Asian Pacific Journal of Reproduction (Jan 2019)
Effect of butylated hydroxytoluene on quality of pre—frozen and frozen buffalo semen
Abstract
Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 °C for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 °C and then frozen to -196 °C in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 ×) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.
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