Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

SIMPLE SUMMARY: Achieving an effective freezing protocol, that is able to preserve the fertilizing ability of turkey semen, is a key aim for the establishment of the first national semen cryobank of autochthonous chicken and turkey breeds within our national project (Tutela della biodiversità nelle razze avicole italiane—TuBAvI). In this regard, we have performed different studies in order to define the best conditions for cryopreservation of turkey semen; namely, we identified an effective freezing protocol which is based on the use of dimethylsulfoxide as a permeant cryoprotectant (CPA) combined with Ficoll as a non-permeant CPA. Here, our purpose was to test this protocol in vivo, by evaluating the effect of two extenders and three inseminating doses. The good fertility and hatching rates achieved here are promising for future studies, in which our cryopreservation protocol will be tested on Italian autochthonous turkey breeds and also to the advantages offered by the extensive use of frozen semen in the turkey breeding industry. ABSTRACT: This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 10(6) sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 10(9) sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 10(6) sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.