Effect of different concentrations of Orvus ES Paste(OEP) and egg-yolk on quality of frozen-thawed spermatozoa in the blue fox Alopex lagopus

摘要

Six high quality male blue foxes from Finland were chosen for collection of semen by digital manipulation and semen samples were mixed and divided into three fractions which were diluted with the first cryopreservative of Tris-fructose-citrate supplemented with different concentration of egg yolk (0%, 10%, 20% v/v). After being equilibrated for 2 h at 5 ℃ , the three fractions diluted semen were divided into four sub-fractions that were added same volume second cryopreservative supplemented with different concentration of OEP (0%, 0.5%, 1%, 2% v/v), respectively. Semen samples were packed in 0.5 ml straws and frozen on a rack at 5 cm above liquid nitrogen and thawed at 70 ℃ for 9 s.Ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM) and quality of frozen-thawed spermatozoa was evaluated in different culture time by fluorescent immunoassay. The results showed that the thickness of two layers of outer acrosome membrane was 0.020 m m and the space between inner acrosome membrane and nuclear membrane was 0.015 m m. The acrosome vesicles were divided into two types during frozen-thawed. In the first type, vesicles were hollow and the average diameter of the vesicle was 1.25 m m hat which was bigger than the second type of vesicles. In the second type, vesicles were solid and full of acrosomal content and its average diameter was 0.83 m m, vesicle numbers were indeterninate. Acrosomal vesiculation was inhibited and types, bulks and numbers of acrosome vesicles were influenced by OEP obviously. Vesicle bulks, total numbers of vesicles and numbers of hollowed vesicles were reduced by appropriate OEP. Quality of frozen-thawed spermatozoa was correlated with doses of OEP and egg yolk. Once supplemented with egg yolk, OEP helped to maintain the integration of plasma membrane(56.3%)and acrosome(57.8%), to improve semen motility obviously(54.7%). The optimal concentrations of egg yolk and OEP were 20% and 1% in cryopreservative of blue fox spermatozoa [ Acta Zoologica Sinica 50(4): 583 - 592 , 2004].