The effect of sex-sorting on stallion semen quality

Abstract

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.