Effects of Cysteamine on Sheep Embryo Cleavage Rates

Öz

Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%). The first group was created as supplemented with cysteamine (Group A) and second group (Group B, control) without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM). After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium), Group AII-CR1aa (Charles Rosencrans medium), Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309), 55.44% (173/312), 65.34% (215/329), 59.34% (200/337) respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

Anahtar Kelimeler

• Sheep, cysteamine, maturation, cleavage rate, embryo

Sisteaminin Koyun Embriyo Bölünme Oranlarına Etkileri

Öz

In vitro kültür esnasında oksidatif stres gametlerin ve embriyoların gelişiminde hasarlara yol açar. Sisteamin, L-askorbik asit, beta merkaptoetanol, sistein, glutatyon gibi birçok antioksidanlar, proteinler, vitaminler oksidatif stresi önlemek için kültür medyumlarına eklenmiştir. Bu çalışma olgunlaşma medyumuna sisteamin ilavesinin koyun embriyonik bölünme oranları üzerine etkisinin saptanması için tasarlandı. Toplam 604 ovaryum 10 kerede toplandı ve 2060 koyun oositi elde edildi, doğrama metodu ile 1818 adet oosit kumulus kompleksleri seçilerek olgunlaşma amacıyla iki gruba ayrıldı (%88,25). İlk grup (Grup A) TCM-199 medyumuna sisteamin eklenen oositler, diğer grup (Grup B, kontrol) TCM-199 medyumuna sisteamin eklenmeyen oositler olarak oluşturuldu. İki grupta 24 saat boyunca 38,8 °C’de %5 CO2 altında, nemli havada in vitro maturasyon (IVM) için inkübe edildi. Oositler, IVM sonrası 50 x 107 / mL taze koç sperması ile BSOF medyumunda 18 saat süreyle döllendi. Döllenme sonrası, olgunlaşma grupları değişik kültür medyumları ile iki alt gruba ayrıldı ve Grup AI-SOF (Sentetik Ovidukt Sıvısı Medyumu), Grup AII-CR1aa (Charles Rosencrans Medyumu), Grup BI-SOF ve Grup BII-CR1aa elde edildi. Bölünme oranları döllenme sonrası 2. günde değerlendirildi. Bölünme oranları gruplar arasında istatistik açıdan önemli fark olmadan (P>0,05) sırasıyla, %59,54 (184/309), %55,44 (173/312), %65,34 (215/329), %59,34 (200/337) olarak saptandı. Sonuç olarak, TCM-199 olgunlaşma medyumuna sisteamin eklenmesinin SOF ve CR1aa kültür medyumlarındaki koyun embriyo bölünme oranları üzerine bir etkisinin olmadığı saptandı.

Anahtar Kelimeler

• Koyun, sisteamin, olgunlaşma, bölünme oranı, embriyo

Kaynakça

1. Arlotto, T., Schwartz, J.L., First, N.L., Leibfield-Rutledge, M.L., 1996. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 45, 943-956.
2. Camargo, L.S.A., Viana, J.H.M., Sa, W.F., Ferreira, A.M., Ramos, A.A., Filho, V.R.V., 2006. Factors influencing in vitro embryo production. Animal Reproduction 3, 19-28.
3. Cognie, Y., Baril, G., Poulin, N., Mermillod, P., 2003. Current status of embryo technologies in sheep and goat. Theriogenology 59, 171-188.
4. Fair, T., Hyttel, P., Greve, T., 1995. Bovine oocyte diameter in maturational relation transcritional activity. Molecular Reproduction and Development 42, 437-442. competence and
5. Farag, I.M., Girgis, S.M., Khalil, W.K.B., Hassan, N.H.A., Sakr, A.A.M., Abd Allah, S.M., Ali, N.I., 2009. Effect of hormones, culture media and oocyte qulity on in vitro maturation of Egyptian Sheep oocytes. Journal of Applied Biosciences 24, 1520-1534.
6. Freitas, V.J.F., Melo, L.M., 2010. In vitro embryo production in small ruminants. Revista Brasileira Zootecnia 39, 409-413.
7. Gardner, D.K., Lane, M., Spitzer, A., Batt, P.A., 1994. Enhanced rates of cleavage and deveolopment for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biology of Reproduction 50, 390-400.
8. Gardner, K.D., Weismann, A., Howles, M.C., Shoham, Z., 2004. Textbook of Assisted reproductive techniques. Informa Health Care, London, pp. 125-161.

9. Gasparrini, B., Boccia, L., Marchandise, J., Di Palo, R., George, F., Donnay, I., 2006. Enrichment of in vitro maturation medium for buffalo (Bubalis bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology 65, 275-287.
10. Gasparrini, B., Sayoud, H., Neglia, G., de Matos, D.G., Donnay, I., Zicarelli, L., 2003. Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development. Theriogenology 60, 943-952.
11. Gomez, M.C., Catt, J.W., Evans, G., Maxwell, W.M.C., 1998. Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization. Theriogenology 49, 1143-1154.
12. Gupta, S., Malhotra, N., Sharma, D., Chandra, A., Ashok, A., 2009. Oxidative stress and its role in female infertility and assisted reproduction: clinical implications. International Journal of Fertility and Sterility 2, 147- 164.
13. Lafleur, M.V., Hoorweg, J.J., Joenje, H., Westmijze, E.J., Retel, J., 1994. The ambivalent role of glutathione in the protection of DNA against singlet oxygen. Free Radical Research 21, 9-17.
14. Lagod, L., Paszkowski, T., Sikorski, R., Rola, R., 2001. The antioxidant-prooxidant complicated by spontaneous abortion. Ginekologia Polska 72, 1073-1078. balance in pregnancy
15. Lapointe, S., Sullivan, R., Sirard, M.A., 1998. Binding of a bovine oviductal fluid catalase to mammalian spermatozoa. Biology of Reproduction 58, 747-753.
16. Livingston, T., Rich, K., MacKenzie, S., Godkin, J.D., 2009. Glutathione content and antioxidant enzyme expression of in vivo matured sheep oocytes. Animal Reproduction Science 116, 265-273.
17. Natarajan, R., Shankar, M.B., Munuswamy, D., 2010a. Effect of α-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage. Journal of Assisted Reproduction and Genetics 27, 483-490.
18. Natarajan, R., Shankar, M.B., Munuswamy, D., 2010b. Effect of L-ascorbic acid supplementation at different gaseous environments on in vitro development of preimplantation sheep embryos to the blastocyst stage. Animal Reproduction Science 7, 21-28.
19. Oyawoye, O., Gadir, A.A., Garner, A., Constantinovici, N., Perrett, C., Hardiman, P., 2003. Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Human Reproduction 18, 2270-2274.
20. Perreault, S.D., Barbee, R.R., Slott, V.L., 1998. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturating hamster oocytes. Development Biology 125, 181-186.
21. Rizos, D., Ward, F., Duffy, P., Boland, M.P., Lonergan, P., 2002. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Molecular Reproduction and Development 61, 234-248.
22. Rosenkrans, C.F., Zeng, G.Q., McNamara, G.T., Schoff, P.K., Fist, N.L., 1993. Development of bovine embryos in vitro is affected by energy substrates. Biology of Reproduction 49, 459-462.
23. Sandal, A.İ., Özdaş, Ö.B., 2012. Modifiye TCM-199 medyumunda olgunlaştırılarak in vitro üretilen sığır embriyolarının dondurulması. Doktora Tezi, İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, İstanbul.
24. Shabankareh, H.K., Zandi, M., 2010. Developmental potential of sheep oocytes cultured in different maturation media: effects of epidermal growth factor, insulin-like growth factor I, and cysteamine. Fertility and Sterility 94, 335-340.
25. Sreenivas, D., Kaladhar, D.S.V.G.K., Yarla, N.S., Thomas, V.M., Palnisamy, A., 2013. Effect of protein supplementation on in vitro maturation of sheep oocytes and in vitro culture of preimplantation with α-tocopherol supplementation in CR1aa medium on sheep embryos to the blasocyst stage. Journal of Allergy and Therapy 4, 2-8.
26. Teleford, N.A., Watson, A.J., Schultz, G.A., 1990. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Molecular Reproduction and Development 26, 90-100.
27. Walker, S.K., Hill, J.L., Kleemann, D.O., Nancarrow, C.D., 1996. Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations. Biology of Reproduction 55, 703- 708.
28. Wani, A.R., Khan, M.Z., Sofi, K.A., Lone, F.A., Malik, A.A., Bhat, F.A., 2012. Effcet of cysteamine and epidermal growth factor (EGF) supplementation in maturation medium on in vitro maturation, fertilization and culturing of embryos in sheep. Small Ruminant Reearch 106, 160-164.