Opti-MEM® temelli oosit matürasyonu ve evreye özgü glikoz takviyesinin koyun embriyolarının in vitro gelişimi üzerine etkileri

Öz

Koyunlarda in vitro embriyo üretimi, yetersiz in vitro matürasyon (İVM) ve kültür koşulları nedeniyle kısıtlı kalmaktadır. Bu çalışmada, Opti-MEM®'in alternatif bir İVM ortamı olarak kullanılabilirliği ve aşamalı glikoz takviyesinin embriyonik gelişim ile blastosist kalitesi üzerine etkileri araştırıldı. Mezbaha kaynaklı ovaryumlardan elde edilen kumulus-oosit kompleksleri, hormon ve serum içeren TCM-199 veya Opti-MEM® ortamında 24 saat matüre edildi. İn vitro fertilizasyonun ardından embriyolar, iki farklı glikoz stratejisi uygulanan amino asit içeren sentetik ovidukt sıvısı (SOFaa) ortamında kültüre edildi: glikossuz ortamdan 1,5 mM glikoza geçiş (G0→G1,5) veya 1,5 mM'den 3,0 mM'e geçiş (G1,5→G3,0). Embriyonik gelişim 8. güne kadar izlendi; blastosist kalitesi, iç hücre kitlesi ve trofektoderm hücrelerinin diferansiyel boyanmasıyla değerlendirildi. Nükleer matürasyon oranları gruplar arasında benzer bulundu; metafaz II oranları TCM-199 ve Opti-MEM® gruplarında sırasıyla %74,62 ve %66,66 olarak saptandı (p>0,05). Embriyonik gelişim hem İVM ortamından hem de glikoz takviye stratejisinden anlamlı düzeyde etkilendi. G1,5→G3,0 protokolü üstün blastosist oluşumu ve kalitesi sağladı; en yüksek blastosist oranı ve iç hücre kitlesi hücre sayısı TCM-199 grubunda elde edildi (p<0,05). Erken dönem glikoz eksikliği blastosist verimini olumsuz etkiledi; bu etki Opti-MEM® kaynaklı embriyolarda daha belirgin gözlemlendi. Sonuç olarak, Opti-MEM® koyun oositlerinde nükleer matürasyonu TCM-199 ile karşılaştırılabilir düzeyde destekledi; ancak optimum blastosist gelişimi, matürasyon ortamına uygun aşamalı glikoz takviyesi gerektirdi.

Anahtar Kelimeler

• blastosist gelişimi
• embriyo kültürü
• glikoz ilavesi
• in vitro olgunlaşma
• Opti-MEM®

Destekleyen Kurum

İstanbul Üniversitesi

Proje Numarası

Project Number: TSG-2017-20659

Etik Beyan

Hayvansal kaynaklı materyallerin kullanıldığı tüm deneysel prosedürler, kurumsal ve ulusal etik düzenlemelere uygun olarak yürütülmüş olup, çalışma İstanbul Üniversitesi Deney Hayvanları Yerel Etik Kurulu tarafından onaylanmıştır (Onay No: 2017-295820).

Teşekkür

Yazarlar, bu çalışma kapsamında teşekkür edilmesi gereken herhangi bir kişi, kurum veya kuruluş bulunmadığını beyan etmektedir.

Effects of Opti-MEM®-based maturation and stage-specific glucose supplementation on ovine embryo development in vitro

Öz

In vitro embryo production (IVEP) in sheep remains constrained by suboptimal in vitro maturation (IVM) and culture conditions. This study evaluated Opti-MEM® as an alternative IVM medium for ovine oocytes and investigated the effects of stage-specific glucose supplementation on embryonic development and blastocyst quality. Cumulus–oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries were matured for 24 h in either TCM-199 or Opti-MEM®, both supplemented with hormones and serum. Following in vitro fertilization, embryos were cultured in a two-step synthetic oviduct fluid medium supplemented with amino acids (SOFaa) under two glucose strategies: glucose-free medium transitioning to 1.5 mM (G0→G1.5), or 1.5 mM transitioning to 3.0 mM glucose (G1.5→G3.0). Development was monitored through Day 8, and blastocyst quality was assessed by differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. Nuclear maturation rates were comparable between groups, with metaphase II rates of 74.62% and 66.66% in TCM-199 and Opti-MEM®, respectively (p>0.05). Embryo development was significantly influenced by both IVM medium and glucose supplementation strategy. The G1.5→G3.0 protocol yielded superior blastocyst formation and quality, with the highest blastocyst rate and ICM cell count recorded in the TCM-199 group (p<0.05). Conversely, early glucose deprivation reduced blastocyst yield, particularly in Opti-MEM®-derived embryos. These findings demonstrate that while Opti-MEM® supports ovine oocyte nuclear maturation comparably to TCM-199, optimal blastocyst development requires stage-specific glucose supplementation tailored to the maturation medium employed.

Anahtar Kelimeler

• blastocyst development
• embryo culture
• glucose supplementation
• in vitro maturation
• Opti-MEM®

Destekleyen Kurum

Istanbul University

Proje Numarası

Project Number: TSG-2017-20659

Etik Beyan

All experimental procedures involving animal-derived materials were conducted in accordance with institutional and national guidelines and were approved by the Experimental Animals Local Ethics Committee of Istanbul University (Approval No. 2017-295820).

Teşekkür

The authors declare that there are no acknowledgements.

Kaynakça

1. Al-Mutary, M., Al-Ghadi, M., Al-Himaidi, A., Iwamoto, D., Al-Anazi, Y., Ammari, A., Ahmad, J., & Al-Khedhairy, A. (2019). Using RT-PCR and glutathione level to study the effect of follicular fluid on in vitro maturation and gene expression of sheep oocytes. Saudi Journal of Biological Sciences, 26, 1216–1222. https://doi.org/10.1016/j.sjbs.2018.01.001
2. Alvites, R. D., Branquinho, M. V., Sousa, A. C., Lopes, B., Sousa, P., Mendonça, C., ... & Maurício, A. C. (2021). Small ruminants and its use in regenerative medicine: Recent works and future perspectives. Biology, 10(3), 249. https://doi.org/10.3390/biology10030249
3. Arıcı, R., Demir, K., Yağcıoğlu, S., Ozturk, G. B., Eser, A., Ersoy, N., ... & Pabuccuoğlu, S. (2025). The efficacy of human recombinant luteinizing hormone for in vitro embryo production in sheep. Turkish Journal of Veterinary Research, 9(1), 19–24. https://doi.org/10.47748/tjvr.1567856
4. Atalla, H., Evecen, M., Pabuccuoğlu, S., & Birler, S. (2018). Effects of equilibration media and co-culture on vitrification of sheep blastocysts derived in vitro. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 66(1), 83–87. https://doi.org/10.1501/Vetfak_0000002891
5. Birler, S., Pabuccuoğlu, S., Ak, K., Alkan, S., et al. (1999). Effects of serum and hormone additions to maturation medium on in vitro maturation of sheep oocytes. İstanbul Üniversitesi Veteriner Fakültesi Dergisi, 25(1), 75–79.
6. Birler, S., Pabuccuoğlu, S., Alkan, S., Özdaş, O. B., Atalla, H., & İleri, İ. K. (2002). Development of in vitro derived sheep embryos to the blastocyst stage. Turkish Journal of Veterinary and Animal Sciences, 26(4), 891–894.
7. Birler, S., Pabuccuoğlu, S., Alkan, S., Ak, K., Evecen, M., & İleri, İ. K. (2003). Effects of supplements to maturation and fertilization media and co-culture with sheep oviductal epithelial cells on in vitro production of sheep embryos. İstanbul Üniversitesi Veteriner Fakültesi Dergisi, 29(1), 157–165.
8. Braga, J. V., Camargo, J. D., Marques, M., Mondadori, R. G., & Lucia, T. (2025). The role of the pentose phosphate pathway on reproductive functions. Animal Reproduction, 22(4), e20240130. https://doi.org/10.1590/1984-3143-ar2024-0130

9. Chi, F., Sharpley, M. S., Nagaraj, R., Roy, S., & Banerjee, U. (2020). Glycolysis-independent glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis. Developmental Cell, 53(1), 9–26. https://doi.org/10.1016/j.devcel.2020.02.015
10. Conaghan, J., Handyside, A. H., Winston, R. M. L., & Leese, H. J. (1993). Effects of pyruvate and glucose on the development of human preimplantation embryo in vitro. Journal of Reproduction and Fertility, 99, 87–95. https://doi.org/10.1530/jrf.0.0990087
11. De Hertogh, R., Vanderheyden, I., Pampfer, S., Robin, D., Dufrasne, E., & Delcourt, J. (1991). Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro. Diabetes, 40, 641–647. https://doi.org/10.2337/diab.40.5.641
12. Guler, A., Poulin, N., Mermillod, P., Terqui, M., & Cognie, Y. (2000). Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes. Theriogenology, 54, 209–218. https://doi.org/10.1016/S0093-691X(00)00342-3
13. Herrick, J. R., Rajput, S., Pasquariello, R., Ermisch, A., Santiquet, N., Schoolcraft, W. B., & Krisher, R. L. (2020). Developmental and molecular response of bovine embryos to reduced nutrients in vitro. Reproduction and Fertility, 1(1), 51–65. https://doi.org/10.1530/RAF-20-0033
14. Krisher, R. L. (2019). Oocyte and embryo metabolomics. Bioscience Proceedings, 19(1). https://doi.org/10.1530/biosciprocs.19.0001
15. Kohri, N., Ota, M., Kousaku, H., Minakawa, E. N., Seki, K., & Tomioka, I. (2023). Optimization of piggyBac transposon-mediated gene transfer method in common marmoset embryos. Plos One, 18(6), e0287065. https://doi.org/10.1371/journal.pone.0287065
16. Lin, J., Xiang, Y., Huang, J., Zeng, H., Zeng, Y., Liu, J., Wu, T., Liang, Q., Liang, X., Li, J., & Zhou, C. (2022). NAT10 Maintains OGA mRNA Stability Through ac4C Modification in Regulating Oocyte Maturation. Frontiers in Endocrinology, 13, 907286. https://doi.org/10.3389/fendo.2022.907286
17. Martin, K. L., & Leese, H. J. (1995). Role of glucose in mouse preimplantation embryo development. Molecular Reproduction and Development, 40(4), 436–443. https://doi.org/10.1002/mrd.1080400407
18. Ramos-Ibeas, P., González-Brusi, L., Used, M. T., Cocero, M. J., Marigorta, P., Alberio, R., & Bermejo-Alvarez, P. (2022). In vitro culture of ovine embryos up to early gastrulating stages. Development, 149(6), dev199743. https://doi.org/10.1242/dev.199743
19. Reed, M. L., Jin, D. I., & Petters, R. M. (1992). Glucose and inorganic phosphate inhibits rat 8-cell embryo development in vitro. Theriogenology, 37, 282. https://doi.org/10.1016/0093-691X(92)90351-Q
20. Shabankareh, H. K., Sarsaifi, K., Mehrannia, T. (2011). In vitro maturation of ovine oocytes using different maturation media: Effect of human menopausal serum. Journal of Assisted Reproduction and Genetics, 28, 531–537. https://doi.org/10.1007/s10815-010-9523-3
21. Shi, L., Yue, W., Zhang, J., Lv, L., Ren, Y., & Yan, P. (2009). Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation. Animal Reproduction Science, 113, 299–304. https://doi.org/10.1016/j.anireprosci.2008.08.002
22. Shirazi, A., Ardali, M. A., Ahmadi, E., Nazari, H., Mamuee, M., & Heidari, B. (2012). The effect of macromolecule source and type of media during in vitro maturation of sheep oocytes on subsequent embryo development. Journal of Reproduction and Infertility, 13(1), 13–19.
23. Summers, M. C., Bhatnagar, P. R., Lawitts, J. A., & Biggers, J. D. (1995). Fertilization in vitro of mouse ova from inbred and outbred strains: Complete preimplantation embryo development in glucose-supplemented KSOM. Biology of Reproduction, 53, 431–437. https://doi.org/10.1095/biolreprod53.2.431
24. Tscherner, A., Brown, A. C., Stalker, L., Kao, J., Dufort, I., Sirard, M. A., & LaMarre, J. (2018). STAT3 signaling stimulates miR-21 expression in bovine cumulus cells during in vitro oocyte maturation. Scientific Reports, 8(1), 11527. https://doi.org/10.1038/s41598-018-29874-w
25. Thompson, J. G. E., Parton, G. A. J., Cruickshank, G. W., Smith, J. F., & Wales, R. G. (1989). Development of sheep preimplantation embryos in media supplemented with glucose and acetate. Theriogenology, 32(2), 323–330. https://doi.org/10.1016/0093-691X(89)90322-1
26. Thompson, J. G. E., Simpson, A. C., Pugh, P. A., Wright, R. W., & Tervit, H. R. (1991). Glucose utilization by sheep embryos derived in vivo and in vitro. Reproduction, Fertility and Development, 3, 571–576. https://doi.org/10.1071/RD9910571
27. Thompson, J. G. E., Simpson, A. C., Pugh, P. A., & Tervit, H. R. (1992). Requirement for glucose during in vitro culture of sheep preimplantation embryos. Molecular Reproduction and Development, 31(4), 253–257. https://doi.org/10.1002/mrd.1080310405
28. Thompson, J. G., McNaughton, C., Gasparrini, B., McGowan, L. T., & Tervit, H. R. (2000). Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. Reproduction, 118(1), 47–55. https://doi.org/10.1530/reprod/118.1.47
29. Thouas, G. A., Korfiatis, N. A., French, A. J., Jones, G. M., & Trounson, A. O. (2001). Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reproductive BioMedicine Online, 3(1), 25–29. https://doi.org/10.1016/S1472-6483(10)61960-8
30. Wani, N. A., Wani, G. M., Khan, M. Z., & Sidiqi, M. A. (1999). Effect of different factors on the recovery rate of oocytes for in vitro maturation and in vitro fertilization procedures in sheep. Small Ruminant Research, 34, 71–76. https://doi.org/10.1016/S0921-4488(99)00046-2
31. Wani, N. A., Wani, G. M., Khan, M. Z., & Salahudin, S. (2000). Effect of oocyte harvesting techniques on in vitro maturation and in vitro fertilization in sheep. Small Ruminant Research, 36, 63–67. https://doi.org/10.1016/S0921-4488(99)00097-8
32. Xie, H. L., Zhu, S., Zhang, J., Wen, J., Yuan, H. J., Pan, L. Z., Luo, M. J., & Tan, J. H. (2018). Glucose metabolism during in vitro maturation of mouse oocytes: A study using RNA interference. Journal of Cellular Physiology, 233(9), 6952–6964. https://doi.org/10.1002/jcp.26484
33. Zhu, J., Moawad, A. R., Wang, C., Li, H., Ren, J., & Dai, Y. (2018). Advances in in vitro production of sheep embryos. International Journal of Veterinary Science and Medicine, 6(1), 15–26. https://doi.org/10.1016/j.ijvsm.2018.02.003