Optimization of ovum pick-up-in vitro fertilization and in vitro growth of immature oocytes in ruminants

Abstract

Due to the strong demand for embryo production from young and genotyped superior animals using ovum-pick up (OPU) combined with in vitro fertilization (IVF), the number of in vitro-produced embryos has exceeded that of in vivo-derived embryos globally since 2016. One of the merits of OPU-IVF is that the administration of follicle-stimulating hormone (FSH) is not essential, while FSH treatment prior to OPU promotes oocyte developmental competence. Thus, investigations are needed to optimize OPU-IVF protocols with and without FSH. In addition, OPU enables oocyte collection from antral follicles in living animals. However, there are numerous immature oocytes in follicles at earlier stages, which are potentially destined to degenerate in ovaries. The technology used to foster acquisition of maturational and developmental competences in these immature oocytes is called in vitro growth (IVG). IVG is expected to contribute to assisted reproductive technologies for livestock, humans, and endangered species. However, no offspring from preantral follicles has been reported using IVG in animals other than in mice. Furthermore, IVG can be used to investigate factors affecting the fertility and developmental competence of oocytes by reconstituting follicle growth at each stage in vitro, which cannot be evaluated in vivo. Here, the technological progress of the optimization of immature bovine oocyte utilization is reviewed alongside findings from a variety of other ruminants.

Keywords: Follicles; In vitro growth; OPU-IVF; Oocytes; Ruminants.

Fig. 1.

Schematic of the relationship between the antral follicle count (AFC) and the interval of ovum-pick up (OPU). In cows with low AFC, the chronically high plasma concentration of follicle-stimulating hormone (FSH) causes a reduction in the production of estradol-17β (E₂) in response to FSH, impairing the negative feedback of E₂ to FSH secretion. This results in a consistent development of middle-sized follicles. In cows with high AFC, the higher E₂ production of the dominant follicle causes the regression of many subordinate follicles 7 days after OPU, which can reduce the fertilization rate of oocytes on OPU at a once a week interval.

Fig. 2.

Schematic of a hypothetical model of the relationship between oocyte competence and the ovarian patterns based on the presence of a dominant follicle (DF) and a corpus luteum (CL). Blood flow in the ovarian arteries is higher in DF+CL, CL, DF, and devoid (without DF and CL) patterns. Higher blood flow into the ovaries may increase the rates of oocyte-cumulus-granulosa complex (OCGC) collection and oocyte maturation in the DF + CL pattern. In the devoid pattern, blood flow into the ovaries is low, but can be relatively evenly distributed, which may be associated with higher maturational competence of the oocytes. In CL and DF patterns, angiogenesis is locally induced in the CL and DF, which may cause areas with poor blood supply and decrease oocyte competence.

Fig. 3.

Follicle size benefits from the coasting period and pre-in vitro maturation (pre-IVM). The coasting period between the last administration of follicle-stimulating hormone and ovum-pick-up (OPU) improves the developmental competence of oocytes that reach approximately 7–10 mm. In contrast, pre-IVM culture improves that of small oocytes (110–115 µm) derived from small antral follicles (2–4 mm).

Fig. 4.

Schematic of the summer heat stress-induced subfertility and low developmental competence in the subsequent cooler autumn. Summer heat stress reduces the amount of reduced glutathione (GSH) levels and alters amino acid metabolism in oocytes of early antral follicles. These follicles grow into ovulatory follicles 30–40 days later with low developmental competence, resulting in poor fertility during the subsequent cooler autumn.

Fig. 5.

Potential amino acids and metabolites as markers of primordial follicle activation and secondary follicle growth. In ovarian cortical culture for primordial follicle activation, higher concentrations of arginine, lysine, and methionine, and a lower concentration of α-aminoadipic acid, a metabolite of lysine, in media are related to higher activation of primordial follicles. In secondary follicle cultures, lower concentrations of histidine, methionine, tyrosine, and hydroxyproline in the media are associated with better growth of secondary follicles.