Öz
Memeli embriyolarının vitrifikasyonu, gametlerin uzun dönem korunması için önemli bir tekniktir. Vitrifikasyon öncesinde en uygun kültür ortamının seçimi, embriyoların çözme sonrası hayatta kalma oranlarını artırmak için hayati önem taşımaktadır. Bu çalışma, Arı sütü (AS) ve fetal sığır serumu (FBS) takviyesinin sığır embriyo gelişimi ve kriyotolerans üzerindeki etkilerini inceleyerek, çözme sonrası canlılık, blastosistlerin hayatta kalma oranları ve hücresel bütünlükleri üzerindeki etkilerini araştırmayı amaçlamaktadır.
Sığır oositleri, kültür ortamında %10 FBS veya %0.625 AS takviyesi ile in vitro olgunlaştırılmıştır. In vitro fertilizasyon (IVF) işleminin ardından oluşan zigotlar 8. güne kadar FBS veya AS ila edilen sentetik oviduktal sıvı (SOF) içinde kültüre edilmiştir. 8. Güne gelen Blastosistler, açık uçlu çekilmiş pipetler (OPS) yöntemi ile etilen glikol (EG) ve dimetil sülfoksit (DMSO) içeren bir kriyoprotektan solüsyon kullanılarak vitrifike edilmiştir. Çözme sonrası hayatta kalma oranları 12 ve 24 saat sonra değerlendirilmiştir. Embriyo kalitesi, blastosist çapı ve toplam hücre sayısı temelinde Hoechst 33258 boyama yöntemi kullanılarak floresan mikroskop altında belirlenmiştir. Elde edilen ANOVA ile analiz edilmiş ve gruplar Tukey HSD testi ile karşılaştırılmıştır.
Blastosist gelişim oranları, AS ile desteklenen grupta (%16.3 ± 3.36) FBS grubuna kıyasla (%24.8 ± 2.58, P < 0.05) anlamlı olarak daha düşük bulunmuştur. Bu durum, AS'nin erken embriyonik gelişimi etkileyebileceğini göstermektedir. Bununla birlikte, azalan blastosist gelişimine rağmen, AS ile kültür edilen blastosistlerin çözme sonrası 12 saatlik hayatta kalma oranları, FBS grubuyla benzer seviyede bulunmuştur (FBS-V: %51.3 ± 3.72 vs. RJ-V: %48.3 ± 4.09, P > 0.05). 24 saatlik hayatta kalma oranları ise AS grubunda sayısal olarak daha yüksek olmasına rağmen (RJ: %50.7 ± 3.41, FBS: %43.5 ± 4.25), bu fark istatistiksel olarak anlamlı bulunmamıştır. Ayrıca, blastosist hücre sayıları ve çapları AS grubunda FBS grubuna kıyasla anlamlı olarak daha düşük bulunmuştur (P < 0.05). Elde edilen sonuçlar, AS'nin embriyolarda yapısal bütünlüğü olumsuz etkilemediğini ve kriyozarara karşı FBS grubu ile benzer etki gösterdiğini düşündürmektedir.
AS takviyesi, blastosist gelişim oranını düşürmesine rağmen kriyotoleransı olumsuz etkilememiştir. Çözme sonrası hayatta kalma oranlarının korunması ve hücresel bütünlüğün benzer kalması, AS'nin embriyolar üzerindeki olası koruyucu etkilerini işaret etmektedir. Bu koruyucu etkiler, AS'nin içerdiği antioksidanlar ve kollajen benzeri proteinler gibi biyoaktif bileşiklere bağlanabilir ve vitrifikasyon sırasında oluşabilecek oksidatif stres ile membran hasarını azaltabilir. Elde edilen bulgular, AS'nin embriyo kalitesini ve donma sonrası hayatta kalma oranları artırmada potansiyel bir alternatif takviye olarak değerlendirilebileceğini göstermektedir. Bununla birlikte, AS'nin optimum konsantrasyonunun belirlenmesi ve vitrifikasyon sürecinde spesifik koruyucu mekanizmalarının aydınlatılması için daha fazla araştırmaya ihtiyaç duyulmaktadır.
Anahtar Kelimeler
Etik Beyan
Etik Kurul İzni Gerkemez
Kaynakça
- Abdulnabi, A. S., & Daham, A. F. (2021). Royal jelly improved in vitro matured bovine oocytes. Annals of the Romanian Society for Cell Biology, 7653.
- Carolan, C., Lonergan, P., Van Langendonckt, A., & Mermillod, P. (1995). Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro. Theriogenology, 43, 1115-1128.
- Choi, Y. H., Carnevale, E. M., Seidel, G. E. Jr., & Squires, E. L. (2001). Effects of gonadotropins on bovine oocytes matured in TCM-199. Theriogenology, 56, 661-670.
- Dorland, M., Gardner, D. K., & Trounson, A. O. (1994). Serum in synthetic oviductal fluid causes mitochondrial degeneration in ovine embryos. Journal of Reproduction and Fertility, Abstract Series, 13, 25.
- Fukui, Y., McGowan, L. T., James, R. W., Asher, G. W., & Tervit, H. R. (1991). Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes. Theriogenology, 35, 499-512.
- Gardner, D. K. (1994). Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biology International, 18, 1163-1170.
- Gardner, D. K., & Lane, M. (1999). Embryo culture systems. In Trounson, A., & Gardner, D. K. (Eds.), Handbook of in vitro fertilization (pp. 85-114). CRS Press, New York.
- Gomez, E., Rodríguez, A., Muñoz, M., Caamaño, J. N., Hidalgo, C. O., Morán, E., Facal, N., & Díez, C. (2007). Serum-free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification. Theriogenology, 69, 1013-1021.
- Kong, I. K., Choi, J. L., Cho, S. G., Bae, I. H., Oh, D. H., Oh, H. J., Kim, H. R., & Kim, J. K. (2001). Effect of addition of royal jelly in tris-buffer extender on the post-thaw viability of canine semen. Theriogenology, 55, 309.
- Kridli, R. T., Husein, M. Q., & Humphrey, W. D. (2003). Effect of royal jelly and GnRH on the estrus synchronization and pregnancy rate in ewes using intravaginal sponges. Small Ruminant Research, 49, 25-30.
- Leibo, S. P., Pollard, J. W., & Martino, A. (1995). Chilling and freezing sensitivity of “reassembled” in vitro derived bovine embryos. Theriogenology, 39, 81-94.
- Lim, J. M., Okitsu, O., Okuda, K., & Niwa, K. (1994). Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro. Theriogenology, 41, 1091-1098.
- Maurer, R. R. (1992). Animal cell culture: A practical approach. In Freshney, R. I. (Ed.), Oxford University Press, Oxford (pp. 15-46).
- Onal, A. G., Guzey, Y. Z., Kariptas, S., & Gocmez, Z. (2006). Effects of honeybee royal jelly on fertilization of matured bovine oocytes. Book of Abstracts of the 57th Annual Meeting of the European Association for Animal Production, No. 6, p. 185.
- Parrish, J. J., Susko-Parrish, J. L., Leibfried-Rutledge, M. L., Crister, E. S., Eyestone, W. H., & First, N. L. (1986). Bovine in vitro fertilization with frozen-thawed semen. Theriogenology, 25, 591-600.
- Schmidt, J. O., & Buchmann, S. L. (1992). Other products of the hive. In Graham, J. M. (Ed.), The Hive and The Honey Bee (pp. 927-988). Dadant and Sons, Hamilton, IL.
- Shamsuddin, M., & Rodriguez-Martinez, H. (1994). Fine structure of bovine blastocysts developed either in serum-free medium or in conventional co-culture with oviduct epithelial cells. Journal of Veterinary Medicine - Series A, 41, 307-316.
- Suo, Z., Zhou, G. B., Meng, Q. G., Yan, C. L., Fan, Z. Q., Zhao, X. M., Fu, X. W., Wang, Y. P., Zhang, Q. J., & Zhu, S. E. (2008). OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development. Zygote, 17, 71–77.
- Tervit, H. R., Whittingham, D. G., & Rowson, L. E. (1972). Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil., 30(3), 493-497.
- Thompson, J. G., Gardner, D. K., Pugh, P. A., McMillan, W. H., & Tervit, H. R. (1995). Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biology of Reproduction, 53, 1385-1391.
- Vajda, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T., & Callesen, H. (1998). Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development, 51, 53-58.
- Vajta, G., Holm, P., Greve, T., & Callesen, H. (1996). Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Animal Reproduction Science, 45, 191-200.
- Van Langendonckt, A., Auquier, P., Donnay, I., Massip, A., & Dessy, F. (1996). Acceleration of in vitro bovine embryo development in the presence of fetal calf serum. Theriogenology, 45, 194-194.
Öz
Vitrification of mammalian embryos is an important process for the long-term preservation of gametes, providing the storage for extended periods. Selection of an optimal culture medium prior to vitrification is also critical to improve post-thaw survival rates. The present study aimed to evaluate the effects of royal jelly (RJ) and fetal bovine serum (FBS) supplementation on bovine embryo development and cryotolerance by evaluating their impact on post-thawing viability, blastocyst survival rates, and cellular integrity following vitrification.
Bovine oocytes were matured in vitro in media supplemented with either 10% FBS or 0.625% RJ. Matured oocytes in vitro fertilized (IVF) and presumptive zygotes were further cultured in synthetic oviductal fluid (SOF) supplemented with their respective treatment (FBS or RJ) until day 8. Blastocysts were vitrified using the open-pulled straw (OPS) method with a cryoprotectant solution containing ethylene glycol (EG) and dimethyl sulfoxide (DMSO). Post-thaw survival rates were assessed at 12 and 24 hours. In order to evaluate embryo quality, blastocysts diameter was recorded and total blastocyst cell numbers were determined using Hoechst 33258 staining under a fluorescence microscope. Data were analysed using one-way ANOVA, with Tukey’s HSD test applied for pairwise comparisons.
Blastocyst development rates were significantly lower in the RJ-supplemented group (16.3% ± 3.36) compared to the FBS group (24.8% ± 2.58, P < 0.05), suggesting that RJ may affect early embryonic development. Despite the decreased blastocyst development, RJ-derived blastocysts exhibited comparable post-thaw survival rates to their FBS counterparts at 12 hours (FBS-V: 51.3% ± 3.72 vs. RJ-V: 48.3% ± 4.09, P > 0.05). Although, survival rates remained numerically higher in the RJ group (50.7% ± 3.41) compared to the FBS group (43.5% ± 4.25) at 24 hours, the difference was not statistically significant. Additionally, blastocyst cell counts and diameters were significantly lower in the RJ group than in the FBS group (P < 0.05) at the post-thaw vitrification, suggesting enhanced structural integrity and resilience remains similar to FBS group against cryoinjury.
RJ supplementation resulted in a reduced blastocyst development and appeared to have no negative effects on the cryotolerance of embryos, as indicated by similar post-thaw survival rates and improved cellular integrity. The potential protective effects of RJ may be attributed to its bioactive compounds, including antioxidants and collagen-like proteins, which may mitigate oxidative stress and membrane damage during vitrification. These findings suggest that RJ could serve as a promising alternative supplement for improving embryo quality and cryosurvival. However, further research is required to optimize RJ concentration and elucidate its specific protective mechanisms in vitrified embryos.
Anahtar Kelimeler
Etik Beyan
No need for Ethical Statement
Kaynakça
- Abdulnabi, A. S., & Daham, A. F. (2021). Royal jelly improved in vitro matured bovine oocytes. Annals of the Romanian Society for Cell Biology, 7653.
- Carolan, C., Lonergan, P., Van Langendonckt, A., & Mermillod, P. (1995). Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro. Theriogenology, 43, 1115-1128.
- Choi, Y. H., Carnevale, E. M., Seidel, G. E. Jr., & Squires, E. L. (2001). Effects of gonadotropins on bovine oocytes matured in TCM-199. Theriogenology, 56, 661-670.
- Dorland, M., Gardner, D. K., & Trounson, A. O. (1994). Serum in synthetic oviductal fluid causes mitochondrial degeneration in ovine embryos. Journal of Reproduction and Fertility, Abstract Series, 13, 25.
- Fukui, Y., McGowan, L. T., James, R. W., Asher, G. W., & Tervit, H. R. (1991). Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes. Theriogenology, 35, 499-512.
- Gardner, D. K. (1994). Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biology International, 18, 1163-1170.
- Gardner, D. K., & Lane, M. (1999). Embryo culture systems. In Trounson, A., & Gardner, D. K. (Eds.), Handbook of in vitro fertilization (pp. 85-114). CRS Press, New York.
- Gomez, E., Rodríguez, A., Muñoz, M., Caamaño, J. N., Hidalgo, C. O., Morán, E., Facal, N., & Díez, C. (2007). Serum-free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification. Theriogenology, 69, 1013-1021.
- Kong, I. K., Choi, J. L., Cho, S. G., Bae, I. H., Oh, D. H., Oh, H. J., Kim, H. R., & Kim, J. K. (2001). Effect of addition of royal jelly in tris-buffer extender on the post-thaw viability of canine semen. Theriogenology, 55, 309.
- Kridli, R. T., Husein, M. Q., & Humphrey, W. D. (2003). Effect of royal jelly and GnRH on the estrus synchronization and pregnancy rate in ewes using intravaginal sponges. Small Ruminant Research, 49, 25-30.
- Leibo, S. P., Pollard, J. W., & Martino, A. (1995). Chilling and freezing sensitivity of “reassembled” in vitro derived bovine embryos. Theriogenology, 39, 81-94.
- Lim, J. M., Okitsu, O., Okuda, K., & Niwa, K. (1994). Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro. Theriogenology, 41, 1091-1098.
- Maurer, R. R. (1992). Animal cell culture: A practical approach. In Freshney, R. I. (Ed.), Oxford University Press, Oxford (pp. 15-46).
- Onal, A. G., Guzey, Y. Z., Kariptas, S., & Gocmez, Z. (2006). Effects of honeybee royal jelly on fertilization of matured bovine oocytes. Book of Abstracts of the 57th Annual Meeting of the European Association for Animal Production, No. 6, p. 185.
- Parrish, J. J., Susko-Parrish, J. L., Leibfried-Rutledge, M. L., Crister, E. S., Eyestone, W. H., & First, N. L. (1986). Bovine in vitro fertilization with frozen-thawed semen. Theriogenology, 25, 591-600.
- Schmidt, J. O., & Buchmann, S. L. (1992). Other products of the hive. In Graham, J. M. (Ed.), The Hive and The Honey Bee (pp. 927-988). Dadant and Sons, Hamilton, IL.
- Shamsuddin, M., & Rodriguez-Martinez, H. (1994). Fine structure of bovine blastocysts developed either in serum-free medium or in conventional co-culture with oviduct epithelial cells. Journal of Veterinary Medicine - Series A, 41, 307-316.
- Suo, Z., Zhou, G. B., Meng, Q. G., Yan, C. L., Fan, Z. Q., Zhao, X. M., Fu, X. W., Wang, Y. P., Zhang, Q. J., & Zhu, S. E. (2008). OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development. Zygote, 17, 71–77.
- Tervit, H. R., Whittingham, D. G., & Rowson, L. E. (1972). Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil., 30(3), 493-497.
- Thompson, J. G., Gardner, D. K., Pugh, P. A., McMillan, W. H., & Tervit, H. R. (1995). Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biology of Reproduction, 53, 1385-1391.
- Vajda, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T., & Callesen, H. (1998). Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development, 51, 53-58.
- Vajta, G., Holm, P., Greve, T., & Callesen, H. (1996). Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Animal Reproduction Science, 45, 191-200.
- Van Langendonckt, A., Auquier, P., Donnay, I., Massip, A., & Dessy, F. (1996). Acceleration of in vitro bovine embryo development in the presence of fetal calf serum. Theriogenology, 45, 194-194.
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Zootekni, Genetik ve Biyoistatistik |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Yayımlanma Tarihi | 30 Haziran 2025 |
| Gönderilme Tarihi | 10 Mart 2025 |
| Kabul Tarihi | 1 Haziran 2025 |
| Yayımlandığı Sayı | Yıl 2025 Cilt: 40 Sayı: 1 |
Kaynak Göster
“Çukurova Üniversitesi Ziraat Fakültesi Dergisi” yayın hayatına 1 Ocak 2016 tarihi itibariyle “Çukurova Tarım ve Gıda Bilimleri Dergisi” adıyla devam etmektedir.
