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Forty years in the field: reproductive biotechnologies shaping genetic progress in cattle in France.

Over the past four decades, reproductive biotechnologies have profoundly transformed cattle breeding by accelerating genetic progress and enabling the dissemination of elite genetics. In this article, I present a perspective based on more than 40 years of practical experience in embryo technologies within Auriva-Elevage, a cooperative organization serving 30,000 farmers in southern France. The development of embryo transfer in France was closely linked to genetic and sanitary challenges, particularly the introduction of North American Holstein genetics and the restrictions on live animal imports due to infectious diseases such as Infectious Bovine Rhinotracheitis. These constraints stimulated the development of national expertise in embryo transfer. Over the years, our team has implemented and adapted a wide range of reproductive technologies including in vivo embryo production and embryo transfer, cryopreservation, embryo sexing, ovum pick-up (OPU), in vitro embryo production (IVP), embryo biopsy, genomic evaluation of embryos, and laser-assisted biopsy techniques. The genomic revolution dramatically increased the strategic value of OPU-IVP for the rapid multiplication of elite donor females. In addition to technological developments, the success of these programs has depended heavily on internal training, collaboration with national organizations such as ELIANCE (previously UNCEIA, ALLICE) and research institutes including INRAE and Toulouse veterinary school, as well as strong international exchanges through scientific networks. Practical examples such as the use of embryo biopsy to prevent genetic diseases demonstrate the applied value of these technologies in breeding programs. This review highlights the technical evolution, organizational structures, and human expertise that have shaped the implementation of reproductive biotechnologies in cattle breeding and discusses the importance of anticipating future needs to ensure continued genetic progress.

OPU-IVP

Surgical and non-surgical transfer of bovine embryos.

Methods of transferring one bovine embryo to the ipsilateral uterine horn have been compared. In heifers mid-ventral surgical laparotomy under general anaesthesia (n=22) was compared with flank surgery with paravertebral anaesthesia (n=21). Pregnancy rate was higher with a midventral approach (P less than 0.05) (77.3 per cent of 42.9 per cent respectively). In heifers mid-ventral surgery (n=36) was compared with two methods of non-surgical transfer either using the Cassou insemination gun (n=36) or a modification of it (n=39) with embryos collected on days 7, 8 or 10 after oestrus. There were no differences in pregnancy rate between methods or different ages of embryos (methods: 55.6, 55.6, 43.6 per cent respectively; Day: 48.8, 55.8, 48.1 per cent respectively) but the condition score of recipients affected success (P less than 0.05). Of 10 cows which each received an embryo using the modified Cassou gun eight became pregnant.

Anesthesia

Nucleotide sequence of the anticodon region of barley embryo phenylalanine transfer RNA.

Highly purified tRNAPhe from barley embryos was completely digested with pancreatic ribonuclease and T1 ribonuclease. The digestion products were separated using DEAE-cellulose chromatography. The Y base-containing fragment of the anticodon region of tRNAPhe has the following nucleotide sequence: Cpm2(2)GppsipCpApGpApCmpUpGmpApApYpAppsipCpUpGp, i.e. the same as in the anticodon region of wheat germ and pea tRNAPhe.

Anticodon

Ultrastructural studies of frozen-thawed 8-cell mouse embryos.

Survival of frozen 8-cell mouse embryos transferred directly upon thawing to the uteri of Day 3 pseudopregnant foster mothers was significantly lower (26%) than the survival of unfrozen 8-cell embryos transferred immediately after collection (73%). When frozen-thawed 8-cell embryos were cultured for 20-24 hr before transfer survival was similar to that of unfrozen 8-cell embryos transferred after 20-24 hr inculture (65% and 73%, respectively). Ultrastructural examination of the frozen-thawed 8-cell embryos revealed no obvious damage to protoplasmic components.

Animals

In vitro culture, storage and transfer of goat embryos.

Goat embryos collected 5 and 7 days after mating, were cultured in vitro at 37 degrees C for 2 days, or stored at 5 degrees C for 1 or 2 days and then cultured for 2 days, or stored in liquid nitrogen (-196 degrees C) for 2-4 weeks and then cultured for 1 day. After culture some of the embryos were transferred to recipient does. Culture and storage was carried out in Dulbecco phosphate buffer enriched with 25% goat serum. 1M glycerol or 2M dimethylsulphoxide (DMSO) was added to the media used for frozen storage. Thirteen of 15 embryos cultured without prior storage showed apparently normal development in culture. Ten of the 13 were transferred and five kids were born. Twenty of 38 embryos stored at 5 degrees C developed in culture and six kids were born following the transfer of 17 embryos. Duration of storage at 5 degrees C had no marked effect upon subsequent development. Six of 48 frozen stored embryos developed in culture. All six were transferred and three kids were born.

Animals

A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching.

BACKGROUND: GATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered. CASE PRESENTATION: The proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband. CONCLUSIONS: NGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

Child

IVF patient subgroups benefit from preimplantation genetic testing for aneuploidy: a prospective multicentre cohort study.

RESEARCH QUESTION: Can preimplantation genetic testing for aneuploidy (PGT-A) improve the ongoing pregnancy rate per transfer and reduce miscarriage rate in patients with advanced maternal age (AMA), recurrent implantation failure (RIF), recurrent pregnancy loss (RPL), or both, without affecting cumulative pregnancy rate? DESIGN: Prospective cohort study of 260 patients undergoing PGT-A aged 36 years or over (AMA group), with a history of three or more blastocyst transfers without birth (RIF group), two or more early pregnancy losses (RPL group), or all. Trophectoderm biopsy was conducted day 5 or 6, and comprehensive chromosome screening was used for PGT-A before single frozen embryo transfer (PGT-A-FET). A total of 3060 patients undergoing single conventional frozen embryo transfer (FET) served as a historical reference group. RESULTS: Patients had increased odds of positive serum beta-HCG after PGT-A-FET compared with FET in the AMA (OR 1.53, 95% CI 1.06 to 2.21) and RIF (OR 2.11, 95% CI 1.41 to 3.14) groups. The PGT-A-FET group significantly improved the odds of ongoing pregnancy in the AMA (OR 2.20, 95% CI 1.52 to 3.18), RIF (OR 3.96, 95% CI 2.60 to 6.04) and RPL (OR 2.81, 95% CI 1.52 to 5.21) groups. The odds of pregnancy loss were significantly reduced with PGT-A-FET in the AMA (OR 0.36, 95% CI 0.21 to 0.64), RIF (OR 0.10, 95% CI 0.04 to 0.26) and RPL (OR 0.14, 95% CI 0.04 to 0.53) groups. Cumulative pregnancy rate did not differ between PGT-A and conventional cycles (RR 0.94, 95% CI 0.80 to 1.10). CONCLUSIONS: PGT-A improved the odds of ongoing pregnancy and reduced the odds of pregnancy loss in all groups. The cumulative pregnancy rate did not differ between PGT-A and conventional cycles. Findings should be interpreted in the context of the observational design.

Adult