Influence of sperm filtration and the addition of glycerol to UHT skimmed milk- and TEST-based extenders on the quality and fertilizing capacity of chilled ram sperm

Abstract

The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, μm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.

Keywords: Cold-stored; Filtration; Skim milk; Sperm; TEST.