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Abstract
Four experiments were conducted in the Buffalo Semen Freezing Center, General Organization for Veterinary Services, Ministry of agriculture, Abbasia and National Research Center at Giza, Egypt, to study some factors affecting the freezability of buffalo bull semen.Semen was collected from five mature buffalo bulls by artificial vagina then the experimental diluents were conducted with each different treatment then the semen was filled in 0.5 ml French straws and sealed automatically then placed on a rack and transferred to a cooled cabinet at 4°C for an equilibration time of 2 h. They were then frozen, at a height of 4 cm over liquid nitrogen surface (in nitrogen vapor, -120 °C) for 10 min, before being plunged into liquid nitrogen (-196°C) for storage. Thawing was done one week after freezing.For this, straws were rapidly immersed in a water bath at 37°C for 30 sec. Each experiment was stated prefreezing and post thawing through assessment of semen quality parameters ; sperm motility, sperm liveability, sperm abnormality, sperm plasma membrane integrity and sperm DNA integrity.The points examined and discussed in this study was : 1- Effect of LDL on freezability of buffalo semen : LDL was extracted by Moussa method (2002).The egg yolk in Tris extender was then replaced by different LDL concentrations (6, 8, 10 and 12%).The results indicated that LDL addition to buffalo bull semen freezing extender offered better protection than egg yolk during semen cryopreservation.This effect was clear specially when added at concentration of 12 %. 2- Effects of disaccharides with different Glycerol concentrations on freezability of buffalo semen : In this category we used trehalose and sucrose in constant amount with different glycerol concentrations (3.68, 5.5 and 7.3%). The improvement in freezability of buffalo semen was found with glycerol concentration in between 5.5 and 7.3%3- Effect of glycerol with different Disaccharides concentrations on freezability of buffalo semen : In this point we investigated the effect of disaccharides (trehalose and sucrose) to reach the optimal concentration of these sugars with constant glycerol concentration at 7.3%.The concentrations examined were (50,100 and 200 mM/L).The results revealed that glycerol with different trehalose concentrations had no remarkable effect on the sperm parameters for both chilled and frozen semen in comparison to Tris medium.However, Tris-Trehalose 50mM/L showed good motility and high intact membrane% prefreezing, whereas, Tris-Trehalose 100 mM/L gave the lower abnormalities% and higher live sperm %. In contrast, Tris-Sucrose 200 mM/L gave better results for chilled semen. whereas, Tris-Sucrose 50 mM/L and Tris medium showed the higher values for frozen semen.4- Effect of some commercially available extenders on freezability of buffalo semen Three diluents were examined; egg yolk based (Triladyl®), milk based (Laiciphos) and soybean based (Bioxcell) extenders.Our finding proved that Laiciphos and Bioxcell resulted in better values for chilled semen comparing to TRIS based diluent.On the contrary, no significant difference between the experimented diluents and TRIS based diluent for frozen semen, however, Triladyl seemed to be the best extender for freezing of buffalo semen.from the present study, it can be concluded that : 1-Low density lipoproteins (LDL) posses good cryoprotective properties for freeze-thaw buffalo bull spermatozoa manifested by better semen characteristics when added to freezing extender at concentration of 12 % than when using 20% egg yolk and this study recommended the use of LDL in Tris extender used for freezing of buffalo bull semen instead of whole egg yolk.2-The optimal concentration of glycerol for cryopreservation of buffalo semen came in between 5.5 and 7.3 %.3- Sucrose and Trehalose could be safely used for cryopreservation of buffalo semen in concentration of 50 and 100mM, respectively.4- Laiciphos and Bioxcell were the best diluents for chilled buffalo semen, whereas, Triladyl came as the extender of choice for freezing of buffalo semen.ReferencesAboagla, E. M. and Terada, T. (2003) : Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing. Biol. Reprod 2003; 69:1245–1250.Aboagla, E. M. and Terada, T. (2004): Effect of the supplementation of trehalose.