Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Abstract

Effect of supplementation of ascorbic acid, caffeine and chloroquine diphosphate in the dilutor on post-thaw quality of spermatozoa was studied. Thirty-two semen ejaculates (8 each from 2 Holstein Friesian and 2 crossbred; 50% Friesian x 50% Haryana) were studied. Ascorbic acid, caffeine and chloroquine diphosphate were added in Tris-egg yolk-glycerol (control) extender at the concentration of 10 mM, 0.54 mM and 7 mM, respectively. The post-thaw semen parameters studied were structural (acrosomal integrity and livability), functional [motility, per cent sperm responding to hypo-osmotic swelling test, i.e. HOS positive sperm and penetrability of sperm in bovine cervical mucus, i.e. sperm penetration distance-SPD] and measurement of activities of Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), Alkaline transaminase (AKP) and Acid transaminase (ACP). Certain enzymes in the seminal plasma. The semen from crossbred bull had significantly lower mass activity (P £ 0.01), initial progressive motility (P £ 0.01), sperm penetration distance (SPD value) (P £ 0.01), HOS positive sperm and seminal plasma GOT activity, but higher abnormal sperm. Incorporation of additives in the dilutor significantly improved the post-thaw semen quality, especially the post-thaw motility, post-thaw per cent live sperm, sperm penetration distance and sperm responding to hypo-osmotic solution. The leakage of all the 4 enzymes (GOT, GPT, ACP and AKP) was significantly less in the samples frozen in presence of ascorbic acid and chloroquine as compared to control. Ascorbic acid resulted in maximum improvement to the quality of frozen-thawed semen followed by chloroquine and caffeine. The study concludes that post-thaw quality of frozen semen of HF and crossbred bulls significantly improves if the dilutor is supplemented with 10 mM concentration of ascorbic acid.