Pregnancy rates of lactating cows after transfer of in vitro produced embryos using X-sorted sperm.

The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were 39.3 ± 3.2% (least-squares mean ± SEM) for TAI nonsorted, 27.3 ± 3.4% for TET nonsorted fresh embryos, and 30.2 ± 3.3% for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles.

Effects of phenazine ethosulfate during culture of bovine embryos on pregnancy rate, prenatal and postnatal development.

The objective was to evaluate the use of phenazine ethosulfate (PES) during culture of embryos on fetal and postnatal calf development. Oocytes collected from abbatoir-derived ovaries were matured and fertilized, and the resulting embryos were cultured in vitro by standard procedures, in a chemically defined medium plus BSA. Day 0 of culture was 18+/-2h after the onset of IVF. From 2.5 to 6.5d, half of the eight-cell embryos served as controls, and the remainder were exposed to 0.3microM PES, which decreases lipid content of embryos. Good-quality blastocysts exposed to PES (n=38) or control (n=35) blastocysts were transferred nonsurgically to synchronized recipients in estrus 6-7.5d earlier, resulting in 9 calves in each group. These in vitro-produced pregnancies were evaluated weekly between 35 and 98d postestrus by ultrasonography, and postnatal development of the calves was monitored for 1 month. Based on a limited number of transfers, use of PES during in vitro culture did not affect pregnancy rates compared to the control at 35 or 98d (P>0.1). Transfer at 7-7.5d after estrus resulted in higher 98-d pregnancy rates than at 6-6.5d (34% vs. 10%; P<0.05). In vitro-derived fetuses that aborted had retarded fetal and placental development compared to those that went to term, but there was no difference in fetal loss between the PES treatment and controls. One calf in the PES group weighing 36kg was born dead at 252d of gestation, and another calf in this group was dead some hours after birth and weighed 22.2kg when parturition was induced at 310d of gestation. It is unclear whether these two abnormal calves were caused by the PES treatment, or were due to in vitro procedures in general. In conclusion, the use of PES during in vitro culture had no effect (P>0.1) on pregnancy rates, conceptus losses between Days 35 and 98 of pregnancy, nor fetal postnatal development in calves born normally.

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting.

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.

Exposure of thawed frozen bull sperm to a synthetic peptide before artificial insemination increases fertility.

We evaluated the effect on fertility of in vitro exposure of thawed frozen bull sperm to synthetic FertPlus peptide prior to artificial insemination (AI). The peptide represented a 60-amino acid sequence within rat prosaposin. Commercial cryopreserved semen was from three Holstein bulls. Onset of estrus in groups of Holstein nulliparous heifers was synchronized via injection of prostaglandin F2-alpha, and heifers were scheduled for AI 8-24 hours after estrus was detected. Semen was thawed, diluted to 2.4 x 10(6) sperm/ml with buffer, and split to provide control and exposed aliquots (0 or 30 microM peptide) that were incubated at 37 degrees C for 10 minutes and then were held at 32 degrees C. The two aliquots of semen then were used on an alternate basis 2-65 minutes later to inseminate females. Each AI (one per female) involved the deposit of approximately 250,000 sperm into each uterine horn. This procedure for AI was used to reduce the pregnancy rate with control semen to below the maximum value for a given bull and to facilitate detection of any beneficial effect of the peptide. For each bull, approximately 32 heifers were inseminated with control semen, and approximately 32 heifers were inseminated with peptide-exposed semen. Pregnancy was evaluated ultrasonically approximately 60 days after AI. After excluding one group of heifers with unusually low fertility, averaged across all animals, a 29% increase in pregnancy rate resulted from exposure of sperm to peptide (P < 0.04; one-tailed chi-square test; means were 48 vs. 62%). Pregnancy rates for the three bulls for control and peptide-exposed semen, respectively, were 42 and 62%, 44 and 64%, and 56 and 61%; means in the first two pairs of values tended to differ (P approximately equal to 0.10). These observations should be confirmed with sperm from other bulls used in a more conventional manner. However, with insemination of a limiting number of cryopreserved sperm, brief exposure of the thawed bull sperm to FertPlus peptide appeared to improve fertility dramatically.

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.