Effects of glutathione on oocyte and embryo development in in vitro fertilization

Abstract

Objective: To evaluate the effect of glutathione (GSH) supplementation in maturation and adaptation media on oocyte development, embryo quality, and oocyte viability after vitrification.

Methods: The GSH concentrations were classified into four groups (0, 0.5, 1.0, and 1.5 mM) which were added to the maturation medium. The maturation process was conducted in vitro for 24 h. Following maturation, oocytes were fertilized with Bali bull semen for 5-6 h and then cultured for 48 h. The morphological quality of ocytes matured with GSH addition and the vitrification method used was evaluated. Parameters assessed included maturation rate, fertilization rate, embryo development, post-vitrification oocyte morphology, and quality of post-vitrification oocytes with GSH added to the adaptation medium.

Results: The addition of GSH to the maturation medium significantly improved oocyte quality and embryo development (P<0.05). Specifically, adding 1.5 mM GSH increased the percentage of oocytes reaching metaphase II from 57.6% without GSH oocytes to 79.0% with 1.5 mM GSH, two-pronuclei fertilization from 47.0% to 72.7%, embryo development from 37.1% to 57.2%, morula formation from 14.6% to 33.7%, and blastocyst formation from 8.1% to 23.8%. Additionally, the survival rate of oocytes post-vitrification increased to 75% with GSH supplementation.

Conclusions: The addition of 0.5-1.5 mM of GSH to the maturation and adaptation media significantly enhanced the metaphase II stage, fertilization rates, cleavage rates, and the survival of oocytes after vitrification. Among the concentrations of 1.5 mM was the most effective in increasing oocyte development and maintaining oocytes viability post-vitrification.

Keywords

Bali cattle / Embryo / Gluthatione / Oocyte / Vitrification

Conflict of interest statement

The authors have no conflict of interst to declare.

Acknowledgements

The authors would like to thank to Universitas Hasanuddin for providing financial support under Penelitian Fundamental Kolaboratif (PFK) (00323/UN4.22/PT.0103/2023), and highest appreciation to the National Research and Innovation Agency (BRIN) and World Class University Program, Indonesia Endowment Fund for Education, Universitas Diponegoro.

Funding

This study was financially supported by Universitas Hasanuddin through Penelitian Fundamental Kolaboratif (PFK) (letter of appointment number: 00323/UN4.22/PT.0103/2023).

Data availability statement

The data of this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Herry Sonjaya, Erni Damayanti, and Hikmayani Iskandar designed the study and wrote the draft article. Erni Damayanti, Hasbi Hasbi, Sudirman Baco, Eka Mulyawati Kaiin, Andi Mujnisa, and Kusumandari Indah Prahesti conducted the experiments and collected the literature. Aulia Uswa Noor Khasanah, Sri Firmiaty, and Asma’ul Fitriana Nurhidayah performed the data for analysis. Rukman Pala collected the oocyte from the slaughterhouse. Daud Samsudewa, Fitra Aji Pamungkas, and Herdis Herdis assisted with the literature collection and data interpretation. All authors read and approved the final version of the manuscript.

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References

[1] Zhou Y, Fu X, Zhou G, Jia B, Fang Y, Hou Y, et al. An efficient method for the sanitary vitrification of bovine oocytes in straws. J Anim Sci Biotechnol 2014; 5(1):1-7.

[2] Rahman NSA, Khan NAMN, Eshak Z, Sarbandi MS, Kamal AAM, Malek MA, et al. Exogenous L-glutathione improves vitrification outcomes in murine preimplantation embryos. Antioxidants 2022; 11(11):1-21.

[3] Mullen SF, Fahy GM. A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep. Theriogenology 2012; 78(8):17091719.

[4] Hwang IS, Hochi S. Recent progress in cryopreservation of bovine oocytes. Biomed Res Int 2014; 2014:570647.

[5] García-Martínez T, Vendrell-Flotats M, Martínez-Rodero I, Ordóñez-León EA, Álvarez-Rodríguez M, López-Béjar M, et al. Glutathione ethyl ester protects in vitro-maturing bovine oocytes against oxidative stress induced by subsequent vitrification/warming. Int J Mol Sci 2020; 21(20):1-26.

[6] Gunawan M, Nuriza N, Kaiin EM, Sjahfirdi L. The effect of glutathione antioxidant addition in maturation medium on the morphology of Garut sheep (Ovis aries) oocytes after vitrification. J Phys Conf Ser 2021; 1725:012062. doi: 10.1088/1742-6596/1725/1/012062.

[7] Iskandar H, Sonjaya H, Yusuf M, Hasbi H. Effect of the addition of insulin transferrin selenium on in vitro maturation and fertilization of Bali cattle oocytes. J Ilmu Tern Vet 2019; 24(3):95-102.

[8] Li F, Cui L, Yu D, Hao H, Liu Y, Zhao X, et al. Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos. J Cell Physiol 2019; 234(5):73847894.

[9] Olexiková L, Dujíčková L, Makarevich AV, Bezdíček J, Sekaninová J, Nesvadbová A, et al. Glutathione during post-thaw recovery culture can mitigate deleterious impact of vitrification on bovine oocytes. Antioxidants 2023; 12(1):1-17.

[10] Mogas T. Update on the vitrification of bovine oocytes and in vitro-produced embryos. Reprod Fertil Dev 2019; 31(1):105-117.

[11] Khazaei M, Aghaz F. Reactive oxygen species generation and use of antioxidants during in vitro maturation of oocytes. Int J Fertil Steril 2017; 11(2):63-70.

[12] Saleh WM. Assessment of different methods of bovine oocytes collection, maturation and in vitro fertilization of abattoir specimens. Iraqi J Vet Sci 2017; 31(1):55-65.

[13] Damayanti E, Sonjaya H, Baco S, Hasbi H. Apoptosis in Bali cattle embryo cells produced in vitro. World’s Vet J 2022; 12(4):462-467.

[14] Hasbi H, Iskandar H, Sonjaya H, Purwantara B, Arifiantini RI, Agil M, et al. Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility. Vet World 2024; 17(3):593-601.

[15] Truong TT, Gardner DK. Antioxidants increase blastocyst cryosurvival and viability post-vitrification. Hum Reprod 2020; 35(1):12-23.

[16] Sharma A, Tiwari M, Gupta A, Pandey AN, Yadav PK, Chaube SK. Journey of oocyte from metaphase- I to metaphase- II stage in mammals. J Cell Physiol 2018; 233(8):5530-5536.

[17] Hasbi H, Sonjaya H, Gustina S. Oocytes population and development competence of Bali cattle embryo in vitro with different ovarian reproductive statuses. Trop Anim Sci J 2022; 45(4):389-396.

[18] Malott KF, Reshel S, Ortiz L, Luderer U. Glutathione deficiency decreases lipid droplet stores and increases reactive oxygen species in mouse oocytes. Biol Reprod 2022; 106(6):1218-1231.

[19] Ren J, Hao Y, Liu Z, Li S, Wang C, Wang B, et al. Effect of exogenous glutathione supplementation on the in vitro developmental competence of ovine oocytes. Theriogenology 2021; 173:144-155.

[20] Sharifi-Rad M, Anil Kumar NV, Zucca P, Varoni EM, Dini L, Panzarini E, et al. Lifestyle, oxidative stress, and antioxidants: Back and forth in the pathophysiology of chronic diseases. Front Physiol 2020; 11:1-21.

[21] Golubkoca A, Leiva T, Snyder K, Schlegel C, Bonvicino SM, Agbaga MP, et al. Response of the glutathione (GSH) antioxidant defense system to oxidative injury in necrotizing enterocolitis Alena. Antioxidants 2023; 12(7):1385.

[22] Nurkarimah DA, Kaiin EM, Karja NWK, Setiadi MA. The effectivity of cysteamine supplementation on improving the in vitro fertilization rate of sheep oocytes. J Vet Sci 2021; 15:118-122.

[23] Pereira BA, Zangeronimo MG, Castillo-Martín M, Gadani B, Chaves BR, Rodríguez-Gil JE, et al. Supplementing maturation medium with insulin growth factor I and vitrification-warming solutions with reduced glutathione enhances survival rates and development ability of in vitro matured vitrified-warmed pig oocytes. Front Physiol 2019; 9:1-13.

[24] Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: Current state. Nutr J 2016; 15(1):1-22.

[25] Chakravarthi S, Jessop CE, Bulleid NJ. The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 2006; 7(3):271-275.

[26] Damayanti E, Sonjaya H, Baco S, Hasbi H, Kaiin EM. Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) profiles in follicular fluid, maturation media and embryo culture of Bali cattle. Adv Anim Vet Sci 2023; 11:1532-1539.

[27] Yusuflu MB, Çizmeci SÜ. The effect of antioxidants added to culture medium on blastocyst development rates. Turkish J Agric Food Sci Technol 2022; 10:1671-1679.

[28] Luberda Z. The role of glutathione in mammalian gametes. Reprod Biol 2005; 5(1):5-17.

[29] Dujíčková L, Makarevich AV, Olexiková L, Kubovičová E, Strejček F. Methodological approaches for vitrification of bovine oocytes. Zygote 2020; 29(1):1-11.

[30] Vendrell-Flotats M, Arcarons N, Barau E, López-Béjar M, Mogas T. Effect of heat stress during in vitro maturation on developmental competence of vitrified bovine oocytes. Reprod Domest Anim 2017; 52:48-51.