In Vitro Fertilization of Immature Bovine Oocytes Matured in a Protein-Free Media and Their Subsequent Development to the Blastocysts

Öz

Fetal calf serum obtained from unborn calves causes pain to the calf. As a result of the ongoing discussions on issues such as less suffering to animals and animal welfare, especially in recent years, there has been a lot of research on the possibilities of using fetal serum substitutes in in vitro embryo and cell culture. However, the use of serum may cause some conflicts, especially in studies investigating single molecular pathways, due to the effects of molecules present in serum and whose concentration cannot be determined precisely on cellular functions. For this purpose, in this study, in which the possibilities of using serum substitutes were investigated, it was determined that the use of knockout serumTM for in vitro maturation significantly increased the number of embryos developing until the first division and blastocyst. On the other hand, it was determined that the use of synthetic serum substitution and serum substitution 2 slightly reduced the number of embryos obtained.

Anahtar Kelimeler

• IVF
• Serum replacement
• Bovine
• Maturation
• Blastocyst
• Oocyte

Protein İçermeyen Medya İle Olgunlaştırılan İmmatür Sığır Oositlerinin İn Vitro Fertilizasyonu ve Blastosiste Kadar Gelişimleri

Öz

Henüz doğmamış buzağılardan elde edilen fötal buzağı serumu, buzağının acı çekmesine neden olmaktadır. Hayvanlara daha az acı çektirilmesi ve hayvan refahı gibi konularda süregelen tartışmalar neticesinde özellikle son yıllarda, in vitro embriyo ve hücre kültüründe fötal serum ikamelerinin kullanılma olanaklarına dair çok sayıda araştırma yapılmaktadır. Bununla birlikte serum içerisinde mevcut olan ve konsatrasyonu tam olarak belirlenemeyen moleküllerin hücresel işlevler üzerine etkilerinden dolayı özellikle tekli moleküler yolakların araştırıldığı çalışmalarda serum kullanımı bazı çakışmalara neden olabilmektedir. Bu amaçla, serum ikamelerinin kullanım olanaklarının araştırıldığı bu çalışmada, in vitro matürasyon için knockout serumTM kullanımının ilk bölünme ve blastosiste kadar gelişen embriyo sayısının önemli oranda artırdığı tespit edilmiştir. Bununla birlikte sentetik serum ikamesi ve serum ikamesi 2 kullanımının ise elde edilen embriyo sayılarını bir miktar düşürdüğü belirlenmiştir.

Anahtar Kelimeler

• IVF
• Serum ikamesi
• Sığır
• Olgunlaştırma
• Blastosist
• Oosit

Kaynakça

1. Ali A, Sirard MA. 2002. Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation. Biol Reprod, 66(4): 901-905. DOI: 10.1095/biolreprod66.4.901.
2. Bavister BD, Rose-Hellekant TA, Pinyopummintr T. 1992. Development of in vitro matured/in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media. Theriogenology, 37(1): 127-146. DOI: 10.1016/0093-691X(92)90251-L.
3. Brackett BG, Zuelke KA. 1993. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39(1): 43-64.
4. Chanson A, Nocera D, Senn A, De Grandi P, Germond M. 2001. Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes. J Assist Reprod Genet, 18(2): 97-105. DOI: 10.1023/a:1026534725483.
5. Çevik M, Şen U, Kocyigit A, Soydan E, Kuran M. 2011. Effects of serum, gonadotropins, epidermal growth factor and estradiol 17-Beta on cumulus expansion and nuclear maturation of bovine oocytes. J Fac Vet Med, University of Kafkas, 17(6): 1009–1014.
6. Cevik M, Kocyigit A, Sen U, Kuran M. 2014. Can commercial human embryo culture media be used in bovine embryo culture? J Fac Vet Med, University of Kafkas, 20(1): 149–153.
7. Duque P, Gomez E, Diaz E, Facal N, Hidalgo C, Diez C. 2003. Use of two replacements of serum during bovine embryo culture in vitro. Theriogenology, 59(3-4): 889-899. DOI: 10.1016/s0093-691x(02)01134-2.
8. George F, Daniaux C, Genicot G, Verhaeghe B, Lambert P, Donnay I. 2008. Set up of a serum-free culture system for bovine embryos: Embryo development and quality before and after transient transfer. Theriogenology, 69(5): 612-623. DOI: 10.1016/j.theriogenology.2007.11.008

9. Grad I, Gajda B, Smorag Z. 2010. Effect of plant protein supplementation on in vitro development of porcine embryos. Animal Sci Papers Rep, 28(3): 271-279.
10. Korhonen K, Kananen K, Ketoja E, Matomaki J, Halmekytö M, Peippo J. 2010. Effects of serum-free in vitro maturation of bovine oocytes on subsequent embryo development and cell allocation in two developmental stages of day 7 blastocysts. Reprod Domest Anim, 45(1): 42-49. DOI: 10.1111/j.1439-0531.2008.01203.x.
11. Lonergan P, Fair T, Forde N, Rizos D. 2016. Embryo development in dairy cattle. Theriogenology, 86(1): 270-277. DOI: 10.1016/j.theriogenology.2016.04.040.
12. Mingoti GZ, Castro VSDC, Meo SC, Barretto LSS, Garcia JM. 2011. The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development. In Vitro Cell Dev Biol Anim, 47(5-6): 361-367. DOI: 10.1007/s11626-011-9400-0.
13. Motohashi HH, Taniguchi R, Sakamoto J, Sankai T, Kada H. 2017. Live, full-term mouse pups from oocytes grown and matured in vitro with serum substitutes. Reprod Biol, 17: 180-184.
14. Parl YH, Gong SP, Kim HY, Kim GA, Choi JH, Ahn JY, Lim HM. 2013. Development of a Serum-free defined system employing growth factors for preantral follicle culture. Mol Reprod Dev, 80(9): 725-733. DOI: 10.1002/mrd.22204.
15. Sağırkaya H, Üstüner B. 2010. Replacement of fetal calf serum with two different synthetic sera in in vitro maturation medium: effects on maturation, fertilization and subsequent development of cattle oocytes in vitro. Uludag Univ J Fac Vet Med, 29(2): 51-56.
16. Sakurai M, Suzuki C, Yoshioka K. 2015. Effect of knockout serum replacement supplementation to culture medium on porcine blastocyst development and piglet production. Theriogenology, 83(4): 679-686. DOI: 10.1016/j.theriogenology.2014.11.003.
17. Smetenina IG, Tatarinova LV, Krivokharchenko AS. 2006. In vitro fertilization of bovine oocytes in protein-free culture system. Ontogenez, 37(6): 438-443.
18. Sen U, Kuran M. 2018. Low incubation temperature successfully supports the in vitro bovine oocyte maturation and subsequent development of embryos. Asian-Australas J Anim Sci, 31(6): 827-834.