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

Abstract

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.

Keywords

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

Supporting Institution

İstanbul Üniversitesi

Project Number

Project Number: TSG-2017-20659

Ethical Statement

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).

Thanks

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

Abstract

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.

Keywords

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

Supporting Institution

Istanbul University

Project Number

Project Number: TSG-2017-20659

Ethical Statement

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).

Thanks

The authors declare that there are no acknowledgements.

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