Effect of Cooling Rates on Cryopreserved Hariana Bull Spermatozoa

This experiment was designed to study the effect cooling rate in Hariana bull spermatozoa during cryopreservation. For this purpose, ejaculates were collected from four Hariana bulls using artificial vagina at biweekly interval. The semen sample which possesses more than 70% progressive motility and above 500 million/ml spermatozoa concentration was subsequently subjected to processing for liquid nitrogen (LN₂) vapour freezing. Semen samples were extended in Tris-egg yolk-glycerol extender and split into 3 parts. These split samples were subjected to cooling from 35°C to 4°C temperature at 2.21^°C/min (rapid), 0.48^°C/min (moderate) and 0.25^°C/min (slow cooling). Each cooled part was than subjected to LN₂ vapour freezing in a programmable biological freezer followed by plunging into LN₂. Samples were evaluated at pre-freeze and post-thaw stages for per cent live spermatozoa, per cent progressively motile spermatozoa, per cent Hypo-osmotic swelling test (HOST) positive spermatozoa and percent spermatozoa with intact acrosome by Fluorescein isothiocyanate-pisum sativum agglutinin (FITC-PSA) method. Maximum damage of cooling was observed in rapid cooling whereas damage found to be minimal in slow cooling protocols.