Twin births in dairy cattle present challenges for producers, resulting in increased prevalence of health issues for both cows and calves, thereby impacting profitability. Genome-wide association study (GWAS) analyses of twinning rate in Holstein cattle have reported the most significant genomic association with twinning rate in a region containing two strong candidate genes, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone chorionic gonadotropin receptor (LHCGR). Coding-sequence variants of these genes were not associated with twinning rate, suggesting that one of the two genes is differentially expressed in association with twinning rate. Granulosa cells were collected from 41 Holstein cows selected to provide similar representation of genotypes for the twinning rate quantitative trait locus (QTL). RNA was extracted from granulosa cells and gene expression was assessed by quantitative PCR with data analyzed by the ΔΔC_T method. QTL genotype was significantly associated with expression of FSHR but was not associated with LHCGR expression. Increased FSHR expression was associated with increasing copy number of the G allele and thus increased twinning rate.

Data are provided as a spreadsheet including quantitative PCR cycle threshold (Ct) values for target genes, FSHR and LHCGR, and reference gene, SF3A1. Imputed SNP genotypes ("CC", "CG" or "GG") for the chromosome 11 twinning rate QTL are provided for each sample.

Cows sampled underwent estrus synchronization for the first insemination following calving using a Double-Ovsynch protocol [21]. Six days after timed artificial insemination (TAI), a dose of gonadotropin-releasing hormone (GnRH) was administered to the cows to induce the luteinization of dominant follicle(s) from the first follicular wave following breeding and initiate a new follicular wave by release of follicle-stimulating hormone (FSH). Consequently, dominant follicle(s) from the new follicular wave developed in a hormonal background of higher progesterone levels, mimicking follicular development during the ovulatory follicular wave. Dominant follicles were aspirated seven days after GnRH administration to collect granulosa cells for RNA extraction and subsequent gene expression analyses. RNAlater was added to the cell pellet (granulosa cells) to preserve RNA. qPCR data were analyzed using the ΔΔC_T method to quantify relative expression of FSHR and LHCGR normalized to the reference gene SF3A1 and to calculate fold‑change differences among QTL genotypes. ΔC_T values were linearly transformed (2^-ΔC_T) to generate gene expression values for statistical analyses. Differences among twinning QTL genotypes (CC, CG, GG) were assessed using the Kruskal–Wallis test. Fold‑change values were computed as the ratio of mean expression between genotypes.