Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extende...Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 ℃ for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 ℃ and then frozen to -196 ℃ in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 × ) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.展开更多
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 c...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.展开更多
Objective:To investigate the effects of non-permeable cryoprotectant,cholesterol-loaded cyclodextrin,when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg y...Objective:To investigate the effects of non-permeable cryoprotectant,cholesterol-loaded cyclodextrin,when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders.Methods:A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina.Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations[0(control),0.75,1.50,2.25 and 3.00 mg/mL]of cholesterol-loaded cyclodextrin.The extended semen samples were cooled to 5曟and then frozen slowly to-196曟in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation.Sperm motility,live sperm,normal sperm morphology,sperm membrane integrity and acrosome morphology were measured.Results:Supplementation of cholesterol-loaded cyclodextrin improved progressive motility,viability,morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre-and post-freezing process(P<0.01).The best concentration was 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.However,greater concentration(3.00 mg/mL)of cholesterol-loaded cyclodextrin had a detrimental effect compared to the control group with the two evaluated extenders(P<0.01).Conclusions:Cholesterol-loaded cyclodextrin supplementation at 1.50-2.25 mg/mL concentration could improve pre-frozen and post-thawed buffalo sperm quality.The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.展开更多
文摘Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 ℃ for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 ℃ and then frozen to -196 ℃ in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 × ) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.
文摘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.
文摘Objective:To investigate the effects of non-permeable cryoprotectant,cholesterol-loaded cyclodextrin,when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders.Methods:A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina.Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations[0(control),0.75,1.50,2.25 and 3.00 mg/mL]of cholesterol-loaded cyclodextrin.The extended semen samples were cooled to 5曟and then frozen slowly to-196曟in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation.Sperm motility,live sperm,normal sperm morphology,sperm membrane integrity and acrosome morphology were measured.Results:Supplementation of cholesterol-loaded cyclodextrin improved progressive motility,viability,morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre-and post-freezing process(P<0.01).The best concentration was 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.However,greater concentration(3.00 mg/mL)of cholesterol-loaded cyclodextrin had a detrimental effect compared to the control group with the two evaluated extenders(P<0.01).Conclusions:Cholesterol-loaded cyclodextrin supplementation at 1.50-2.25 mg/mL concentration could improve pre-frozen and post-thawed buffalo sperm quality.The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.
