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. 2022 Dec;35(12):1827-1838.
doi: 10.5713/ab.21.0504. Epub 2022 Mar 3.

Genome-wide association study for frozen-thawed sperm motility in stallions across various horse breeds

Elena V Nikitkina  1 , Natalia V Dementieva  1 , Yuri S Shcherbakov  1 , Mikhail M Atroshchenko  2 , Andrei A Kudinov  1 , Oleg I Samoylov  1 , Marina V Pozovnikova  1 , Artem P Dysin  1 , Anna A Krutikova  1 , Artem A Musidray  1 , Olga V Mitrofanova  1 , Kirill V Plemyashov  1 , Darren K Griffin  3 , Michael N Romanov  3
Affiliations

Genome-wide association study for frozen-thawed sperm motility in stallions across various horse breeds

Elena V Nikitkina et al. Anim Biosci. 2022 Dec.

Abstract

Objective: The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds.

Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom Equine Genotyping Array.

Results: We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds. As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME/NM23 family member 8 (NME8), olfactory receptor family 2 subfamily AP member 1 (OR2AP1), and olfactory receptor family 6 subfamily C member 4 (OR6C4). Potential implications of effects of these genes on sperm motility are herein discussed.

Conclusion: The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system.

Keywords: Candidate Genes; Cryopreservation; Equus caballus; Single Nucleotide Polymorphisms (SNPs); Sperm; Stallion.

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Conflict of interest statement

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Figures

Figure 1
Figure 1
Examples of individual stallions used in the present genome-wide association study for frozen-thawed sperm motility: (A) Khitmos, of the Trakehner breed, dark bay color; (B) Santrek, of the Trakehner breed, bay color; (C) Logotip, of the Orlov Trotter, black color; (D) Vympel, of the Orlov Trotter, black color; and (E) Zhasmin, of the Arabian breed, white color.
Figure 2
Figure 2
Example of the computer-assisted semen analysis (CASA) using a stallion semen sample. Sperm tracks generated by CASA: PR, progressive (green) tracks of individual sperms; NP, non-progressive (blue) tracks; and IM, immotile (or dead) sperm (red dots).
Figure 3
Figure 3
Quality parameters in stallion semen samples before and after freezing: (A) PM, fresh sperm progressive motility; (B) FPM, post-thaw forward progressive motility; and (C) DPM, difference between PM and FPM values. Breeds grouped: Heavy draft – Russian Heavy Draft, Soviet Heavy Draft; Purebreds – Akhal-Teke, Arabian, Bashkir, Budyonny, Hanoverian, Karabakh, Rhenish German Coldblood, Tersk, and Thoroughbred; Trotters – French Trotter, and Standardbred; and Warmblooded – Holsteiner, Orlov Trotter, Russian Riding, Trakehner, and Welsh Pony.
Figure 4
Figure 4
Principal component analysis (PCA) plots based on the sperm progressive motility data (A and C) and correlations between PC1 and tested factors (B). For (A) and (B), three factors were used: progressive motility (PM), post-thaw forward progressive motility (FPM), and difference (DPM) between PM and FPM values. For (C), the breed factor was also tested as the fourth eigenvector. Breeds analyzed: Akhal-Teke, Arabian, Bashkir, Budyonny, Holsteiner, Karabakh, Rhenish, Selle Français, Standardbred, Tersk, Thoroughbred, Trakehner, Welsh Pony; and breed group: Heavy draft (Russian Heavy Draft + Soviet Heavy Draft).
Figure 5
Figure 5
Manhattan (A) and quantile-quantile (Q-Q; B) plots resulted from the genome-wide association study for sperm DPM. Blue line corresponds to the threshold of chromosome-wide suggestive levels for a SNP effect (p<3.262409e-6), and red line represents the threshold of genome-wide significance (p<1.631204e-7). The Q-Q plot (B) shows the observed p-value plotted against the expected one. PM, progressive motility; FPM, post-thaw forward progressive motility; DPM, difference between PM and FPM values; SNP, single nucleotide polymorphism.
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