Sex-sorted canine sperm cryopreservation: Limits and procedural considerations

Abstract

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.

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted, frozen–thawed spermatozoa in field conditions

“…After thawing, the percentages of total motile spermatozoa and acrosome‐intact spermatozoa were determined as described by Merlo et al . (). In brief, it was evaluated for sperm forward progressive motility (SPM – on a scale from zero to five, where five represented the best SPM) and sperm motility (SM – from 0 to 100%, in increments of 5%), using subjective analysis under a microscope (×100; Olympus Co., Tokyo, Japan).…”

“…Semen straws were thawed in a water bath at 70°C for 8 s, and semen was then placed into plastic tubes containing 2 mL of Tris buffer (198 mmol/L Tris, 73 mmol/L citric acid, 44 mmol/L glucose, 0.05% streptomycin sulfate, 500 UI benzylpenicillin/mL) at 37°C and then maintained at 37°C until assessment. After thawing, the percentages of total motile spermatozoa and acrosome-intact spermatozoa were determined as described by Merlo et al (2015). In brief, it was evaluated for sperm forward progressive motility (SPMon a scale from zero to five, where five represented the best SPM) and sperm motility (SMfrom 0 to 100%, in increments of 5%), using subjective analysis under a microscope (×100; Olympus Co., Tokyo, Japan).…”

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes’ zona pellucida

“…The sperm-rich fraction was evaluated before freezing for volume, concentration, and rate of morphologically normal spermatozoa. The volume was measured by a calibrated micropipette, and sperm concentration was determined with a Bürker chamber, after dilution of the sperm suspension 1:40 with 10% formol buffered saline to immobilize spermatozoa and counted using a phase contrast microscope (400x; Axiolab; Zeiss, Italy) equipped with a warming plate (37°C; Thermo Plate; Tokai Hit, Japan) as reported in [14]. Percentages of morphologically normal spermatozoa were determined at the same microscope (1000x) after dilution of semen 1:1 with 10% formol buffered saline, and at least 200 spermatozoa per sample were examined.…”

“…After centrifugation at 300 g for 10 min [14], performed to concentrate sperm, the supernatant was removed and sperm pellets were resuspended in two steps in freezing extenders reaching a final concentration of 200 × 10 6 spermatozoa/mL.…”

“…The slides were then observed using a Nikon Eclipse E 600 epifluorescence microscope (Nikon Europe BV, Badhoeverdop, The Netherlands) and at least 200 spermatozoa per sample were scored. The presence of a green acrosomal fluorescence was considered indicative of an intact acrosome, while a partial or total absence of fluorescence was considered to indicate acrosome disruption or acrosome reaction [14].…”