In vitro embryo production (IVEP) refers to the generation of embryos outside the maternal organism through the retrieval of oocytes, their fertilization with sperm under laboratory conditions, and the subsequent culture of the developing embryos within a regulated environment. The initial successful birth of offspring from in vitro-derived embryos represented a pivotal advancement, highlighting the essential contribution of in vitro embryo technologies to the progress of the goat industry [1]. Ensuring embryo viability during the in vitro culture process remains a cornerstone for the effective application of in vitro embryo production (IVEP) methodologies [2]. In vitro embryo culture media are critical to the successful development of goat embryos, encompassing a wide range of formulations—from chemically defined media containing basic salts to complex, undefined media enriched with serum and other uncharacterized components. In the majority of studies on in vitro embryo production (IVEP) in goats, presumptive zygotes cultured in vitro using complex media and serum supplementation [3]. The efficiency of blastocyst formation and overall embryonic development is significantly affected by the composition of the in vitro culture media. In the context of goat embryo production, commonly employed media include Brackett and Oliphant (BO), Tyrode’s Albumin Lactate Pyruvate (TALP), synthetic oviduct fluid (SOF), and Charles Rosenkrans (CR) medium. These formulations are routinely enriched with fetal calf serum (FCS), amino acids, and antioxidants to promote optimal embryonic growth and viability.

Fetal calf serum (FCS) remains the most widely employed growth supplement in cell culture systems due to its rich composition of bioactive components, including growth factors, hormones, amino acids, proteins, vitamins, inorganic salts, and antibodies [4]. The precise composition of fetal calf serum (FCS) remains incompletely characterized, and its biochemical variability arising from differences between individual donor foetuses can significantly influence cell culture outcomes [5]. Fetal calf serum (FCS) is commonly integrated into in vitro embryo culture media owing to its abundant repertoire of bioactive compounds such as antioxidants, growth factors, and essential nutrients—that facilitate cellular proliferation and enhance embryonic development [6].

Pre-implantation stage embryos can be cultured in a variety of media, ranging from simple formulations containing balanced salt solutions and carbohydrates to more complex compositions, such as those used in tissue culture systems [2]. Numerous studies have substantiated the use of sequential embryo culture media as an effective strategy to accommodate the evolving physiological requirements of embryos during both early and later stages of development. The selection of culture media, along with targeted supplementation at specific developmental phases, plays a critical role in optimizing the efficiency of in vitro blastocyst production. While fetal calf serum (FCS) has been shown to improve blastocyst development and hatching rates [7], its use remains subject to variability due to undefined components. Exposure of early-stage embryos to fetal calf serum (FCS) has been demonstrated to negatively impact blastocyst quality [8], as serum exhibits biphasic effects significantly inhibiting the initial cleavage division while markedly enhancing development at later stages [9]. Moreover, the inclusion of fetal calf serum (FCS) in in vitro embryo culture has been associated with elevated lipid accumulation in embryos, which negatively impacts cryopreservation outcomes. Consequently, the use of FCS in in vitro embryo culture remains a subject of ongoing debate.

The Cỏ goat is an indigenous breed native to Vietnam and represents a valuable genetic resource for the conservation of the country’s goat biodiversity. Characterized by their small stature, Cỏ goats are widely recognized for their disease resistance, high reproductive efficiency, and strong adaptability to Vietnam’s climatic conditions [10]. This study aimed to produce high-quality in vitro Cỏ goat embryos suitable for cryopreservation and subsequent embryo transfer.

All experimental procedures conducted in this study complied with Vietnamese legislation and adhered to Decision No. 2056/QĐ-BNN-KHCN, issued by the Ministry of Agriculture and Rural Development of Vietnam on June 28, 2024.

Experiment1: Influence of fetal calf serum supplementation timing onin vitroCỏ goat embryo production

As presented in Table 1, no statistically significant differences were observed in the cleavage rates of Cỏ goat oocytes among the 0-hour, 48-hour, 120-hour, and control groups following in vitro fertilization (P > 0.05). In contrast, the 0-hour group exhibited a significantly higher blastocyst formation rate and greater average cell number per blastocyst compared to the other groups (Table 1; P < 0.05). Remarkably, hatching blastocysts were detected exclusively in the 0-hour group (Table 1).

Table 1.The effect of FCS supplementation timing on in vitro Cỏ goat embryo production.

Six replications were performed. Percentage data are shown as mean ± SEM. Values with different superscripts in the same column differ significantly (P < 0.05).

Experiment2: Influence ofin vitroculture medium onin vitroCỏ goat embryo production

Based on the findings from Experiments1, in Experiments2 fetal calf serum (FCS) will be supplemented into the in vitro culture medium for Cỏ goat embryos at 0 hours post-IVF. As shown in Table 2, there was no statistically significant difference in the cleavage rate of Cỏ goat oocytes following in vitro fertilization among the SOFaa and CR1aa groups (P > 0.05). Although both media were supplemented with fetal calf serum (FCS), however, the SOFaa group exhibited a significantly higher blastocyst formation rate and average cell number per blastocyst compared to the CR1aa group (37.04% vs. 24.98% and 136.08 vs. 125.37, respectively; P < 0.05).

Table 2.The effect of in vitro culture medium on in vitro Cỏ goat embryo production.

Six replications were performed. Percentage data are shown as mean ± SEM. Values with different superscripts in the same column differ significantly (P < 0.05). 1): SOFaa + 2.5% FCS (v/v, at 0 hour post IVF); 2): CR1aa + 2.5% FCS (v/v, at 0 hour post IVF).

The utilization of oocytes derived from 5 - 6-month-old prepubertal goats may be advantageous compared to those obtained from older animals, as prepubertal does yield a greater number of oocytes, and their in vitro developmental competence being comparable to that of adult goats. Therefore, in this study, oocytes obtained from 5 - 6-month-old prepubertal goats were utilized. As shown in Table 1, supplementation of fetal calf serum (FCS) in the in vitro culture medium for Cỏ goat embryos significantly improved both the blastocyst formation rate and the occurrence of hatching blastocysts. However, no statistically significant differences in cleavage rates were observed among groups supplemented with FCS at 0, 48, or 120 hours post-IVF, or in the absence of FCS. These results align with previous findings by Abdel-Wahan et al. (2018) [14] and Goel et al. (2016) [2]. Abdel-Wahan et al. (2018) [14] reported no significant variation in cleavage rates of bovine oocytes when FCS was added at either 20 or 42 hours post-IVF. Similarly, Goel et al. (2016) [2] found that FCS supplementation during the cleavage phase of goat oocytes post-IVF did not significantly affect cleavage rates (Figure 1). Notably, Goel et al. (2016) [2] demonstrated that FCS supplementation during the post-IVF culture phase led to a significantly higher blastocyst yield (P < 0.05) compared to media lacking FCS or supplemented at later stages. Therefore, the findings of this study suggest that the supplementation of fetal calf serum (FCS) to the in vitro culture medium at 0 hours post-IVF account for the higher rate of hatching blastocysts observed, compared to supplementation at later time points (Table 1).

Figure 1. Cỏ goat embryos at the 2 - 4 cell stage, cultured in SOFaa medium supplemented with 2.5% fetal calf serum (FCS) at 0 hours post-in vitro fertilization (IVF), were observed on Day 2 post-IVF.

Extensive researches has explored strategies to improve culture media for in vitro embryo production in domestic animals, with particular emphasis on the supplementation of sera, hormones, and somatic cells [15]. Among these, serum supplementation during oocyte maturation has demonstrated considerable benefits, largely attributed to its high concentration of growth factors that play a crucial role in modulating oocyte maturation. In addition to its antioxidant properties and its capacity to prevent zona pellucida hardening, serum also provides a rich source of essential components—including hormones, minerals, trace elements, lipids, and detoxifying agents—that collectively contribute to enhanced embryonic development [16]. According to Sena-Netto et al. (2020) [17], supplementation of fetal calf serum (FCS) to the embryo culture medium at 0 hours post-IVF significantly accelerates the progression to the morula stage. While the exact mechanisms by which FCS influences embryonic development are not yet fully elucidated, its positive effects are presumed to be linked to its rich content of growth factors, amino acids, and antioxidant properties. These components contribute to an increased blastocyst formation rate and enhance the overall quality of embryos cultured in FCS supplemented media compared to those cultured without FCS.

Although fetal calf serum (FCS) is known to support the development of in vitro embryos, Sudano et al. (2011) [18] reported that its inclusion in embryo culture media led to an increase in lipid accumulation within embryos, which negatively affected their suitability for cryopreservation. Furthermore, Ghaedrahmati et al. (2024) [19] found no significant differences in blastocyst formation rates when using varying concentrations of FCS (2.5%, 5%, and 10%) in the culture medium. Based on these findings, fetal calf serum (FCS) was incorporated at a concentration of 2.5% (v/v) into the in vitro culture medium for Cỏ goat embryo development, with the objective of enhancing embryo cryosurvival without compromising blastocyst yield.

Blastocyst quality is a critical determinant of pregnancy success following embryo transfer [8]. In this study, embryo quality was assessed using two key indicators: the percentage of hatching blastocysts (Figure 2) and the average cell number per blastocyst (Figure 3). The highest hatching rate (8.06%) was recorded in embryos cultured in Synthetic Oviductal Fluid supplemented with amino acids (SOFaa) and fetal bovine serum (FCS) administered at 0 hours post-fertilization. Furthermore, embryos cultured under this condition exhibited a significantly greater average cell number per blastocyst compared to those cultured with FCS supplementation at later time points.

The results of Table 2 in this study are in agreement with those of Wan et al. (2008) [20], who demonstrated that Synthetic Oviductal Fluid supplemented with amino acids (SOFaa) is more conducive to the in vitro development of ovine IVF embryos compared to CR1aa medium. SOFaa is widely utilized for cattle, sheep and goat embryo culture due to its composition, which closely mimics oviduct fluid and includes essential components such as hormones, growth factors, and amino acids. These constituents enhance the developmental competence of embryos prior to transfer. SOFaa medium represents a flexible culture system that can be tailored to support various species and embryonic developmental stages through the incorporation of specific additives such as amino acids, serum, and growth factors.

The CR1aa medium is composed of inorganic salts (NaCl, KCl), a buffering agent (NaHCO₃), and metabolic energy substrates including lactic acid, sodium

Figure 2. Cỏ goat blastocysts and hatching blastocysts, cultured in SOFaa medium supplemented with 2.5% fetal calf serum (FCS) at 0 hours post-in vitro fertilization (IVF), were observed on Day 7 post-IVF.

Figure 3.Cỏ goat hatching blastocyst stained with Hoechst 33342.

pyruvate, and glutamine. Moreover, the medium provides both essential and nonessential amino acids, which are critical for supporting embryonic development [21]. Notably, CR1aa medium lacks glucose but is supplemented with a complete profile of essential and nonessential amino acids to sustain embryo viability and growth. During the transition from the 8-cell stage to the blastocyst, nonessential amino acids and glutamine promote blastocyst formation and facilitate hatching. Conversely, essential amino acids have been shown to enhance blastocyst cell numbers and facilitate cellular differentiation processes that promote the formation of the inner cell mass (ICM) [22].

The developmental competence of mammalian embryos post-implantation is significantly influenced by cellular death mechanisms, particularly apoptosis, occurring during the preimplantation stages. Apoptotic activity has been documented in both in vivo and in vitro embryos across a range of domestic species, including equine, porcine, ovine, caprine, and bovine [23]. Suboptimal culture conditions that compromise cell proliferation and elevate apoptosis rates in early-stage embryos are correlated with reduced implantation success, increased fetal resorption, and lower birth weights during post-implantation development [24]. Wan et al. (2008) [20] further reported that embryos cultured in CR1aa medium exhibited a significantly higher incidence of apoptotic signals and greater numbers of apoptotic cells per embryo compared to those cultured in SOFaa medium. Although the underlying mechanism remains unclear, it is hypothesized that certain components in CR1aa medium may induce embryonic cell apoptosis. The findings reported by Wan et al. (2008) are consistent with our results. Although apoptosis was not assessed in this study, the observation that blastocysts cultured in CR1aa medium contained fewer cells than those cultured in SOFaa medium supports this hypothesis. These findings may explain the enhanced performance of the SOFaa medium relative to the CR1aa medium, as evidenced by improved blastocyst formation, higher hatching rates, and increased average cell numbers per blastocyst observed in this study (Table 2).

In conclusion, supplementing SOFaa medium with fetal calf serum at 0 hours post-fertilization is effective for enhancing the in vitro production of Cỏ goat embryos.

The research was carried out at the Key Laboratory of Animal Cell Technology. The authors would like to thank the Vietnamese Ministry of Science and Technology for supporting the project.