Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production.

Abstract Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F 2a treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n ¼ 25) or the dominant phase (Day 7, n ¼ 14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, useable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (AES.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the useable oocytes and embryos, this difference was significant (9:6 AE 1:4 and 3:5 AE 0:6 versus 3:9 AE 0:6 and 1:1 AE 0:3; P < 0:01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of useable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P < 0:01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all useable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8:0 AE 1:2 versus 3:8 AE 2:4; P ¼ 0:012 and 30.3% versus 14.9%; P < 0:01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.

Supplementary Material for: Aneuploidy Detection and mtDNA Quantification in Bovine Embryos with Different Cleavage Onset Using a Next-Generation Sequencing-Based Protocol

<p>Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.</p