172 PRE-IMPLANTATION GENETIC DIAGNOSIS COMBINED WITH FREEZING AND TRANSFER OF IN VITRO-PRODUCED EMBRYOS ALLOWS CREATING GENETIC RESOURCES FROM A MOSAIC BULL

Abstract

The polled and multisystemic syndrome (PMS) is a genetic abnormality observed in the progeny of a unique bull affected by a large chromosomal deletion and cellular mosaicism. Hemizygous females, representing 15% of total progeny, are hornless and show organ malformations, including of the ovaries. Hemizygous males are assumed to die in early fetal development. This study was initiated to produce sufficiently affected fetuses using the semen of the sire as a unique genetic resource. Oocytes from slaughterhouse ovaries were in vitro matured, fertilized with the same bull and cultured in SOF medium using standard in vitro procedures currently performed in the laboratory. On Day 7, grade 1 to 3 embryos were biopsied (5 to 10 cells) and frozen using a conventional glycerol procedure. Whole-biopsy amplification of genomic DNA was performed using a Qiagen Repli-g^® Mini Kit (Qiagen, Valencia, CA, USA). Sex determination was done by PCR (UNCEIA Sexing Kit, UNCEIA, Paris, France). The PMS status was indirectly determined using both tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism procedures to genotype a single SNP located within the deleted region. Because the sire was both homo- and hemizygous for this SNP, 3 categories of progeny were defined according to their genotypes: unaffected (heterozygous), potentially affected (homo- or hemizygous for the same allele as their sire) and affected (hemizygous for the alternate allele). Thirteen affected and 21 potentially affected female embryos were thawed and transferred (1 ≤ n ≤ 4) into 17 Day 7 recipients. Pregnant females were slaughtered on Day 9 and fetuses were recovered; sex and PMS status were then verified. From 2133 inseminated oocytes (7 replicates), 64% cleaved and 10% (n = 216) developed to the blastocyst stage on Day 7. This was significantly lower than the 87% cleavage and 25% blastocyst development rates observed with a control bull used to inseminate 368 oocytes from the same batches. Finally, 174 embryos were biopsied and 169 were frozen. Fifty percent were sexed as female (n = 87). Among them, 15% (n = 13) were affected and 29% (n = 25) were potentially affected. The 24-h survival rate averaged 67% from 54 thawed, unaffected embryos, whereas the hatching rate at 72 h was 59%. Seven female fetuses were recovered from 6 recipient cows. Clinical examination revealed an absence of horn buds for 3 of them. This status was subsequently confirmed by a non-Mendelian inheritance study based on Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA, USA) genotyping data. This combination of in vitro procedures allowed us to increase the number of affected fetuses from 15 to 43% (3/7) in the mosaic bull progeny. In addition, it enabled us to produce valuable material from a very limited resource to perform clinical and functional studies. Finally, it demonstrates the feasibility of a pre-implantation genetic diagnosis combined with freezing and transfer of IVP embryos.