed for plasma and peripheral white blood cell (PWBC) extraction from Angus/Simmental cross heifers. Samples were collected across two breeding seasons (2017-2018, 2020-2021) and at three different research locations, the Black Belt Research and Extension Center (BBREC) of the Alabama Agricultural Experiment Station in Marion Junction, AL, U.S.A., the Wiregrass Research and Extension Center (WREC) in Headland, AL, U.S.A. and the College of Veterinary Medicine North Auburn Beef Unit (NABU) in Auburn, AL, U.S.A. Also, at this timepoint, heifers were evaluated for their adjusted weaning weights and age. Heifers then underwent a fixed-time AI program at approximately 14 months of age followed by pregnancy status evaluation via ultrasound. All heifers used in this study were evaluated for uniformity in age and body condition before admission into the study. Blood plasma from NABU (AI-Pregnant N = 18, open N = 28) was analyzed using ultra-performance liquid chromatography to generate amino acid profiles at weaning. The blood plasma from all three research stations (AI-Pregnant N = 9, open N = 6) was then analyzed for target microRNAs related to inflammation and fertility. Next, small RNA was isolated from the PWBC (AI-Pregnant N = 7, open N = 7) collected from BBREC at the time of weaning and sequenced for miRNA profiles and the most significant miRNA was cross validated in the blood plasma. Amino acid profiles were used to find differences between heifers that conceived to first service AI and those that remained open after AI. Two of the 20 amino acids were significantly different between the two groups (p < 0.05). Histidine (p = 0.023) and Cystine (p = 0.034) were both significantly upregulated in heifers that conceived to AI. For miRNA RT-qPCR analysis and validation of several miRNAs, bta-miR-125b, bta-miR-145, and bta-miR-26a-5p were targeted. Difference in the expression levels of bta-miR-125b, bta-miR-145, and bta-miR-26a-5p were non-significant (p > 0.05) between the two groups. Small RNA sequencing results showed 11 and 5 miRNAs significantly downregulated (p < 0.05) in heifers that remained open and conceived to AI respectively. Among 16 differentially expressed miRNAs, the top significant miRNA was bta-miR-92b (padj = 0.005). Bta-miR-92b was selected for further validation in the blood plasma and showed no significant difference (p > 0.05) between the two groups. In summary, histidine, cystine, and bta-miR-92b in the PWBC showed differences between heifers with differing reproductive outcomes at the time of weaning. Validation of these results in more samples and different time points would help to build a framework to improve the reproductive efficiency. Furthermore, validation of these amino acids and miRNAs in reproductive tissues and cells would help to identify the therapeutic targets for fertility.