Sex-sorted canine sperm cryopreservation: limits and procedural considerations

The aim of this study was to define a protocol to store dog sperm before and after sorting to obtain an insemination dose sufficient to allow the conception by artificial insemination. Experiment 1 and 2 were performed to evaluate the more appropriate extender for preserving at room temperature dog sperm before and after sorting. Four extenders were tested: (1) Tris-fructose-citrate (TFC), (2) Tris-glucose-citrate (TGC), (3) modified Tyrode's albumin lactate pyruvate medium (mTALP), and (4) third fraction of the ejaculate (after centrifugation at 5000× g for 10 minutes; III FRAC). Experiment 3 and 4 were performed to evaluate the ability of dog semen to withstand sex sorting and freezing/thawing. Modified Tyrode's albumin lactate pyruvate medium was the best extender for canine sperm storage at room temperature (20 °C-25 °C) before (total motility: TFC, 8.3 ± 1.7; TGC, 50.0 ± 11.5; mTALP, 70.0 ± 0.1; III FRAC, 25.0 ± 1 0.4; P < 0.05) and after sorting (total motility: TFC, 7.3 ± 1.5; TGC, 10.3 ± 1.5; mTALP, 33.3 ± 6.7; III FRAC, 8.7 ± 5.8; P < 0.05), even if at 24-hour sorted sperm quality was impaired in all extenders tested herein. Sperm quality decreased after sorting (total motility: control, 92.5 ± 0.9; sorted, 52.9 ± 6.0; P < 0.05) and, especially, after freezing/thawing (total motility: frozen control, 25.7 ± 4.1; frozen sorted, 2.4 ± 1.2; P < 0.05). In conclusion, mTALP is an appropriate medium for canine sperm storage before and soon after sorting (hours), but a long storage period of sexed sperm at room temperature is not adequate. Cryopreservation greatly impaired sperm quality, and further studies are needed to optimize the freezing protocol for sexed dog sperm.