Effect of carboxylated poly l-Lysine as a cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull semen

Abstract

Buffalo bull sperm are more prone to cryo-injuries. Glycerol being the most common permeable cryoprotectant exerts cytotoxic effects on sperm which cause a reduction in fertility. Thus, the exploration of new cryoprotectant is needed. For this purpose, we investigated the effect of carboxylated poly l-Lysine (CPLL) as cryoprotectant used with different concentrations of glycerol on post-thaw sperm motility, kinematics, plasma membrane integrity, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), catalase concentration and in vivo fertility of Nili Ravi buffalo bull semen. In experiment 1, semen samples (n = 15, bulls = 3) were diluted with Tris-citrate-egg yolk extender containing different concentration of CPLL [0% (C0), 0.25% (C0.25), 0.5% (C0.5), 0.75% (C0.75), 1% (C1)]. Each concentration of CPLL was added in extender containing either 7% (G7) or 5% (G5) glycerol. Diluted semen samples were cooled and cryopreserved using standard procedures. Post-thaw total and progressive motility, plasma membrane integrity, acrosome integrity, and MMP were found higher (P < 0.05) in group (G5C0.75) containing 0.75% CPLL and 5% glycerol as compared to the control group (G7C0) and other groups while LPO was recorded lower (P < 0.05) in the same group (G5C0.75). In experiment 2, in vivo fertility was compared between G5C0.75 (5% Glycerol+ 0.75% CPLL; depicted better post-thaw quality) and control group G7C0. Buffaloes were inseminated after 24 h of onset of estrus. Pregnancy diagnosis was performed per rectum at least 60 days post insemination. The fertility rates [56% (58/102) vs. 36% (37/103)] were higher (P < 0.05) in G5C0.75 as compared to the control group G7C0. Based upon these results, this study concludes that the addition of 0.75% CPLL in combination with 5% glycerol in freezing extender improves the post-thaw structure, function and in vivo fertility of Nili Ravi buffalo bull semen.

Keywords: Buffalo semen; Carboxylated Poly l-Lysine; Cryopreservation; Fertility; Post-thaw semen quality.