Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis)

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, regucalcin is reportedly a putative cold-tolerance gene, and a cryoprotective role for regucalcin against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN is abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 µg/ml), as a supplement for Tris-citric acid-fructose-egg yolk-glycerol semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 µg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% versus 40.6 ± 2.6%; P<0.01), acrosome integrity (53.3 ± 7.4 versus 75.6 ± 6.8; P<0.05), and zona pellucida binding (31.6 ± 14.0 versus 191.9 ± 12.3 bound spermatozoa; P<0.01) compared to control conditions without RGN. Thus ∼1 µM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender containing Tris, citric acid, fructose, egg yolk, and glycerol. This article is protected by copyright. All rights reserved