Effect of different cryoprotectants, equilibration time, and warming regimens on canine spermatozoa after vitrification using coconut water extender
Boyana Ivanova, Anton Antonov
Abstract: The aim of the present investigation was to evaluate the effect of different cryoprotectants, equilibration time and warming regimens on canine sperm quality after vitrification using coconut water extender. Ten ejaculates were collected separately by digital manipulation from 10 adult dogs. Only the second fraction of the ejaculate was used in this study. It was evaluated about volume, concetration, viability, total and progressive motility, kinetic parameters and morphology, divided into two aliquots and diluted with 2 types of coconut water based extender until final concentration of 100x106 spermatozoa/ml. Base vitrification media (BVM) was prepared using 50% (v/v) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution. Extender A consisted of BVM with addition of soy lecithin and fructose at 1% and 0.25 M sucrose and Extender B consisted of BVM with 20% (v/v) egg yolk and 1% fructose. Both of the extended samples were divided into three aliquots and each of them was processed at different regimens: without equilibration (E0), 5ºC for 30 min (E30) and 5ºC for 60 min (E60) and then vitrified by dropping 33 μl of sperm suspension directly into liquid nitrogen. Sperm pellets were devitrified at least one week later and warming was done at 37ºC or 42ºC for 2 min. Sperm motility parameters were assayed using a computer-aided sperm analysis (CASA) system, viability-by supravital staining technique and morphology parameters were evaluated in Haemacolor® stained semen samples. In conclusion, our results demonstrate that when vitrification and coconut water extender were used, egg yolk as a cryoprotectant, presence of equilibration time of 60 min and warming at 42°C for 2 min provided the best canine sperm quality results.
Keywords: coconut water; dog; semen; vitrification
Citation: Ivanova, B. & Antonov, A. (2023). Effect of different cryoprotectants, equilibration time, and warming regimens on canine spermatozoa after vitrification using coconut water extender. Bulg. J. Agric. Sci., 29(6), 1120–1127.
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| Date published: 2023-12-15
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