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The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls.

The double staining Trypan blue/Giemsa has been used to evaluate the semen of two stallions (A and B) bred in the same farm. In order to compare the fertility rate of the last mating season with the results of this staining technique, semen samples were collected at 10 days intervals and stained according to Kovács and Foote (1992). Sperm cells were then classified as: live with intact acrosome (VCA), live with acrosome reacted (VAR), dead with intact acrosome (MCA), dead with acrosome reacted (MAR), dead with damaged acrosome (MAD), only sperm head (ST). Mating data and pregnancy rate at 40 days of gestation were collected in order to evaluate the first cycle pregnancy rate and the seasonal pregnancy rate. Differences of pregnancy rate between stallions were analyzed by Chi Square test. Differences of sperm cells percentage in the staining classes were analyzed by ANOVA, after the angular transformations. Seasonal pregnancy rate were different between stallions (85,7% vs 52,9% in A vs B; P