F. K. Hollinshead

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 degrees 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 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) 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 x 10(6) than 5 or 20 x 10(6) 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.

Flow cytometric sorting of frozen-thawed spermatozoa in sheep and non-human primates.

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen-thawed spermatozoa. The efficacy of sorting frozen-thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85-92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen-thawed samples (FT-NEAT: 32.7 +/- 2.5%; FT-WASH: 32.2 +/- 3.3%; FT-GRADIENT: 73.9 +/- 3.7%) compared with FRESH (83.3 +/- 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 +/- 5.1% and 27.2 +/- 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 +/- 0.9% and 83.3 +/- 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 +/- 2.4% and 71.7 +/- 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 +/- 1.3% and 77.2 +/- 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 +/- 1.7 mm) compared with FT-WASH (29.1 +/- 3.8 mm) and FT-GRADIENT (28.4 +/- 2.0 mm) and similar (P < 0.05) to FRESH (23.7 +/- 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86-97% purity) of frozen-thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 +/- 5.5%; baboon: 37.5 +/- 2.5%; chimpanzee: 73.0 +/- 2.0%), acrosome intact (marmoset: 68.5 +/- 7.5%; baboon: 89.5 +/- 1.5%; chimpanzee: 84.0 +/- 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen-thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.

Preservation and artificial insemination of sexed semen in sheep

Sperm-sexing technology using flow cytometry is in advanced stages of development for the sperm of several species. The sorting process could compromise sperm viability and sperm require specific handling procedures both before and after sorting to maintain the integrity and function of the sorted sperm. Standard freezing protocols have been modified for post-sorting cryopreservation of sperm and frozen sperm have been successfully thawed, sorted, refrozen and subsequently used to produce offspring. The relatively low numbers of available sorted sperm have, in some cases, led to modification of artificial insemination techniques to maximise efficiency of use. Multiple ovulation and embryo transfer, or in vitro fertilisation and associated technology, may lead to the more efficient use of sexed sperm.