Effect of different pre-freezing rates on buffalo sperm motility and ultrastructure preservation

Aim of this work has been the study of different pre-freezing rates on sperm motility and ultrastructure preservation. It has been used semen coming from two and five years old breeding-buffaloes. Motility and concentration were assessed to evaluate fresh semen characteristics. Semen was diluted 1:4 and two cooling shapes were tested: 1) fast shape and 2) soft shape. Equilibrature periods of 6 h and overnight at + 5 °C were also tested. Samples of fresh and cooled semen were used also to evaluate ultrastructural morphology and acrosomal integrity. The results show that motility remains constant until 6 h of cooling-off period, while decreases after overnight equilibration: this decrement is more evident for fast shape cooling samples than slow shape cooling ones. At the thawing only overnight equilibration samples show a significant decrement of motility. Vitality is always >90%; only for overnight equilibration samples it is at the thawing about 70%. Acrosomal integrity is about 90% in all samples and in any way shows significant differences. Fresh and 2 h equilibrated cells show a normal ultrastructural morphology: after 6 h equilibration, a partial detachment of acrosomale membrane is observed; after overnight equilibration we can observe different deformed and swollen heads. In conclusion, during cooling-off period, soft cooled samples show a higher motility. Moreover at thawing, while there are not significant differences between cooling shapes regard total motility, a better forward motility can be observed in samples obtained with a soft cooling rate.