(326) High A4 Cows are Associated with a Novel FSHR SNP

Mackenzie D. Stohlmann¹; Brooke E. Rudloff¹; Rachel R. Reith¹; Corrine F. Monaco¹; Ashlyn L. Fread¹; Scott G. Kurz¹; Andrea S. Cupp¹ 

1. Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE, 68583-0908, USA

Abstract Text:

A population of females in the University of Nebraska-Lincoln research herd exhibit naturally occurring androgen excess (androstenedione; A4; High A4) in the follicular fluid of dominant follicles. These High A4 cows are often anovulatory and have a 17% reduction in calving rate. Whole genome sequencing (WGS) of 35 animals that are High A4 or control revealed 22 single nucleotide polymorphisms (SNPs) in the Follicle Stimulating Hormone Receptor (FSHR) gene. Several of these FSHR SNPs tend (Chi-Square test, p=0.05) to segregate into the High A4 population, including a novel variant designated as FSHR SNP2 ((chr11:31404255G >C (rs21971504)). We also have conducted DNA genotyping with the Illumina BovineSNP50 v2 or GGP Bovine 100K SNP panels but the FSHR SNP2 is not on either of these genotyping arrays. The FSHR SNP2 was chosen because it was associated with our heifer pubertal classifications that had delayed or discontinuous/irregular cyclicity puberty compared to typical or early puberty classifications. Originally, we had developed these puberty classifications to determine if how a heifer attained puberty may affect whether they developed androgen excess or the High A4 phenotype. Women with androgen excess or polycystic ovary syndrome (PCOS) often have delayed or irregular cyclicity. Thus, we hypothesized that High A4 cows will have one or two copies of the novel FSHR SNP2, allowing it to be utilized to identify the High A4 cow population in our herd. To test this hypothesis, the DNA from heifers born between 2017 and 2023 was extracted from buffy coat on weekly blood plasma samples (total n=72; Control=32, High A4=40). Because this FSHR SNP2 is not on either genotyping array that was used, we used Kompetative Allele Specific PCR (KASP), an assay utilizing primers made of two competing alleles based on the provided SNP sequence, to determine FSHR SNP2 genotypes in these 72 females. These samples were amplified using PCR. Positive and negative controls from heifer DNA (with known haplotypes from WGS) were used on each plate as quality and experimental controls. High A4 status was determined after ovariectomy for each animal, based on the amount of A4 in the medium of ovarian cortex cultures (High A4 being > 1ng/ml/day average over 7 days of culture). A Chi-Square test was used to test if there was significant association between two categorical variables (A4 Status and FSHR SNP2 genotype). There was a higher frequency (p=0.02) of High A4 females possessing one or two copies of the alternate FSHR SNP2 compared to the control group. The position of FSHR SNP2 is in a region that can be epigenetically regulated and may serve as an enhancer of transcription factors that are downstream of FSHR. At this point, we are uncertain what those gene(s) may be but a gene downstream of FSHR is Luteinizing Hormone Chorionic Gonadotropin Receptor (LHCGR). The abundance of mRNA for LHCGR was increased in both theca and granulosa cells of High A4 cows, so this is a potential target to be evaluated. Ultimately, FSHR SNP2 could serve as a potential marker for identifying High A4 animals, which could be used to select against them in order to increase the reproductive efficiency of the herd.