Breeding techniques utilizing genome selection can enhance the frequency of desirable traits in livestock and improve the efficiency of animal production. Reproductive biotechnologies, such as in vitro production (IVP) using genetically superior animals (donor and sire), can significantly accelerate genetic improvement (Botigelli et al., 2023; Landecker and Clark, 2023). However, the production efficiency of IVP remains low, and there are qualitative differences compared to embryos derived in vivo.

ESCs, derived from the ICM of blastocysts, can proliferate indefinitely and differentiate into any cell type. The derivation of stable ESCs from domestic animals enables the effective use of various genetic improvement and reproduction tools, such as embryoids, genomic selection, and gene editing (Soto and Ross, 2016). Bovine ESCs were successfully derived and maintained using a customized mTeSR1 medium supplemented with FGF2 and IWR-1 on inactivated MEFs (Bogliotti et al., 2018). Bogliotti et al. (2018) achieved a 50% efficiency in establishing bESCs, regardless of the embryo source or ICM isolation method. However, when using ovum pick-up (OPU) derived blastocysts, bovine ESCs were established with 100% (6/6) efficiency. Therefore, the efficiency of ESC establishment may be influenced by the quality of the blastocysts.

Numerous studies have utilized various additives, such as fetal bovine serum (FBS), antioxidants, cytokines, and growth factors, to enhance the efficiency and quality of IVP. Conditioned medium, which contains growth factors and cytokines secreted by stem cells, has been shown to improve the development rate and quality of blastocysts in cattle and pigs (Bhang et al., 2014; Kwon et al., 2015; Uhm, 2023). Additionally, the use of stem cell conditioned media (SCM) in blastocyst production has demonstrated significant improvements. Furthermore, treatments with stem cell culture media, such as N2B27 during the post-morula stage, have been reported to enhance blastocyst development and quality (Ramos-Ibeas et al., 2023). These findings suggest that incorporating stem cell culture media or SCM into the IVP system can effectively enhance the production of high-quality blastocysts.

This study aimed to develop techniques to enhance the productivity and quality of IVF bovine embryos. mTeSR1, a culture medium formulated for the long-term maintenance of human stem cells, contains growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-β). We investigated whether supplementing with mTeSR1 could improve the productivity and quality of IVF bovine embryos. Additionally, the resulting blastocysts were cultured to assess their potential for establishing bovine ESCs and their efficiency was evaluated.

In vitro embryo production

The ovaries of cows were obtained at a slaughterhouse, placed in a saline solution. Only oocytes with homogeneous cytoplasm and cumulus cells were used. For IVM, cumulus oocytes complexes (COCs) were placed in TCM-199 (Gibco-BRL, NY, USA) with 10% FBS (Gibco-BRL), 0.2 mM Na-pyruvate (Sigma, St. Louis, MO, USA), 50 μg/mL gentamycin (Sigma) and hormones (FSH 0.02U/mL, estradiol 1 μg/mL; Sigma), followed by incubation at 38.5℃, with 5% CO₂ for 20-22 h.

Matured oocytes were subjected to IVF using IVF 100 medium (Research Institute for the Functional Peptides, Yamagata, Japan) with freeze-thawing semen. Motile spermatozoa were obtained using the Percoll method and were added to droplets containing COCs at a final concentration of 1 × 10⁶ spermatozoa mL^-1. Sperm and oocytes were coincubated for 18 h at 38.5℃ with 5% CO₂, and the day of in vitro insemination was designated as day 0.

After coincubation, the presumptive was washed and transferred to 200 mL drops of IVC (modified synthetic oviductal fluid medium with amino acids; mSOFaa) medium, supplemented with 5% FBS (control) and mTeSR1 5X supplement (STEMCELL, 85850; 1% mTeSR1, 2% mTeSR1, or 5% mTeSR1, respectively), and then cultured at 38.5℃ with 5% CO₂ and 5% O₂ for 9 days. Embryos were evaluated on day 2 post insemination (pi) for cleavage rate and day 9 pi for the blastocyst and hatching rate.

For outgrowth culture, zona pellucida of blastocysts was removed using 18G needle under a stereo-microscope and seeded on a monolayer of gamma-irradiated MEFs with outgrowth culture media (mTeSR1-based media), as described previously (Zhao et al., 2021). Media was changed every other day and outgrowths were fixed at day 7 of culture.

Apoptotic cell number in blastocysts and immunostaining

Blastocysts and outgrowths were fixed in PBS containing 4% (w/v) paraformaldehyde for 1 h and permeabilized in 0.5% Triton X-100 solution for 30 min at 37℃. For TUNEL assay, fixed blastocysts were incubated in TUNEL reaction medium (in situ cell death detection kit, TMR red; Roche, Penzberg, Germany) for 1 h at 38.5℃ in darkness. Following the washing of TUNEL reagent, samples were incubated in DyLight™ 550 conjugated SOX2 antibody (Invitrogen). For DNA counterstaining, all samples were incubated in Hoechst 33342 (1 μg/mL) for 30 min at 37℃ and then mounted on glass slides in VECTASHIELD antifade medium (Vector Laboratories, Newark, CA, USA) under cover slides. Images of each blastocyst were captured under an inverted epifluorescence microscope at a magnification of 200 X. The number of apoptotic nuclei and thetotal number of nuclei were examined.

Quantitative PCR of blastocysts

RNA extraction and transcription of single blastocysts were performed using FastLane Cell cDNA Kit (Qiagen N.V., Hilden, Germany) following the manufacturer’s protocols. Quantitative PCR was performed using the Power SYBR^® Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) on the QuantStudio Real-Time PCR System (Applied Biosystems). The conditions for RT-qPCR were as follows: 95℃, 5 min, followed by 35 amplification cycles (95℃, 5 s; 60℃, 10 s). The reaction was terminated by an elongation and data acquisition step at 72℃ for 30 s. The primer sets shown in Table 1 were used.

Table 1. Primer sets for RT-qPCR.

Gene	Primer sequences (5’ to 3’)	Product size (bps)	References
Sox2	F-GGTTGACATCGTTGGTAATTTATAATAGC R-CACAGTAATTTCATGTTGGTTTTTCA	88	NM_001105463
Cdx2	F-GCCACCATGTACGTGAGCTAC R-ACATGGTATCCGCCGTAGTC	140	XM_871005
Bax	F-GCAGAGGATGATCGCAGCTG R-CCAATGTCCAGCCCATGATG	197	U92569
Bcl2l1	F-CGTGGAAAGCGTAGACAAGGAG R-GTAGAGTTCCACAAAAGTGTC	133	AB238936
18S rRNA	F-GACTCATTGGCCCTGTAATTGGAATGAGTC R-GCTGCTGGCACCAGACTTG	87	AF176811.1

Statistical analysis

Data were analyzed using IBM SPSS Statistics package (ver. 22.0). Embryo development, apoptosis, and gene expression data were analyzed by one way analysis of variance and Duncan’s multiple range test. Cell number in blastocysts were compared by Student’s t-test. A statistically significant difference was determined as p < 0.05.

This study was conducted to examine the effect of the addition of mTeSR1 supplement on the in vitro development of bovine preimplantation embryos (Table 2). The cleavage rate on day 2 pi was 53.51% in the control, and ranged from 58.12% to 66.58% in all mTeSR1 treatment groups. On day 7 pi, the development to the morula stage in the 2% mTeSR1 group was 40.96% (55/133), which was significantly higher than the 28.38% (44/165) observed in the control (p < 0.05). The developmental rate was further analyzed by assessing the number of blastocysts formed on day 9 pi. The blastocyst development rate in the 2% mTeSR1 was 36.1% (50/133), significantly higher than the 26.06% (44/165) in the control (p < 0.05). The hatching rate on day 9 pi in the control was significantly lower than in the 2% mTeSR1 (p < 0.05). No significant differences were observed among the various mTeSR1 treatment groups.

Table 2. Effect of mTeSR1 on in vitro development of embryos.

Treat	No. of embryos	No. (% ± SD) of embryos developed to
		2-Cell	Morula	Blastocyst	Hatched blastocyst
Control	165	90 (53.51 ± 8.47)a	47 (28.38 ± 4.82)a	44 (26.06 ± 3.94)a	28 (17.29 ± 4.35)a
1% mTeSR1	128	75 (58.12 ± 7.17)ab	47 (36.13 ± 5.02)ab	41 (31.94 ± 4.91)ab	25 (20 ± 4.02)ab
2% mTeSR1	133	89 (66.58 ± 8.21)b	55 (40.96 ± 8.44)b	50 (36.1 ± 10.63)b	40 (28.82 ± 8.44)b
5% mTeSR1	131	84 (63.92 ± 7.79)a	39 (29.83 ± 8.02)a	39 (29.83 ± 8.02)a	29 (22.28 ± 9.57)ab

^a,bValues with different superscripts are significantly different (p < 0.05)..

As evidenced by early embryonic development results, the 2% mTeSR1 treatment had a beneficial effect on the development and hatching of blastocysts compared to the control. TUNEL analysis was performed on day 9 pi blastocysts to examine apoptotic cells, and the total number of cells in the blastocysts was also analyzed (Table 3 and Fig. 1). The total number of cells and ICMin the blastocysts did not significantly differ among groups. However, the apoptotic index in the 2% mTeSR1 (6.17 ± 0.58) was significantly lower than in the control (p < 0.05), with no significant differences observed among the mTeSR1 treatment groups.

Table 3. Effect of mTeSR1 on cell number and apoptosis of blastocysts.

Treatment	No. of blastocysts	Total cell number (± SD)	ICM cell number (± SD)	ICM: total cell ratio (± SD)	TUNEL positive cells (± SD)
Control	12	394.75 ± 28.91	83.67 ± 6.06	21.25 ± 0.52	8.31 ± 0.87a
1% mTeSR1	14	426.5 ± 17.6	92.86 ± 4.83	21.82 ± 0.83	6.85 ± 0.79ab
2% mTeSR1	17	424.35 ± 12.21	95.65 ± 3.29	22.58 ± 0.58	6.17 ± 0.58b
5% mTeSR1	18	343.28 ± 29.81	77.33 ± 6.87	22.54 ± 0.61	7.6 ± 0.55ab

^a,bValues with different superscripts are significantly different (p < 0.05). Cell number of blastocysts were determined at day 9 of IVC..

Figure 1.Images of embryos, ICM with SOX2-positive cells (green), and TUNEL-positive cells in blastocysts at day 9 pi. Apoptotic cells in blastocysts were analyzed using the TUNEL assay, with DNA and fragmented DNA stained using DAPI (blue) and TdT (red), respectively.

RT-qPCR was utilized to confirm the expression of genes associated with embryo quality (Fig. 2). Sox2 gene expression in the 5% mTeSR1 was significantly higher than in the control (p < 0.05), whereas no significant expression differences were noted in the other mTeSR1 treatment groups. Expression of caudal type homeobox 2 (Cdx2) and B-cell lymphoma 2 like 1 (Bcl2l1) genes did not differ among groups. However, the 5% mTeSR1 exhibited significantly higher Bax expression compared to the control and other mTeSR1 treatment groups (p < 0.05).

Figure 2.Relative gene expression in bovine blastocysts derived from different culture conditions. Significant differences (p < 0.05) are indicated by different letters (a-d), and error bars represent the standard error of the mean (± SEM).

Blastocysts from the control (n = 30) and 2% mTeSR1 (n = 20) on day 9 pi were subjected to outgrowth culture for 7 days to examine their outgrowth morphology (Fig. 3A). Outgrowths formed dome-shaped colonies with higher efficiency in the 2% mTeSR1 treatment than in the control (61.53% versus 36.36%, respectively, Fig. 3B).

Figure 3.The effect of mTeSR1 on the morphologies (A) and outgrowth rates (B) of blastocysts. Major morphologies (flat and dome shapes) of blastocyst outgrowth at day 7 of outgrowth culture are shown. Outgrowth rates were calculated as the ratio of dome-shaped colonies to plated blastocysts. Error bars represent the standard error of the mean (± SEM).

This study aimed to investigate the effects of mTeSR1 supplementation (1%, 2%, and 5%) on the in vitro development of bovine preimplantation embryos. mTeSR1, a serum-free and animal product-free medium, was originally developed for the derivation and long-term feeder-independent culture of human ESCs (Ludwig et al., 2006). mTeSR1 contains various bioactive substances, including growth hormones and proteins (Vallier et al., 2004). These substances include those with beneficial effects on embryo development, such as bFGF, TGF-β, pipecolic acid (PA), hypoxanthine, and thymidine, as well as those with harmful effects, such as gamma-aminobutyrate (GABA). PA, a product of lysine metabolism, reduces mitochondrial activity and reactive oxygen species (ROS) in oocytes, functioning as a ROS scavenger and thereby improving subsequent embryo development (Treleaven et al., 2021). Hypoxanthine and thymidine prevent early pregnancy loss associated with vitamin B6 deficiency, thereby enhancing blastocyst development. However, it has been reported that the number of cells in the blastocyst decreases (Kwong et al., 2010). GABA is a major inhibitory neurotransmitter in the central nervous system. In IVP, GABA inhibited early embryonic development, resulting in a significant reduction in the rate of blastocyst formation dose-dependently (Tian et al., 2020).

In the present study, addition of mTeSR1 in the IVC medium significantly enhanced the cleavage rates and blastocyst formation of fertilized bovine oocytes. Although bFGF is not detected at the 8-cell to 16-cell stages of bovine embryos, it is known to be expressed in conceptuses and the uterine epithelium (Martal et al., 1998). They reported that bFGF induces the transition from morula to blastocyst, improves the hatching rate, and increases the number of blastomeres in ICM. Treating embryos with TGF-β at high concentrations (50-100 ng/mL) shortly after fertilization (48 h) had no effect on early embryonic development but increased the expression of genes associated with blastocyst quality, such as Nanog and DNA methyltransferase 3A (Dnmt3a) (Barrera et al., 2018). The combined addition of bFGF and TGF-β to the IVC medium has been reported to enhance bovine embryo development due to their synergistic effect (Neira et al., 2010). However, TGF- β inhibitor did not affect the growth rate of blastocysts, but resulted in a decrease in the total number of cells in the bovine blastocysts (Hajian et al., 2016). In the present study, 2% mTeSR1 treatment enhanced early embryo development and hatching rates of blastocysts, but total number of cells and the ICM in the blastocysts did not differ among groups. Therefore, it is postulated that the addition of mTeSR1 at low concentrations is effective in the early embryonic development and quality improvement of bovine IVF embryos.

SOX2 is crucial for maintaining pluripotency within the ICM (Zhu and Zernicka-Goetz, 2020). It helps sustain the expression of key pluripotency genes such as octamer-binding transcription factor 4 (OCT4) and NANOG, which are essential for the development of the ICM into the epiblast and primitive endoderm (PE). Within the ICM, SOX2 is involved in the segregation of cells into the epiblast and the PE (Khan et al., 2012). The role of SOX2 for regulation of gene expression is crucial for this differentiation process. In bovine embryos, SOX2 expression begins at the 8-cell stage and gradually accumulates in the ICM cells as the blastocyst expands, eventually becoming restricted to the ICM in late blastocysts by day 8.5 pi (Luo et al., 2022). In this study, the blastocyst on day 9 pi was stained using SOX2 antibody to identify ICM cells. The ratio of ICM cells to total cells in a blastocyst did not differ among groups. When embryos were exposed to FGF2, outgrowths contained PE colonies were increased compared to control (Yang et al., 2010). In the present study, dome-shaped colonies of SOX2-positive cells were more frequent in the mTeSR1 group, which indicates that the ICM status of the blastocyst developed in the mTeSR1 group is more suitable for the establishment of bovine ESCs. Thus, these results cannot be attributed to the growth hormones present in mTeSR1, as the concentration of mTeSR1 used here is very low. Therefore, the positive effects are likely due to the various other constituents contained in mTeSR1.

The expression of the Sox2, a marker for PE, was higher in the 5% group compared to the control group. However, there was no difference in the number of ICM. Therefore, it appears that Sox2 was more strongly expressed in the ICM of the treatment group. This suggests that the addition of mTeSR1 could qualitatively enhance the ICM. The ICM/total cell ratio was lowest in the control, showing a tendency to increase in the order of 1%, 5%, and 2% groups. This trend was similar to the expression pattern of the Cdx2. CDX2 was used as a trophectoderm (TB) marker and is reported to play a role in maintaining TE integrity, although it is not directly associated with TE formation during bovine development (Goissis and Cibelli, 2014). Blastocyst hatching is known to be influenced by the condition of the TE (Isaac et al., 2024). In the present study, the hatching rate of blastocysts was significantly higher in the 2% group compared to the control group. However, the expression of the Cdx2, a marker for TB, showed no differences across all groups. Taken together, these results suggest that the addition of mTeSR1 does not contribute to an increase in the number of cells in blastocysts, but it may lead to qualitative improvement in blastomeres in embryos.

BCL2L1 is considered an inhibitor of apoptosis, while BAX is regarded as a pro-apoptotic protein. Significantly higher Bax expression was observed in the 5% mTeSR1 group. However, TUNEL-positive cells were lowest in the 2% mTeSR1 group and highest in the control, differing from the Bax/Bcl2l1 expression pattern. Overall, the number of TUNEL-positive cells was lower in all mTeSR1 treatment groups compared to the control group, which is likely due to the reduction of ROS by PA contained in mTeSR1. The higher number of TUNEL-positive cells in the 5% mTeSR1 group might be influenced by a Bax-independent pathway, such as GABA-induced KCl deficiency (Ikonomovic et al., 1997). Therefore, it is postulated that the addition of 2% mTeSR1 supplement to the IVC medium could reduce DNA fragmentation without harmful effects.

In conclusion, this study confirmed that adding mTeSR1 to IVC media enhances the quality and production efficiency of bovine blastocysts. The resulting blastocysts efficiently formed dome-shaped colonies with strongly positive SOX2 cells in outgrowth culture. These findings indicate that this IVP method could facilitate the establishment of bovine ESCs and outgrowth method could serve as an efficient and effective tool for evaluating blastocysts from IVF without the need for embryo transfer. Although, the efficiency of offspring production following embryo transfer of blastocysts produced by this method is necessary to be verified, this method could effectively be applied to various research resulting in understanding of the roles of key genes and signaling pathways associated with the quality of bovine embryos.

None.

Conceptualization, D-J.K., H.L., and D.K.; methodology, D.K., and D-J.K.; Investigation, D-J.K., and D.K.; data curation, D-J.K.; writing-original draft preparation, D.K., and D-J.K.; writing-review and editing, H.L., and D-J.K.; supervision, D-J.K.; project administration, D-J.K.; funding acquisition, H.L.

This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01620003)” Rural Development Administration, Republic of Korea, and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A1A01070671).

All procedures were approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (Permit No: NON2022-087).

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No potential conflict of interest relevant to this article was reported.