Czech J. Anim. Sci., 2026, 71(7):319-329 | DOI: 10.17221/61/2026-CJAS

Improved cryotolerance of bovine blastocysts by 1α,25-dihydroxyvitamin D3 supplementation during in vitro culture and post-thaw recoveryOriginal Paper

HakKyo Lee ORCID...1,2, Do-Hyun Kim ORCID...1, DaeJin Kwon ORCID...1
Department of Animal Biotechnology, Jeonbuk National University, Jeonju, Republic of Korea
International Agricultural Development and Cooperation Center, Jeonbuk National University, Jeonju, Republic of Korea

In vitro embryo production (IVP) is a valuable technique for advancing cattle breeding and reproductive biotechnology. Nevertheless, embryos produced in vitro often display reduced developmental competence and cryotolerance compared with their in vivo counterparts. Vitamin D is known to regulate many cellular functions, including growth and antioxidant defence. This study evaluated the impact of vitamin D supplementation during in vitro culture on bovine embryo development and post-thaw recovery. Oocytes were matured and fertilised in vitro and presumptive zygotes were cultured with 0 (control), 25, or 50 ng/ml of 1α,25-dihydroxyvitamin D3. Supplementation of 25 ng/ml vitamin D did not significantly alter cleavage or blastocyst rates; however, it increased the expression of IGF2R and SOD2. In contrast, 50 ng/ml impaired embryonic development. the VD/VD group showed higher blastocyst hatching rates, lower mitochondrial reactive oxygen species (ROS) levels, increased IGF2R expression and reduced BAX expression and BAX/BCL2L1 ratio (P < 0.05). Overall, these findings suggest that the low-dose vitamin D supplementation may support embryonic recovery from cryo-induced stress, potentially through the modulation of the oxidative balance and growth-related pathways, rather than by enhancing the pre-freezing developmental competence.

Keywords: apoptosis; cattle; cryopreservation; embryo quality

Received: May 14, 2026; Accepted: June 18, 2026; Prepublished online: July 24, 2026; Published: July 28, 2026  Show citation

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HakKyo L, Do-Hyun K, Kwon D. Improved cryotolerance of bovine blastocysts by 1α,25-dihydroxyvitamin D3 supplementation during in vitro culture and post-thaw recovery. Czech Journal of Animal Science. 2026;71(7):319-329. doi: 10.17221/61/2026-CJAS.
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    References

    1. Arias ME, Sanchez R, Felmer R. Evaluation of different culture systems with low oxygen tension on the development, quality and oxidative stress-related genes of bovine embryos produced in vitro. Zygote. 2012 Aug;20(3):209-17. Go to original source... Go to PubMed...
    2. Averaimo S, Milton RH, Duchen MR, Mazzanti M. Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress. FEBS Lett. 2010 May 17;584(10):2076-84. Go to original source... Go to PubMed...
    3. Banker M, Sorathiya D, Shah S. Vitamin D deficiency does not influence reproductive outcomes of IVF-ICSI: A study of oocyte donors and recipients. J Hum Reprod Sci. 2017 Apr-Jun;10(2):79-85. Go to original source... Go to PubMed...
    4. Banliat C, Mahe C, Lavigne R, Com E, Pineau C, Labas V, Guyonnet B, Mermillod P, Saint-Dizier M. The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo. BMC Genomics. 2022 Dec 19;23(1):839. Go to original source... Go to PubMed...
    5. Bo GA, Mapletoft RJ. Evaluation and classification of bovine embryos. Anim Reprod. 2013;10(3):344-8.
    6. Bognar Z, Csabai-Tanics TJ, Gorgey E, Miko E, Horvath-Szalai Z, Szekeres-Bartho J. The effect of calcitriol on the development and implantation capacity of embryos from hyper-stimulated mice. Front Immunol. 2023 Jul 21;14:1200704. Go to original source... Go to PubMed...
    7. Cagnone G, Sirard MA. The embryonic stress response to in vitro culture: Insight from genomic analysis. Reproduction. 2016 Dec;152(6):R247-61. Go to original source... Go to PubMed...
    8. Cajas YN, Canon-Beltran K, Ladron de Guevara M, Millan de la Blanca MG, Ramos-Ibeas P, Gutierrez-Adan A, Rizos D, Gonzalez EM. Antioxidant nobiletin enhances oocyte maturation and subsequent embryo development and quality. Int J Mol Sci. 2020 Jul 27;21(15):5340. Go to original source... Go to PubMed...
    9. Cao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. Oxidative stress and oocyte cryopreservation: Recent advances in mitigation strategies involving antioxidants. Cells. 2022 Nov 11;11(22):3573. Go to original source... Go to PubMed...
    10. Chen Z, Robbins KM, Wells KD, Rivera RM. Large offspring syndrome: A bovine model for the human loss-of-imprinting overgrowth syndrome Beckwith-Wiedemann. Epigenetics. 2013 Jun;8(6):591-601. Go to original source...
    11. Christakos S, Dhawan P, Verstuyf A, Verlinden L, Carmeliet G. Vitamin D: Metabolism, molecular mechanism of action, and pleiotropic effects. Physiol Rev. 2016 Jan;96(1):365-408. Go to original source... Go to PubMed...
    12. Consiglio M, Viano M, Casarin S, Castagnoli C, Pescarmona G, Silvagno F. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes. Exp Dermatol. 2015 Oct;24(10):748-53. Go to original source...
    13. de Souza VP, Jensen J, Whitler W, Estill CT, Bishop CV. Increasing vitamin D levels to improve fertilization rates in cattle. J Anim Sci. 2022 Jul 1;100(7):skac168. Go to original source...
    14. Ferre LB, Kjelland ME, Strobech LB, Hyttel P, Mermillod P, Ross PJ. Review: Recent advances in bovine in vitro embryo production: Reproductive biotechnology history and methods. Animal. 2020 May;14(5):991-1004. Go to original source...
    15. Grzechocinska B, Dabrowski FA, Cyganek A, Wielgos M. The role of vitamin D in impaired fertility treatment. Neuro Endocrinol Lett. 2013;34(8):756-62.
    16. Gualtieri R, Kalthur G, Barbato V, Di Nardo M, Adiga SK, Talevi R. Mitochondrial dysfunction and oxidative stress caused by cryopreservation in reproductive cells. Antioxidants (Basel). 2021 Feb 24;10(3):337. Go to original source... Go to PubMed...
    17. Janati Idrissi S, Le Bourhis D, Lefevre A, Emond P, Le Berre L, Desnoes O, Joly T, Buff S, Maillard V, Schibler L, Salvetti P, Elis S. Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process. Sci Rep. 2021 Jun 2;11(1):11618. Go to original source... Go to PubMed...
    18. Kageyama M, Ito J, Shirasuna K, Kuwayama T, Iwata H. Mitochondrial reactive oxygen species regulate mitochondrial biogenesis in porcine embryos. J Reprod Dev. 2021 Apr 21;67(2):141-7. Go to original source...
    19. Keane KN, Cruzat VF, Calton EK, Hart PH, Soares MJ, Newsholme P, Yovich JL. Molecular actions of vitamin D in reproductive cell biology. Reproduction. 2017 Jan 1;153(1):R29-R42. Go to original source... Go to PubMed...
    20. Krisher RL, Herrick JR. Bovine embryo production in vitro: Evolution of culture media and commercial perspectives. Anim Reprod. 2024 Sep 23;21(3):e20240051. Go to original source...
    21. Kwon DJ, Park KD, Lee H, Oh JD. Lipid-rich bovine serum albumin is beneficial for the early development of bovine embryos, but induces lipid droplet formation in the blastocyst. Czech J Anim Sci. 2023 Dec 1;68(11):443-50. Go to original source...
    22. Li M, Hu S, Sun J, Zhang Y. The role of vitamin D3 in follicle development. J Ovarian Res. 2024 Jul 17;17(1):148. Go to original source... Go to PubMed...
    23. Li M, Li L, Zhang L, Hu W, Shen J, Xiao Z, Wu X, Chan FL, Cho CH. 1,25-Dihydroxyvitamin D3 suppresses gastric cancer cell growth through VDR- and mutant p53-mediated induction of p21. Life Sci. 2017 Jun 15;179:88-97. Go to original source... Go to PubMed...
    24. Liu Y, Du Y, Lin L, Li J, Kragh PM, Kuwayama M, Bolund L, Yang H, Vajta G. Comparison of efficiency of open pulled straw (OPS) and Cryotop vitrification for cryopreservation of in vitro matured pig oocytes. CryoLetters. 2008 Jul-Aug;29(4):315-20.
    25. Lopera-Vasquez R, Hamdi M, Maillo V, Gutierrez-Adan A, Bermejo-Alvarez P, Ramirez MA, Yanez-Mo M, Rizos D. Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro. Reproduction. 2017 Apr;153(4):461-70. Go to original source... Go to PubMed...
    26. Ludwig T, Eggenschwiler J, Fisher P, D'Ercole AJ, Davenport ML, Efstratiadis A. Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol. 1996 Aug 1;177(2):517-35. Go to original source... Go to PubMed...
    27. Marsico TV, de Camargo J, Valente RS, Sudano MJ. Embryo competence and cryosurvival: Molecular and cellular features. Anim Reprod. 2019 Oct 23;16(3):423-39. Go to original source... Go to PubMed...
    28. Plasschaert RN, Bartolomei MS. Tissue-specific regulation and function of Grb10 during growth and neuronal commitment. Proc Natl Acad Sci U S A. 2015 Jun 2;112(22):6841-7. Go to original source... Go to PubMed...
    29. Ren L, Xuan L, Han F, Zhang J, Gong L, Lv Y, Zhang W, Yang S, Xu B, Yan Y, Guo L, Liu H, Wan Z, Liu L. Vitamin D supplementation rescues simvastatin induced myopathy in mice via improving mitochondrial cristae shape. Toxicol Appl Pharmacol. 2020 Aug 15;401:115076. Go to original source... Go to PubMed...
    30. Sirard MA. 40 years of bovine IVF in the new genomic selection context. Reproduction. 2018 Jul;156(1):R1-7. Go to original source... Go to PubMed...
    31. Smith LC, Therrien J, Filion F, Bressan F, Meirelles FV. Epigenetic consequences of artificial reproductive technologies to the bovine imprinted genes SNRPN, H19/IGF2, and IGF2R. Front Genet. 2015 Feb 25;6:58. Go to original source... Go to PubMed...
    32. Tveden-Nyborg PY, Alexopoulos NI, Cooney MA, French AJ, Tecirlioglu RT, Holland MK, D'Cruz NT. Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos. Theriogenology. 2008 Oct 15;70(7):1119-28. Go to original source... Go to PubMed...
    33. Wetscher F, Havlicek V, Huber T, Gilles M, Tesfaye D, Griese J, Wimmers K, Schellander K, Muller M, Brem G, Besenfelder U. Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle. Theriogenology. 2005 Jul 1;64(1):30-40. Go to original source... Go to PubMed...
    34. Xu F, Wolf S, Green O, Xu J. Vitamin D in follicular development and oocyte maturation. Reproduction. 2021 May 5;161(6):R129-37. Go to original source... Go to PubMed...
    35. Xu J, Lawson MS, Xu F, Du Y, Tkachenko OY, Bishop CV, Pejovic-Nezhat L, Seifer DB, Hennebold JD. Vitamin D3 regulates follicular development and intrafollicular vitamin D biosynthesis and signaling in the primate ovary. Front Physiol. 2018 Nov 14;9:1600. Go to original source... Go to PubMed...
    36. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, Broadbent PJ, Robinson JJ, Wilmut I, Sinclair KD. Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat Genet. 2001 Feb;27(2):153-4. Go to original source... Go to PubMed...

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