An alternative method to circumvent poor semen cooling and fertility of stallions

Abstract

Cooled-shipped semen is routinely used in the equine breeding industry. Although most stallions present satisfactory semen quality and fertility after cooling and transportation, a population of stallions offer a suboptimal sperm cooling tolerance even when strategies to enhance stallion semen cooling are applied. Therefore, it is critical to developing alternative methods to improve the post-cooling semen quality from this population of stallions. This study assessed sperm parameters and fertility of cooled-stored stallion semen processed and resuspended in three extenders. In experiment 1, semen was collected from 21 stallions classified as having good (“Good coolers,” n=8) or poor (“Bad coolers,” n=13) sperm characteristics during cooled-storage after semen dilution in milk-based extender (SM, BotuSemen®). Immediately after semen collection, semen was extended at 50 million/mL in SM extender and stored at 5°C for 24h. Thereafter, cooled-stored semen was processed by SpermFilter® or conventional centrifugation (600 × g/10 min), and the resulting pellets were resuspended in SM, SM containing pentoxifylline (SM-P, BotuSemen Turbo®), or egg yolk-based extender containing cryoprotectants (EY, BotuCrio®). Unprocessed cooled semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. In experiment 2, 66 cooled semen doses from stallions (n=9) classified as Bad coolers were randomly used to inseminate18 mares in 66 cycles (Unprocessed, n=22; SpermFilter®/SM-P, n=16; or SpermFilter®/EY, n=28 cycles). Data were analyzed with a mixed model, Tukey's, and logistic regression. Overall, resuspending centrifuged (Total motility [TM], 73±14%; progressive motility [PM], 36±13%) or filtered (TM, 75±15%; PM, 37±13%) semen in EY enhanced (P<0.05) sperm motility compared to unprocessed semen (TM, 50±33%; PM, 23±18%) and the other groups. However, when semen from only Bad Coolers was analyzed, motility of semen processed by SpermFilter® and resuspended in SM-P was enhanced (TM, 52±19%; PM, 22±14%) compared to unprocessed semen (TM, 30±26%; PM, 10±10%) and similar (P>0.05) to semen resuspended in EY (TM, 69±16%; PM, 34±14%). Pellet resuspension with EY (52±25%) and SM-P (49±25%) improved the HMMP of Bad cooler stallions (P<0.05) compared to unprocessed semen. Semen processed by SpermFilter® had superior PMI (72±8%) than centrifuged semen (64±11%; P<0.05) overall. In experiment 2, pregnancy rate of mares inseminated with SpermFilter®/SM-P (50%) or SpermFilter®/-EY (68%) was greater than with unprocessed semen (14%) (P<0.05). In conclusion, semen parameters and fertility of stallions with poor semen cooling can be enhanced by SpermFilter® and resuspension of the pellets with EY or SM-P. Acknowledgments: The authors thank FAPESP (grant #2017/13883-9) for the financial support.