John Schenk

Insemination of heifers with sexed sperm

Data from inseminating 1,000 heifers consecutively with sexed sperm and 370 heifers with control sperm in 11 small field trials are summarized. Semen was from 22 bulls of unknown fertility of various beef and dairy breeds, and 6 inseminators participated. Freshly collected sperm were sexed using a MoFlo flow cytometer/cell sorter after staining sperm with the DNA-binding dye Hoechst 33342; the principle is that the bovine X chromosome has 3.8% more DNA than the Y chromosome. Accuracy approaching 90% males or females was achieved. There was little difference in pregnancy rates between sexed, unfrozen and sexed, frozen sperm. In 5 of 6 field trials, there was little difference in pregnancy rates between insemination doses of 1.0 to 1.5 x 10(6) versus 3.0 x 10(6) sexed, frozen sperm. In the most recent trials, pregnancy rates with sexed, frozen sperm were within 90% of unsexed, frozen controls that had 7 to 20 times more sperm/insemination dose; however, in a few trials, control pregnancy rates were substantially higher than with low doses of sexed sperm. There were too few inseminations per bull to test bull differences in pregnancy rates rigorously. Insemination of sexed, frozen sperm bilaterally into the uterine horns produced pregnancy rates similar to insemination into the uterine body in 4 of 5 field trials. Pregnancy rates among inseminators did not differ significantly. There was no excess embryonic death between 1 and 2 months of gestation with pregnancies from sexed sperm, and very few abortions occurred between 2 months of gestation and term. Although rigorous epidemiological studies remain to be done, calves resulting from sexed sperm appear to exhibit no more abnormalities than controls.

Cryopreservation of flow-sorted bovine spermatozoa

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm.

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO(2) in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10(6) sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.

PREGNANCY RATES IN HEIFERS AND COWS WITH CRYOPRESERVED SEXED SPERM: EFFECTS OF SPERM NUMBERS PER INSEMINATE, SORTING PRESSURE AND SPERM STORAGE BEFORE SORTING

Field trials were conducted to increase fertility with AI of flow-sorted, sexed bovine sperm. In the first trial, a novel competitive fertilization approach was used to compare pressures (30psi vs 50psi) for sorting sperm. Both X- and Y-sperm were sorted to approximately 95% purity at 30 and at 50psi; X-50+Y-30 (and the converse) were mixed in equal numbers for AI of heifers. Fetal sex divulged which treatment produced the pregnancy; 82% of pregnancies resulted from the 30psi treatment (P<0.05). Based on a similar approach, a new-pulsed laser did not damage sperm any more than the previous standard continuous wave laser. In a large field trial, sorting sperm at 40psi increased pregnancy rates in heifers relative to 50psi (42.3% vs 34.1%, n=367/group, P<0.05). Storing sperm for 20h before sorting at 40psi decreased pregnancy rates from 42.3% (n=367) to 36.8% (n=368; P<0.05). Breeding heifers with sexed sperm 55-56h after CIDR removal and PGF(2alpha) resulted in 34% (n=32) pregnant, compared to 49% (n=35) with fixed-time insemination 67-68h after CIDR removal (P>0.1). Lactating dairy cows pre-screened for normal reproductive tracts when OvSynch injections (GnRH, prostaglandin, GnRH) were initiated, had similar (P>0.1) pregnancy rates to timed AI, with 10x10(6) sexed sperm (43.9%, n=57), 2x10(6) sexed sperm (40.5%, n=57) and 10x10(6) unsexed control sperm (55.6%, n=58). A final field trial with unselected, lactating dairy cows resulted in similar pregnancy rates for 2x10(6) sexed sperm in 0.25mL straws (25.0%, n=708) and 0.5mL straws (24.4%, n=776), but lower (P<0.05) than unsexed control sperm (37.7%, n=713). Younger cows and those >84 days in milk had the highest pregnancy rates for both sexed and unsexed sperm. These studies improved sperm sexing procedures, and provided insight into appropriate commercial use of sexed sperm.

Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition.

In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.