Post thawing motility and morphology of bovine spermatozoa frozen in ethylene glycol without sugar

The study examined the pre-freezing and post thawing motility of bovine spermatozoa frozen in ethylene glycol in the absence of sugar as compared to that of the current method of freezing spermatozoa using glycerol with fructose. Semen samples were collected from a three-year old Holstein Freesian bull with the use of an artificial vagina. The samples were initially examined for fresh motility right after collection. The samples were then divided into control and treatment groups. Semen samples for the control were extended with Tris egg yolk extender containing fructose and 7.37% (IM) glycerol. Samples for the treatments were extended in the Tris-egg yolk extender devoid of sugar with 5.57% (IM) ethylene glycol. Motilities of extended samples were examined. They were then packaged in 0.5 ml French straws which were sealed with polyvinyl chloride powder after which straws were subjected to equilibrium. The control group was equilibrated for four hours in a water bath placed inside a refrigerator. The treatment groups were first cooled in a 20 deg C water bath for 10 minutes. They were first equilibrated in a 4 deg C water bath for 10 minutes (Treatment 2), 30 minutes (Treatment 3) and 1 hour (Treatment 4). Just prior to freezing, a sample from each treatment group as well as the control was taken for evaluation of pre-freezing motility. The control group was subjected to the standard method of freezing (slow freezing) while treatment were first suspended in liquid nitrogen vapor for 2 minutes then submerged in liquid nitrogen. The straws were then thawed in 35 deg C water bath and evaluated for post-thaw motility. Smears of samples for fresh and extended semen, the control and all other treatments were stained using Giemsa and evaluated by counting and classifying abnormalities in 100 cells under oil immersion. This study showed that it is possible to freeze semen with viable post-thaw motility using ethylene glycol without sugar. It established the optimum equilibrium time (30 minutes) for rapid freezing of bovine spermatozoa extended with ethylene glycol devoid of sugar. The use of ethylene glycol as a cryoprotectant, regardless of equilibrium time, was found to show better results that glycerol. Finally, this study also demonstrated that morphology of semen frozen and thawed using ethylene glycol as a cryoprotectant is comparable to fresh semen. The best measure of the efficiency of this alternative technique in rapid freezing of bovine spermatozoa would be the rate of conception, as is being investigated in a concurrent study, since post-thawing motility and morphology may not be enough to confirm its effectiveness.