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.

Since the birth of Louise Brown, the world’s first “test tube baby”, was reported in 1978 [1], the number of children conceived through in vitro fertilization (IVF) is estimated to be at least 12 million [2]. This technology is widely feasible and has resulted in the generation of offspring from cattle [3], horses [4], sheep [5], goats [6], pigs [5], dogs [7], cats [8], fowls [9], dromedary camels [10], llamas [11], buffaloes [12], deer [13], mice [14], rabbits [15], hamsters [16], rats [17], baboons [18], rhesus monkeys [19], cynomolgus monkeys [20], and marmosets [21]. In particular, IVF is now widely used commercially in cattle and has exceeded in vivo embryo production in terms of the number of embryos produced at the global level since 2016 [22]. A major reason for this extreme increase in IVF-derived embryos is the practical application of genomic selection, in which the breeding values of progeny are estimated based on their parents’ single-nucleotide polymorphism information at early stages [23, 24].

Ultrasound-guided transvaginal ovum-pick up (OPU) combined with IVF (OPU-IVF) enables oocyte collection and production of embryos from young heifers genotyped for genomic evaluation even before puberty [25]. Furthermore, laparoscopic OPU in calves is also applicable for embryo production [26,27,28]. In addition, OPU can be conducted more frequently at shorter intervals than in vivo embryo production, which consists of multiple ovulations and uterine flushing, requiring a longer interval for uterus recovery. Then the efficiency of embryo production by OPU-IVF is higher than that of in vivo embryo production [29]. Another advantage of OPU-IVF is that the administration of follicle-stimulating hormone (FSH) is not essential. For in vivo embryo production, only one or a few embryos can be obtained without FSH administration, because cattle are mono-ovulatory species. In contrast, multiple embryos can be produced in OPU-IVF without FSH, because oocytes in immature antral follicles before pre-ovulatory size have acquired developmental competence for blastocyst development, while the size of the follicles from which oocytes are derived significantly affects embryo productivity [30]. Although FSH-free OPU-IVF is feasible, FSH administration prior to OPU is known to improve the developmental competence of oocytes, and has been applied to OPU in cattle [31] and other ruminants (sheep [32], goats [33], water buffaloes [34], and camels [35]). Therefore, optimization of OPU-IVF with and without FSH is necessary.

Although IVF technology has enabled the utilization of immature oocytes, there are still numerous oocytes in the ovaries that are potentially destined to degenerate. The technology for fostering these oocytes in immature follicles before the acquisition of maturational and developmental competence in vitro is called in vitro growth (IVG) [36, 37]. IVG is expected to contribute to the reproduction of animals, including humans, for example, in cancer patient fertility preservation [38]; however, it can also be utilized as an experimental model to reconstitute follicular development at early stages, which cannot be evaluated in vivo. In mice, it is feasible to generate pups from primordial follicles [39, 40], primordial germ cells (PGCs) [41, 42], pluripotent stem cell-derived PGC-like cells [43], and male pluripotent stem cells [44] using in vitro culture systems. However, offspring from secondary follicles or earlier stages using IVG have not yet been obtained in other animals, including sheep [45], goats [46], and water buffaloes [47], and no mature oocytes have been reported in cattle [48, 49]. The earliest stage of follicles from which the birth of calves from IVG-derived oocytes has been reported is the early antral follicle stage (0.4–1.0 mm diameter) [50,51,52]. Indeed, oocytes derived from early antral follicles do not have maturational competence but can acquire fertilizability and developmental competence after approximately 2 weeks of IVG [51, 53]. Therefore, the early antral follicle stage is a critical phase for oocytes to acquire maturational and developmental competence. The IVG of oocytes from these follicles can be utilized as an experimental model to investigate the impact of factors affecting fertility and embryo productivity [54]. Consequently, IVG studies as follicular growth models can provide significant insights to improve the efficiency of OPU and fertility.

In this review, the technological progression of the optimization of immature bovine oocyte utilization is reviewed alongside the findings from a variety of other ruminants. Emphasis will be on the optimal timing and frequency of oocyte collection by OPU, and on the utilization of IVG culture systems as follicular growth models.

Effects of follicular waves and antral follicle count on the optimal OPU interval

In cattle and most other ruminants, multiple antral follicles start to grow simultaneously; only the dominant follicle (DF) is selected for ovulation, while other subordinate follicles regress. These well-documented wave-like follicular growth dynamics are called follicular waves [55, 56]. Therefore, the number of antral follicles observed by ultrasonography, which depends on the timing of follicular waves, such as at recruitment, deviation, and ovulation, is varied considerably [57]. Furthermore, even if the follicle sizes observed at the same time point are similar, these follicles can be in both the growing and regressing phase [58,59,60,61]. Thus, the quality of oocytes collected by OPU at random times is not consistent, and the outcome of embryo productivity varies considerably. The removal of antral follicles in ovaries by aspiration, known as ablation, causes the reduction of estrasiole-17β (E₂) and inhibin plasma levels, and the emergence of a new follicular wave occurs [55]. Considering that the duration of a follicular wave is 9 to 10 days in 2-wave animals and 8 to 9 days in 3-wave animals [62], OPU at a once a week interval enables the continuous collection of oocytes from newly recruited follicles. In contrast, one study suggested that the optimal interval for OPU was affected by the number of antral follicles in the ovaries, known as the antral follicle count (AFC). Nagai et al. [63] divided cows into two groups based on the maximum number of antral follicles in continuous OPU sessions: twice a week (3 or 4 days) or once a week (7 days). The rate of normal fertilized oocytes was higher in high AFC cows (≥ 30, 29.0%) than in low AFC cows (< 30, 19.7%) with OPU-IVF at a twice a week interval (P < 0.05). Conversely, the low AFC cows (21.3%) showed a higher rate of normally fertilized oocytes than the high AFC cows (8.3%) at a once a week interval (P < 0.05).

To clarify the reason why the relationship between AFC and oocyte fertilizability is dependent on the OPU interval, follicular growth dynamics and hormone concentrations were investigated in the plasma and follicular fluids from two groups of cows (high AFC: ≥ 30, low AFC < 30) based on the maximum number of antral follicles during follicular waves [64]. In cows with high AFC, the number of recruited antral follicles (4–8 mm) was higher than that in low AFC cows (P < 0.05) and showed typical wave-like dynamics, with increases followed by decreases over 7 days (P < 0.05), whereas this number was stable in low AFC cows. This suggests that a higher proportion of follicles are in the regression phase in high AFC cows on OPU at once a week interval. In addition, concentrations of E₂ in plasma during the estrous cycle and ovulatory follicles were higher in cows with high AFC than in cows with low AFC (P < 0.05). The plasma concentration of testosterone, a precursor of E₂, was also higher in cows with high AFC (P < 0.05). Conversely, the plasma concentration of FSH, which originally promotes the production of E₂ in granulosa cells, was higher in cows with low AFC (P < 0.05).

Furthermore, an IVG culture of oocyte-cumulus-granulosa complexes (OCGCs) from early antral follicles (0.5–1.0 mm) was conducted from ovaries with high AFC (≥ 25) or low AFC (< 25) for 12 days as a model of growing early antral follicles to the ovulatory phase [65]. Consistent with the in vivo follicular fluid of ovulatory follicles, E₂ production was higher in the high AFC group than that in the low AFC group (P < 0.05), and the nuclear maturation rate of IVG oocytes was higher in the high AFC group (78.9%) than in the low AFC group (66.4%, P < 0.05). These results indicate that E₂ production in response to FSH is impaired by chronically high blood FSH levels in cows with low AFC, which suppresses the negative feedback of E₂ on FSH secretion. However, most subordinate follicles are in the regression phase due to the higher concentration of E₂ in a DF of high AFC cows. This promotes maturational competence of the inside oocyte, but results in lower oocyte fertilization in subordinate follicles on OPU at a once a week interval (Fig. 1).

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.

Therefore, the optimal interval of OPU depends on the number of antral follicles in the ovaries, and OPU at short intervals of 3 to 4 days (twice a week) may improve embryo productivity in animals with high AFC. However, such intensive OPU programs may not be applicable to farmers and veterinarians who do not routinely conduct OPU. In such cases, controlling follicular waves using hormonal treatments can be useful. The combination of a progesterone (P₄)-releasing device with E₂ can regress antral follicles regardless of their developmental stage, resulting in the emergence of newly recruited follicles [66]. Furthermore, administration of prostaglandin F_2α simultaneously with E₂ and P₄ induces corpus luteum (CL) regression, which can physically interfere with follicle aspiration and improve blastocyst rate (45.8%) compared with a control without any hormonal treatment (38.5%, P < 0.05) [67]. However, another study indicated that this protocol only seems to be effective in animals with high AFC [68], which supports the previous findings on fertilization, follicular growth dynamics, and hormone concentrations [64].

Effects of large follicles and CL in the ovaries on subordinate follicles

In cases of random OPU during estrous cycles, there is a random DF and/or a CL in the ovaries, even in slaughterhouse-derived ovaries. In mono-ovulatory species, the presence of a DF and/or a CL in the ovaries can be divided into four patterns: DF+CL (an ovary with both DF and CL), DF (an ovary with DF alone), CL (an ovary with CL alone), and devoid (an ovary with neither DF nor CL) [69, 70], which are known to affect ovarian function and follicle growth. The blood flow of the ovarian artery is higher in the DF+CL, CL, DF, and devoid patterns, in descending order [71]. The frequency of the DF+CL pattern is higher in the right ovary than in the left [71], and the blood flow in the ovaries with the DF+CL pattern is higher in the right ovary than in the left [69]. These differences between the right and left ovaries are thought to increase the likelihood of ovulation occurring in the right ovary than in the left [70]. Moreover, the blood flow of DF, CL, and antral follicles (6–6.9 mm) is known to be higher in the DF+CL pattern than in the other patterns [72], which may affect the blood flow of smaller follicles and the developmental competence of oocytes. Although the relationship between ovarian patterns and developmental competence of oocytes from antral follicles has been investigated, findings have been inconsistent [73,74,75]. Because oocytes acquire maturational and developmental competence during the growth from early antral follicles smaller than 1 mm [51], the effect of the presence of a DF and CL in ovaries on the growth of early antral follicles (0.5–1.0 mm) was investigated using IVG of OCGCs as a model [76]. The collection rate of OCGCs from early antral follicles with the devoid pattern (47.5%) was similar to that of the DF+CL pattern (43.1%), and higher than that of the DF (37.9%) and CL (37.5%) patterns (P < 0.05). The nuclear maturation rate of DF+CL pattern oocytes (81.8%) was similar to devoid oocytes (81.3%) and higher than CL oocytes (63.3%) (P < 0.05). The ratio of E₂ to P₄, an indicator of healthy follicle growth [54], was the highest in DF+CL among all patterns (P < 0.05). The blood flow of the ovarian artery was the highest in the DF+CL pattern, followed by the CL, DF, and devoid patterns [71]. Therefore, the higher maturation rate of oocytes and the ratio of E₂ to P₄ observed in DF+CL could be due to higher blood flow into the entire ovary. In contrast, blood supply can be evenly distributed in the ovaries of the devoid type because of the absence of DF and CL, both of which induce angiogenesis at local levels [77, 78], resulting in a higher oocyte maturation rate (Fig. 2). These results indicate that the developmental competence of oocytes can be affected by the ovarian patterns classified by the presence of DF and CL at the early stage of follicles that are not of a collectable size by OPU.

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.

Coasting period between the last FSH administration and OPU, and application of pre-IVM for small follicle oocyte rescue

The interval between the last FSH administration and OPU, known as the coasting period, affects the developmental competence of oocytes [79, 80]. During this period, FSH withdrawal causes a reduction in the plasma concentration of FSH, similar to in the proestrus phase [81], and the pulsate secretion of luteinizing hormone (LH) increases [82]. Subsequently, LH-dependent follicular growth accelerates, and progressive hypoxia and apoptosis, and inflammation, of granulosa cells increase in the follicles, leading to an environment similar to the pre-ovulatory phase of DF [83]. Nivet et al. [80] investigated the optimal duration of the coasting period for OPU with multiple FSH administrations during the luteal phase. A coasting period of 44 h (71%) or 68 h (63%) resulted in a higher developmental competence of oocytes, whereas extending the period until 92 h decreased developmental competence. Oliveira et al. [84] compared the blastocyst rate between a non-stimulated control and FSH administration followed by 44 h of coasting that showed the high blastocyst rate in the study by Nivet et al. [80]. Using 35 Holstein cows, they found no benefit of coasting on the blastocyst rate (17.1%) compared to the control (12.2%, P = 0.13) [84]. The most critical difference between these two studies was the aspirated follicle diameter. In the study by Oliveira et al. [84], in which there was no effect of the coasting period, oocytes were collected from follicles of at least 2 mm. However, in the study by Nivet et al. [80], the findings of which suggested a benefit of the coasting period, the aspirated follicles were at least 5 mm. In addition, during the optimal coasting period (44 and 68 h), they most commonly observed follicles of 7–10 mm in diameter, corresponding to the phase for the acquisition of responsiveness to LH. These results combined suggest that oocytes in small antral follicles (2–5 mm), which do not acquire responsiveness to LH, do not benefit from the coasting period (Fig. 3).

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).

Generally, there are positive correlations between follicle diameter, oocyte diameter, and oocyte developmental competence. Furthermore, oocytes derived from antral follicles approximately 2 mm already have maturational competence, but do not acquire developmental competence [85, 86]. Thus, they begin meiosis after aspiration without ovulatory stimulation, such as in the LH surge, known as precocious meiotic resumption, resulting in lower developmental competence [87, 88]. Pre-in vitro maturation (pre-IVM) is a method for rescuing oocytes from small follicles, especially in slaughterhouse-derived ovaries or for OPU without FSH [88, 89]. Meiosis is arrested by the maintenance of cyclic adenosine monophosphate (cAMP) in oocytes, which is either produced by the oocytes themselves [90, 91] or supplied by cumulus cells via gap junctions [92, 93]. Pre-IVM is the culture system controlling the cAMP level of oocytes prior to IVM to prevent precocious meiotic resumption and improve developmental competence [31, 89]. Two target enzymes control the cAMP level in oocytes during the pre-IVM period. Adenylate cyclase promotes cAMP synthesis, its activators include forskolin and low-dose FSH [31]. Phosphodiesterase (PDE) degrades cAMP to 5´-AMP, its inhibitors include 3-isobutyl-1-methylxanthine (IBMX) and cilostamide [31]. The combination of these activators of adenylate cyclase and inhibitors of PDE has been widely tested for use in pre-IVM for bovine in vitro embryo production [31].

It was previously hypothesized that the optimal timing of pre-IVM administration depends on oocyte diameter; therefore, the effect of oocyte diameter and pre-IVM length on developmental competence was investigated [94]. Oocytes were classified into small-sized (110–115 μm) and large-sized (≥ 115 μm) according to their diameters, and cultured for 0, 5, or 10 h prior to IVM with low-dose FSH (2 × 10^–6 units/ml) and IBMX (500 μM). In both groups, approximately 90% of oocytes were arrested at the germinal vesicle stage after 5 h of pre-IVM, but approximately half of the oocytes resumed meiosis after 10 h. The nuclear maturation rate after pre-IVM, followed by IVM, was higher in the large-sized group than in the small-sized group (P < 0.05). The percentage of blastocysts was higher in the large-sized group, which was not affected by the pre-IVM period (31%), than that in the small-sized group (P < 0.05). In contrast, the percentage of blastocysts in the small-sized group was higher after 5 h of pre-IVM (16%) than after 0 h (9%) and 10 h (8%) (P < 0.05). These results suggest that pre-IVM for 5 h does not diminish the developmental competence of large-sized oocytes but promotes that of their smaller counterparts (Fig. 3). This was supported by the findings of Desenzi et al. [95] who collected oocytes regardless of follicular diameter. There was no reported developmental competence benefit of 2 h of pre-IVM, with a pre-maturation medium containing 500 μM of IBMX and 1 mM of dibutyryl-cAMP, which was optimized using oocytes from small follicles (2–4 mm in diameter) [96]. The combination of OPU and pre-IVM should be investigated on a large scale to ensure its effectiveness.

Validation of the optimal coasting period for a single FSH administration

Although FSH administration improves the developmental competence of oocytes, the treatment consists of multiple administrations of FSH for 3 to 4 days, which is labor-intensive and can be stressful for animals. Various single FSH administration treatments have been reported for in vivo embryo production, which can be divided into two categories [97]. One is the use of specific solvents such as polyvinylpyrrolidone (PVP) [98, 99], aluminum hydroxide gel [100], and hyaluronan [101, 102], to slowly release FSH from the administration site into the blood. The other is FSH administration at the site where drugs are transferred into the blood more slowly than intramuscular administration, such as subcutaneous [99, 103, 104] and epidural administration [105, 106], using physiological saline as a solvent. After a single administration, plasma FSH concentrations are maintained at a higher level than at the basal level (PVP [99], aluminum hydroxide gel [100], hyaluronan [107], subcutaneous [104], and epidural [106]), and the growth of multiple ovulatory follicles is induced. These single FSH administration protocols have also been applied to ovarian stimulation for OPU-IVF. Most studies have reported that the efficiency of these treatments for developmental competence does not differ from that of non-stimulated animals or is lower than that of multiple FSH administration [31]. However, the optimal coasting period between each treatment of a single administration of FSH and OPU has not yet been evaluated.

Blondin et al. [108] administered FSH alone to beef heifers, followed by slaughter at 24, 48, or 72 h after FSH administration and oocyte collection. The developmental competence of the oocytes was higher at 48 h than at 24 and 72 h. Furthermore, oocytes collected 4 to 5 h after slaughter showed greater developmental competence than those collected immediately after slaughter (1–2 h). However, these results [108] cannot be applied directly to other protocols because plasma concentrations of FSH are different after intramuscular and subcutaneous administration [97], and the release of FSH from an administration site into the plasma is also affected by the type of animal, breed, parity, and body condition of the donors. For example, a single subcutaneous administration of FSH induces the growth of multiple ovulatory follicles in beef breeds [103, 104], whereas the same treatment is ineffective in Holstein heifers [99]. In addition, a single epidural FSH administration induces multiple ovulatory follicles in dromedary camels, but is not as effective as a conventional superovulation protocol (equine chorionic gonadotrophin and multiple intramuscular FSH administration) [109]. To optimize the OPU protocol with a single FSH administration, more efforts are needed to identify the optimal timing for OPU after each treatment, considering the potential differences among donors.

Effects of summer heat stress on growing immature oocytes

Owing to global warming, the average temperature in Japan has increased by over 2°C in 120 years, from 1900 to 2020 [110]. Dairy cows are unable to maintain their body temperature when the outside temperature exceeds approximately 25°C [111], and increasing body temperature is thought to reduce food intake and increase oxidative stress, resulting in reduced reproductive performance [112]. Oocytes that mature in vitro and in vivo, as well as those derived from immature follicles (2–8 mm) commonly used in IVF, show reduced developmental competence due to heat stress [113]. Moreover, reduced oocyte developmental competence is observed in the subsequent cooler autumn, suggesting a continuous effect of summer heat stress [114]. It takes approximately 1 to 2 months for the reproductive performance of heat-stressed cows to recover [115, 116], and the development of early antral follicles (<1 mm) into ovulatory follicles takes approximately 30 to 40 days [117]. This suggests that oocytes in small antral follicles are also damaged by heat stress during summer, causing a continuous reduction in oocyte developmental competence and fertility during the subsequent cooler autumn.

However, the effect of heat stress on early antral follicle development remains unclear. Therefore, an IVG culture system of bovine OCGCs derived from early antral follicles (0.5–1.0 mm) [54] was used to evaluate the effect of heat stress on the growth of bovine early antral follicles [118]. OCGCs from slaughterhouse-derived ovaries were divided into a control group cultured at 38.5°C, similar to the normal body temperature of cows, and a heat shock group cultured at a temperature cycle mimicking those of dairy cows in a hot environment (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, 39.5°C: 9 h), and cultured for 12 days in vitro [118]. The nuclear maturation rate of oocytes did not differ between groups (control: 62.1%, heat shock: 51.9%); however, the blastocyst development rate was higher in the control group (27.7%) than in the heat shock group (0.0%) (P < 0.05). In addition, the level of reduced glutathione (GSH), which protects cells against oxidative stress, was higher in the control group than in the heat shock group (P < 0.05). Furthermore, the addition of cysteine, which promotes GSH production, to the heat shock group increased GSH levels and improved the blastocyst rate (27.9%) compared to without cysteine (6.1%, P < 0.05). Heat exposure of OCGCs isolated from early antral follicles has also been shown to alter the metabolism of amino acids (isoleucine, leucine, valine, aspartic acid, alanine, glycine, and ornithine), some of which (isoleucine, leucine, valine, and alanine) are also known to affect mature oocytes and early stage embryos with low developmental competence [119]. These results suggest that oxidative stress reduces the amount of GSH and alters amino acid metabolism in the oocytes of cows exposed to heat stress during the summer, resulting in continuously low developmental competence and poor fertility during the subsequent cooler autumn (Fig. 4). Furthermore, these results indicate that measures for the management of animals exposed to heat stress for more than a month are needed to maintain reproductive performance from summer to autumn. In addition, oocyte developmental competence recovers more quickly when OPU is repeated twice a week compared to once every 3 weeks during summer and autumn [114]. Therefore, intensive OPU twice a week may remove heat stress-damaged oocytes and result in quicker recruitment of healthy follicles with higher developmental competence.

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.

Amino acid metabolism of preantral follicles

In postpartum dairy cows, metabolic abnormalities due to a negative energy balance (the energy required for milk production exceeds the energy from dietary intake) affect ovarian function [120]. Feeding high-energy diets for 12 weeks after parturition does not affect the first postpartum ovulation [121], but it does occur earlier in cows fed high-energy diets for 3 weeks prior to parturition [122]. These results imply that follicle metabolism at earlier stages is important for the recovery of ovarian function. Many metabolic factors such as glucose, proteins, and lipids affect the development of antral follicles [123]. However, the significance of metabolic factors has not yet been evaluated in preantral follicles, which cannot be evaluated in vivo currently. Among the many metabolic factors, amino acids are important metabolic parameters that play vital physiological roles as both substrates and regulators in several metabolic pathways [124,125,126]. They also are utilized as markers of maturational and developmental competence in oocytes [127, 128]. Therefore, the metabolism of preantral follicles using ovarian cortical cultures was compared between primordial follicle activation and secondary follicle culture [129, 130]. Amino acids and their metabolites were compared in spent media based on the degree of follicle activation (high vs. low) in cortical cultures and diameter growth (growth vs. degeneration) in secondary follicle cultures. The concentrations of arginine, lysine, and methionine were significantly higher in the high activation group, while α-aminoadipic acid, a metabolite of lysine, was significantly lower in the low activation group (P < 0.05, Fig. 5). In secondary follicle culture, the concentrations of histidine, methionine, tyrosine, and hydroxyproline were higher in the degenerate group than in the growth group (Fig. 5). These amino acids and metabolites can be useful as markers of the healthy growth of follicles in vitro and may provide insights for improving the culture system and feeding management.

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.

Conclusions and perspectives

Although the number of in vitro-produced bovine embryos has increased globally, optimization of the utilization of immature oocytes can be improved further. For OPU-IVF without FSH administration, the optimal interval between each session is affected by AFC, and a twice a week interval shows better fertilizability than a once a week interval in high AFC cows, in which a high proportion of subordinate follicles regresses 7 days after OPU. The presence of the DF and CL also affects the maturational competence of oocytes; however, these effects can start from the stage of early antral follicles, which are too early and small to be collected by OPU.

Although FSH administration is not essential for OPU-IVF, it promotes the developmental competence of oocytes. However, FSH administration during the optimal coasting period for OPU-IVF does not benefit oocytes from small antral follicles, and pre-IVM culture support them in improving developmental competence. In addition, the optimal coasting period for a single FSH administration needs to be investigated, considering the plasma concentration of FSH after each treatment.

IVG studies suggest that heat stress and metabolic factors can affect the growth of follicles at early stages, implying the need to take measures in the management of breeding programs over a period of several months.

Making mature oocytes from preantral follicles grown in vitro remains the biggest challenge in the reproduction of mono-ovulatory species. Studies from various perspectives on ovarian physiology and pathology will provide insights into improving IVG culture systems and promote their widespread application across species.

Conflict of interests

The author declares no conflict of interest.