Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean Buffalo (B.bubalis) spermatozoa

The composition of the extender in which semen is diluted before freezing p lays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy duri ng capacitation and fertilization. Since cryopreservation procedures for Bu ffalo spermatozoa have not been adequately defined, the aim of the study wa s to improve the survival rate of buffalo (Bubalus bubalis) spermatozoa aft er cryopreservation by optimizing the timing for adding glycerol and by enr iching the cryoprotectant extender with an energy source substrate. Semen w as collected with an artificial vagina from 5 bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for v iability, pooled and held at room temperature (28 degrees C) for 1h. Then a liquots of pooled semen were subjected to dilution and equilibration in tri plicate as follows: Experiment 1. Glycerol (3%) in a commercial extender wa s added to the semen at 28 degrees C and cooled to 5 degrees C for 1 h; the n extender with 11% glycerol was added before further equilibration (initia l glycerol addition; IGA) and the samples held at 5 degrees C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before free zing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5 de grees C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n = 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiment s 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (TPA and LPA samples). The effect of addition time (initi al vs late) of glycerol and pyruvate was evaluated by measuring sperm motil ity, progressively forward motility and viability. After freezing-thawing t he percentage of motile spermatozoa was significantly higher (0.001<P<0.01) after a late addition of glycerol and pyruvate (LGA 5 and LPA 5). The optimizing of the timing of the glycerol addition and the presence in t he extender of an energy source rendered a higher efficiency in thawed sper matozoa. (C) 2000 by Elsevier Science Inc.