Post‐cooling semen processing and sperm re‐suspension as an alternative method to circumvent poor semen cooling in stallions

Background Artificial insemination with cooled‐shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. Objective To assess sperm quality parameters and fertility of cooled‐stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. Study design Controlled and field study. Methods In Experiment 1, semen was collected from 21 stallions classified as having good (‘ Good‐coolers ’, n = 8) or poor (‘ Bad‐coolers ’, n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk‐based (SM) diluent, and refrigerated for 24 h. Then, the cooled‐stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM‐P), or an egg yolk‐based (EY) extender. Unprocessed cooled‐stored 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. Experiment 2, cooled semen from 9 stallions classified as Bad‐cooler s was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM‐P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. Results Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM‐P ( p < 0.0001). Semen processed by SpermFilter® resuspended in SM‐P was similar to EY ( p > 0.05). Pellet resuspension with EY and SM‐P improved the HMMP of Bad‐cooler stallions ( p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen ( p < 0.0001). Mares inseminated with SpermFilter®/SM‐P (50%, 8/16) or SpermFilter®/‐EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) ( p < 0.001). Main limitations Low number of mares in the fertility trial. Conclusion Sperm quality and fertility of Bad‐cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM‐P.