Comparison between Low/Programmable Freezing and Fast Freezing Protocols of Hungarian Guinea Fowl Semen

Semen cryopreservation is a practical method for banking germplasm from valuable indigenous species which have an increasing risk of extinction. Many scientific publications have described different protocols of avian sperm cryopreservation for both domesticated and non-domesticated avian species, involving cryoprotectant type and packaging method, as well as freezing and thawing rates. In this research, a comparative approach was used to evaluate two freezing protocols for guinea fowls: the modified slow programmable method using 10% ethylene glycol (EG), and the newly applied fast freezing method (pellet formation) with 6% dimethylacetamide (DMA). The efficiency of two protocols is measured by both in vitro sperm evaluation assay (determination of sperm concentration and motility, morphological and live/dead sperm analysis) and in vivo sperm evaluation assay (artificial insemination, determination of fertility rate and embryonic death). For fertility determination, candling of incubated eggs was used, extended by checking of the ratio of early embryonic mortality. In vitro qualification showed that the survival rate of live and intact spermatozoa was significantly higher after pelletation than after slow protocol (28.59% vs 23.53%, p=0.022, Mann-Whitney U test). In vivo qualification showed that artificial insemination of frozenthawed semen yielded 50.2% fertility rate with the pellet, while only 25.5% with the slow programmable method (while it was 84.8% in control, p=0.027, Kruskal-Wallis ANOVA test). The simple in vitro examinations used in this research were not able to detect injuries which caused embryonic death during freezing procedure, therefore, despite the seemingly acceptable sperm surviving rate, the ratio of eggs containing a normal embryo in case of slow, programmable frozen-thawed semen artificial insemination was significantly lower than in case of fresh semen artificial insemination (5.88% and 70.46%, while it was 28.37% in case of pellets, p=0.038, Kruskal Wallis ANOVA test).