Effect of extender on motility and sperm DNA fragmentation of stallion and jack semen after storage at 4°C

Abstract

Cooled semen is widely used, it has become part of the routine breeding management, achieving higher fertility rates than frozen semen (Pagl et al. Theriogenology. 2006; 66:1115-1122). During storage, sperms undergo oxidative stress, leading to an impair in semen quality, affecting sperm functionality (Del Prete et al. Animal Reproduction Science. 2019; 210:106195). Sperm DNA fragmentation (sDF) is a parameter that affects the fertilizing capacity and adequate embryo development. It doesn't fully correlate with conventional parameters, so its evaluation gives a completer and more objective estimate about the stallion fertility (López-Fernández et al. Theriogenology. 2007; 68:1240-1250). There is little information about the sperm DNA quality in jacks. The aim of this study was to assess the total (TM) and progressive (PM) motility, through SCA^Ⓡ, and sDF by the SCD test using the Halomax Kit^Ⓡ Equus-halomax HT-EC40. Two ejaculates from three stallions and three jacks (4 to 13 years) of proven fertility, located at the Equine Reproduction Center of the UNAM, Tequisquiapan, Querétaro, were included in the study. Semen collection was performed with a Hannover type artificial vaginal during July, all ejaculates contained a minimum of 60% progressive motile sperms. Immediately after collection, semen was divided and diluted with INRA 96 (IMV Technologies, L'Aigle, France) or Botusemen (Botupharma^Ⓡ, Botucatu, SP, Brazil) to obtain a final concentration of 50 × 10⁶ sperms/ml and storage at 4°C. Motility and sDF was evaluated at 0, 12, 24 and 36 hours of storage. Statistical analyses were performed using the binomial distribution and the logit link function. The model included extender, species, and time, as well as double interactions and triple interactions. The Bonferroni method was used for multiple comparisons. The significance level was set at P<0.05. Sperm motility decrease and sDF increase during storage at 4°C in stallions and jacks sperms extended either in INRA 96 or Botusemen. There are no differential effects in TM or sDF between extenders in any of the species, however, PM on stallion semen was affected depending on the extender used, being better with INRA 96. It is likely that the similarity of the results between extenders is due to the similar composition between these skimmed milk-based extenders. Impairment of motility occur faster in jack sperm than in stallion sperm, while sDF is greater in stallion than in jack sperm.