120 Fetal development in lactating dairy cows following timed embryo transfer with in vitro-produced embryos derived from conventional or sex-sorted sperm

Abnormally large offspring (LO) have been associated with in vitro-produced (IVP) bovine embryos. The use of sex-sorted semen for IVP involves further manipulation of the gametes, potentially increasing the risk of LO. Although the number of IVP embryos transferred globally has markedly increased in recent years, data on fetal development and calf birthweight following IVF with sexed semen is scarce. The aim of this study was to determine the influence of semen type on the morphometric parameters of both dairy and beef breed IVP fetuses. Oocytes were collected once per week for up to 4 successive weeks from Holstein-Friesian dairy donors (ET-DAIRY; n = 53) and Angus beef donors (ET-BEEF; n = 37) using transvaginal ovum pickup. Oocytes were matured in vitro and fertilized using conventional (CV) or X-sorted sperm (SS) from a panel of sires, and 7 days later a single grade 1 blastocyst was transferred fresh to recipient lactating dairy cows (n = 510) in 7 herds. In a small number of cases, when an insufficient number of fresh embryos were available on the day of transfer, frozen embryos from a previous study were used. Recipients were synchronized using a 10-day Progesterone-Ovsynch protocol, and randomly assigned to receive CV-DAIRY, SS-DAIRY, CV-BEEF, or SS-BEEF on Day 7 after presumptive oestrus. In total 471 recipients received a fresh embryo and 39 received a frozen embryo. For all cows that had not returned to oestrus, pregnancy status was diagnosed on Days 32 to 38 by transrectal ultrasonography using an IMV Easi-Scan:Go equipped with a fixed 128-crystal linear probe. All cows diagnosed pregnant were re-examined on Day 63; in pregnant cows (n = 243, SS = 131, CV = 112), fetal sex (male, n = 70, female, n = 173) was determined based on the position of the genital tubercle. The percentage of male fetuses resulting from CV and SS was 50.0% and 10.7%, respectively. A video of each fetus was recorded at the 8-cm depth setting, and thoracic diameter (TD) and biparietal diameter (BPD) were subsequently measured using ImageJ’s freehand line function. The effect of semen type on the TD and BPD was determined using ANOVA with PROC GLM for SAS. The model included fetal sex, oocyte donor, sire, semen type, embryo breed, donor age, recipient parity, and two-way interactions as fixed effects. Only significant effects were retained in the final model. Sire (P < 0.0001) and fetal sex (P = 0.02) affected TD, but there was no significant interaction between these two variables. TD was greater in males than females (19.3 ± 1.06 vs 18.8 ± 1.11 mm, respectively). Semen type (CV vs SS) did not affect TD or BPD. The BPD was not affected by any other variable and was not correlated with TD (r = 0.12, P = 0.13), potentially because of noise in the BPD measurement. In conclusion, IVP embryos derived from oocytes fertilized with SS or CV semen resulted in similar size fetuses at 63 days of gestation, although sexual dimorphism was already apparent at this time. Future studies will correlate Day 63 fetal measures with calf birthweight and maternal blood transcriptome analysis.

The research was supported by the Irish Department of Agriculture, Food and the Marine (2021R665). MBR was supported by a H2020-MSCA-Individual Fellowship (101021311).