Evaluation of carboxylated poly l-lysine for cryopreservation of Labeo rohita sperm.

Carboxylated poly l-lysine (CPLL) is an anti-freeze agent having pronounced non-permeating yet membrane stabilizing cryoprotective capabilities. The objective was to evaluate the CPLL supplementation in extender in terms of post thaw quality (sperm), total antioxidant activity (milt) and fertilization potential of cryopreserved Labeo rohita sperm. For this purpose, male brood fish reared at fish seed hatchery, Rawal Town Islamabad, Pakistan were captured from different rearing ponds and acclimatized in hatchery ponds for 6 hours. The brooder were injected with Ovaprim (0.2 ml/kg), milt was collected after 8 hours in cooled sterilized falcon tubes, maintained at 4°C and evaluated for sperm motility. The milt collected from three brooders (n=3) was diluted in extenders viz., modified Kurokura-2 extender having 10% methanol (control); experimental extenders with CPLL supplementation at the rate of 0.5%, 1% and 1.5%. Diluted milt was filled in 0.5ml straws, exposed to liquid nitrogen vapors and cryopreserved. Cryopreserved milt was thawed at 25 C and assessed for post-thaw sperm quality. Sperm motility, motility duration, viability, total antioxidant capacity and DNA integrity was significantly higher (P<0.05) in extender having 1.5% CPLL than control. To evaluate the fertilization rates, male and female brooders were injected with Ovaprim at 0.2ml/Kg and 0.5ml/Kg body weight respectively. Fresh eggs and milt were collected through abdominal stripping. Batches of 10g of eggs from each female (n=2) were fertilized with one straw, each from frozen sperm with KE+methanol (control), KE+methanol+1.5% CPLL and 50 μl fresh milt (negative control). After 1.5 hours of fertilization, eggs were collected from all jars and a total of 200 eggs were counted. The fertilized eggs appeared clear and transparent while unfertilized eggs looked opaque with disintegrated nucleus. Sperm fertilization rate (%) was higher (P<0.05) in extender KE+methanol+1.5%CPLL (78.7±0.5) compared to control (KE+methanol) (52.0±0.4) however, it was lower compared to that of negative control, the fresh milt (85.2±0.6). In conclusion, supplementation of carboxylated poly l-lysine (1.5%) to modified Kurokura-2 extender having 10% methanol improves post thaw motility, motility duration, viability, DNA integrity, anti-oxidant capacity (milt) and fertilizing ability of cryopreserved L. rohita sperm.