Cryopreservation of boar semen with special emphasis on cryodamage and its mitigation

Abstract

A total of 92 sperm-rich fraction of ejaculates were collected from two boars each newlineof Hampshire (HS), Hampshire x Khasi Local (HS x KL) with 87.5 per cent exotic newlineinheritance and HS x KL with 75 per cent exotic inheritance maintained at Livestock newlineResearch Farm, Division of Livestock Production, ICAR Research Complex for NEH newlineRegion, Umiam, Meghalaya by gloved/bared hand technique once or twice in a week. The newlineseminal ejaculates thus obtained were frozen in 0.5 ml straws in liquid nitrogen using 3 newlineper cent glycerol in BTSLEYG extender allowing 3 hours holding time and 1 hour newlineequilibration period to study the quality of semen before and after freezing to find out the newlineextent of cryodamage which was sought to be mitigated by resorting to different freezing newlinemethods, addition of different antioxidants, and incorporation of cholesterol-loaded newlineMethyl-and#946;-cyclodextrin (CLC) either alone or in combination with antioxidant in the newlineextender. newlineSixty ejaculates were used to study the effect of different freezing methods. Semen newlinewas frozen either by conventional method or by controlled freezing method adopting newlinecooling at 3and#730;C/min from 5 to and#8722;6and#730;C, 1 minute holding at and#8722;6and#730;C and then freezing either at newline20and#730;C, 40and#730;C or 60and#730;C/min from -6and#730;C to -140and#730;C before plunging into liquid nitrogen. Equal newlinenumber of ejaculates were used in different freezing methods. newlineSixteen ejaculates were subjected to study the effect of supplementation of newlineGlutathione (GSH, 1 mM), Vitamin E (Trolox, 0.2 mM) and Butylated hydroxytoluene newline(BHT, 0.2 mM) as antioxidants in the first fraction of the extender as compared to without newlinesupplementation. Another sixteen ejaculates were used to find out effect of addition of newlineCLC either alone or in combination with BHT in the extender. In both the experiments newlinesplit sample technique of the ejaculates was followed. Quality of semen was estimated by newlineevaluating the following sperm parameters: motile sperm by subjective method, live sperm newlineby Eosin- Nigrosin staining, live intact acrosome by Nigrosin-Eosin-Giemsa staining, newlineplasma membrane intact s