In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing

The effect of sex sorting and freeze–thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen–thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen–thawed spermatozoa (60.9 ± 2.9% v. 57.0 ± 3.3% and 4.0 ± 0.1 v. 3.5 ± 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37°C). Sorted and non-sorted (control) frozen–thawed spermatozoa had similar acrosome integrity (73.7 ± 1.8% v. 75.2 ± 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 ± 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 ± 2.6% B pattern) before freezing. Overall, more sorted frozen–thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 ± 0.7%; P < 0.01) and less were uncapacitated (35.5 ± 0.6%; P < 0.05) than non-sorted (control) frozen–thawed spermatozoa (7.7 ± 0.8% and 38.6 ± 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen–thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 ± 11.9 v. 73.9 ± 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 ± 7.8 v. 38.6 ± 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen–thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen–thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen–thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 × 106 (commercial control) frozen–thawed spermatozoa (59%) than for 5, 10, 20 and 40 × 106 total sorted frozen–thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 × 106 resulted in a higher pregnancy rate (31%) than 106 (17%; P < 0.05), but was similar to ewes that received 4 × 106 sorted frozen–thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 × 106 than 5 or 20 × 106 non-sorted (control) or sorted frozen–thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen–thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen–thawed spermatozoa.