Africanized honeybee (Apis mellifera) semen freezing using Tris-based and Collins extenders

This study is aimed at evaluating the effect of different extenders on the cryopreservation of semen from Africanized honeybees ( A. mellifera ). Semen from honeybee drones from 10 different colonies was obtained by endophallus exposure technique and immediately evaluated for motility, viability using fluorescent probes, functional membrane integrity using the water test, and morphology. Samples from each colony were divided in three aliquots and subjected to a dilution ratio of 12:1 (diluent: semen) using Tris, Tris + egg yolk (Tris+EY), and Collins extender. Samples were cryopreserved and stored in liquid nitrogen for one week and then rewarmed and reevaluated. Immediate dilution provoked no significant effect on sperm motility and functional membrane integrity, regardless of the extender used; however, the greatest values ( P < 0.05) for normal sperm morphology were found at the use of isolate Tris (69.3 ± 1.9%). After thawing, there were no significant differences among extenders with relation to the preservation of sperm motility, viability, and functional membrane integrity, but the Tris extender provided the highest post-thawing values ( P < 0.05) for sperm normal morphology (49.2 ± 4.9%) while the Collins extender provoked the highest amounts ( P < 0.05) of curled tail defects (67.5 ± 3.2%). Moreover, the Tris was the only extender at preserving the proportion of normal sperm after thawing similar to what was verified for fresh samples. In summary, we suggest the use of a Tris-based extender for the cryopreservation of Africanized honeybee semen.