Impact of replacing egg yolk with lecithin on quality of pre-freeze and post-thaw buffalo spermatozoa

摘要

Objective:To estimate the result of egg yolk replacement with alternative cryopreservatives such as plant-derived lecithin from soybean on sperm quality parameters pre and post freezing in buffalo bulls.Methods: The control cryopreservation extender was tris-citric acid-fructose-egg yolk-glycerol (TCFYG) diluent. Semen samples were extended gradually 1:10 with TCFYG control extender and tris-citric acid-fructose-glycerol (TCFG) extender plus variable concentrations of soybean lecithin (0.5％, 1.0％, 1.5％, 2.0％, 2.5％ and 3.0％) to ensure 60 million active spermatozoa/mL of the extended semen. The diluted semen samples were refrigerated slowly (roughly for 2 h) up to 5℃ and equilibrated for 2 h. Semen was filled into 0.25 mL polyvinyl French straws (IMV, France). After equilibration period, the straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen for 10 min and were then dipped stored in liquid nitrogen at -196℃. Results:The respective overall percentages of forward motile spermatozoa, live spermatozoa, morphologically normal spermatozoa, acrosome integrity and hypo-osmotic swelling reactivity observed primarily in fresh semen, after equilibration (pre-freeze stage) and post freezing (post-thaw stage) in TCFYG (control) extended semen declined progressively and statically (P<0.01) during these periods of study. Pre-freezing stage: replacement of egg yolk into TCFG with soybean lecithin at concentrations of 1.0％ and 1.5％ significantly (P<0.01) ameliorated the maintenance of (motility, viability, acrosome and membrane integrity ％), meanwhile it had significantly (P<0.01) reduced the abnormality ％ of spermatozoa to the lowest value compared to control TCFYG and to some other concentrations in use. Post-thaw stage: the replacement of egg yolk with 1.0％ soybean lecithin (SL) showed significantly (P<0.01) higher percentage of sperm progressive motility compared to 1.5％ SL and TCFYG control. These values were significantly (P<0.01) higher than 0.5％, 2.0％, 2.5％ and 3.0％ SL. The post thawing live sperm percentage mean values were significantly (P<0.01) higher in 1.0％ SL and 1.5％ SL compared to control. These values were significantly (P<0.01) higher than in 0.5％, 2.0％, 2.5％ and 3.0％ SL. The mean values of post-thaw morphological normal sperm percentage did not differ between 1.0％ SL and control groups but significantly (P<0.01) higher than 0.5％, 1.5％, 2.0％, 2.5％ and 3.0％ SL. The respective percentage mean values of post-thaw sperm with head, mid-piece and tail abnormalities were significantly (P<0.01) lower in 1.0％ SL than all other SL concentrations. Concerning the post-thaw percentages of acrosome and sperm membrane integrity, the respective mean values were significantly (P<0.01) higher in 1.0％ SL and 1.5％ SL as compared to control. Mean values of both parameters in the 0.5％ SL were intermediate between 1.0％ and 1.5％ SL versus control groups. The previously mentioned mean values in acrosome/membrane integrity were significantly (P<0.01) higher than 2.0％ SL, 2.5％ SL and 3.0％ SL.Conclusions: Lecithin-based diluent can be a potent proper alternative extender for preservation of spermatozoa during pre- and post-freezing process. SL 1.5％ extenders have supplied an optimal environment and condition for ameliorating the quality of pre-freezing and post-thaw buffalo spermatozoa by means of improved motility, viability, functional acrosome, sperm membrane integrity and morphologically normal spermatozoa.