Thoroughbred stallion fertility is significantly associated with FKBP6 genotype but not with inbreeding or the contribution of a leading sire.

This is a follow-up study to validate the previously detected association of the FKBP6 gene with stallion subfertility. Using a select cohort of 150 Thoroughbred stallions with detailed breeding records, we confirm significant association (P < 0.0001) between low per-cycle pregnancy rates (≤50%) and a combined A/A-A/A genotype of SNPs chr13:11 353 372G>A and chr13:11 353 436A>C in FKBP6 exon 5. We also show that stallion subfertility and the combined genotype A/A-A/A are not associated with the level of genetic diversity based on 12 autosomal microsatellite markers, or with pedigree-based inbreeding rate, or the extent of contribution of a leading Thoroughbred sire, Northern Dancer, in a stallion's pedigree. We develop a TaqMan allelic discrimination assay for the two SNPs to facilitate accurate and high-throughput genotyping. We determine allele, genotype and combined genotype frequencies of FKBP6 exon 5 SNPs in a global cohort of 518 Thoroughbreds (76% stallions or geldings and 24% mares) and show that the frequency of the A/A-A/A genotype is 4%. Because there is no similar association between the FKBP6 exon 5 genotype and stallion subfertility in Hanoverians, we suggest that the two SNPs are not causative but rather tagging a breed-specific haplotype with genetic variants unique to Thoroughbreds. Further WGS-based research is needed to identify the molecular causes underlying the observed genotype-phenotype association in Thoroughbred stallions.

The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen.

Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (P < 0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706 ±â€¯244) and MERO (1576 ±â€¯1076) treatment groups than in TICAR-CLAV (4678 ±â€¯1388) or PIP-TAZ (8108 ±â€¯3198) treatment groups (P < 0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478 ±â€¯4374; P < 0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; P < 0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0-40% and 0-15% for K. pneumonia and P. aeruginosa, respectively; P < 0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen.