ANALYSIS OF INDICATORS OF FERTILITY OF PORCINE OOCYTES THAT HAVE FINISHED GROWTH PHASE IN VIVO ASPIRATED FROM THE FOLLICLES OF DIFFERENT DIAMETERS

  • T. I. Kuzmina Russian Research Institute for Farm Animal Genetics & Breeding (St.Petersburg–Pushkin, Russia)
  • S. I. Kovtun Institute of Animal Breeding and Genetics nd. a. M.V.Zubets of NAAS (Chubynske, Ukraine)
  • E. C. Usenbekov Kazakh National Agrarian University (Almaty, Kazakhstan)
  • O. A. Epishko Grodno State Agrarian University (Grodno, Belarus)
  • V. N. Stefanova Institute of Cytology of the Russian Academy of Sciences (St.Petersburg-Pushkin, Russia)
Keywords: oocyte, follicle, embryos, Sus Scrofa Domesticus, BCB-test, in vitro

Abstract

The selection of competent oocytes to completion of meiosis in vitro, fertilization or reconstructing (cloning, transgenesis) is the initial stage of cell reproductive technologies in animal husbandry. The development of effective methods of early prediction prospective potencies for extracorporeal maturation and fertilization of oocyte is the actual problem of rapidly developing embryo technologies. Numerous factors determined developmental competence of the oocytes. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species, including pigs (Ericsson S. et al, Theriogenology, 39(1): p.214, 1993). BCB determines the intracellular activity of glucose-6-phosphate dehydrogenase, which plays an important role in cell growth, as a key enzyme in the pentose phosphate cycle. The enzyme activity in the growing oocyte increases, opposite in the oocytes that have finished growth phase it decreases (Alm et al., 2005). BCB - diagnostics of the initial population of oocytes based on staining with vital dye brilliant cresyl blue have proposed as an effective indicator of completion of oocyte growth phase.  

The aim of the present study was to evaluate the developmental competence of porcine oocytes that have finished growth phase (BCB+) in vivo depending on diameter (d) of follicles (d <3 mm, 3 –5 mm, <6 mm).

Before in vitro maturation compact cumulus oocyte complexes were incubated in BCB solution (13 μM) for 90 minutes. Treated oocytes were divided into BCB­-­ (colourless cytoplasm) and BCB+ (coloured cytoplasm). We have found that different diameter follicles contain both growing oocytes and oocytes that have finished growth phase in vivo (follicles d <3 mm – 71%; follicles d 3 - 5 mm – 86%; follicles d 6 – 8mm – 86%). Only BCB+ oocytes were used in the experiments. The medium used for oocyte maturation was NCSU 23 supplemented with 10% follicular fluid, 0.1 mg/ml cysteine,10 IU/ml eCG and 10 IU/ml hCG. Follicular fluid was collected from follicles with 3 - 6 mm in diameter. Oocyte cumulus complexes were cultured in maturation medium with pieces of wall (600 – 900 µmin length) from non athretic healthy follicles (d 3 – 6mm). After 20 – 22 h of culture, oocyte cumulus complexes and pieces of wall were washed and transferred into the same maturation medium but without hormonal supplements for another 20-22 h of culture. After in vitro maturation, oocytes were fertilized in vitro and embryos were cultured by standard protocols (Kuzmina et al., 2008). We have estimated oocyte maturation, quality of early embryos including status of chromatin (Tarkowsky, 1966). All chemicals used in this study were purchased from Sigma-Aldrich. Data were analyzed by Chi2 – test.

Oocytes that have finished their growth phase of examined species have shown high potency to maturation in all groups of experiment (follicles d <3 mm – 78%; follicles d 3 –5mm – 79%; follicles d 6 – 8 mm– 85%). Level of oocyte with degenerative chromatin had not significant differences in all groups of experiments. We did not find significant differences between the level of cleavage and blastocyst in all groups of experiments. Percentages of cleavage and blastocyst in the groups were: follicles d <3 mm– 43% (27/63) and 29% (18/63); follicles d 3 – 5 mm– 46% (45/98) and 35% (34/98); follicles d < 6 – 8 mm–48% (28/58) and 28% (16/58) (χ² test). Analysis of morphology and chromatin abnormalities in embryos has not shown significant differences between the groups of experiment.

Developmental competence of Sus Scrofa Domesticus oocytes that have finished growth phase in vivo, isolated from the follicles of various diameters (<3 mm, 3 – 5mm and 6 – 8mm) was analyzed. There were no significant differences in the level of cleavage and embryos on the blastocyst stage and their morphological characteristics. The findings suggest the equal potency to the maturation and fertilization of oocytes that have finished growth phase in vivo, independently of diameter of follicles.

References

1. Kuzmina, T. I., D. A. Novichkova, and N. A. Volkova. 2013. Modelirovanie sistem sozrevaniya ootsitov sviney in vitro – Modelling of maturation systems for porcine oocytes in vitro. Sel'skokhozyaystvennaya biologiya – Agricultural Biology. 2:52–57.
2. Kurome, M., H. Ueda, R. Tomii, K. Naruse, and H. Nagashima. 2006. Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer. Transgenic Research. 15:229–240.
3. Alm, H., H. Torner, and B. Lohrke. 2005. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology. 63:2194–2205.
4. Rodriguez-Gonzale E., M. Lopez-Bejar, E. Velilla, and M. T. Paramio. 2002. Selection of prepubertal goat oocytes using the brilliant cresyl blue test. Theriogenology. 57:1397–1409.
5. Roca, J., E. Martinez, J. M. Vazquez, and X. Lucas. 1998. Selection of immature pig oocytes for homologous in vitro penetration assays with the brilliant cresyl blue test. Reproduction, Fertility and Development. 10:479–486.
6. Alcoba, D. D., M. Conzatti, G. D. Ferreira, A.M. Pimentel , Kussler AP, E. Capp , H. von Eye Corleta, and I. S. Brum. 2016. Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells. Zygote. 24(1):83–88
7. Egerszegi, I., H. Alm, and J. Ritky. 2010. Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities. Reprod Fertil. Dev. 22:830–838.
8. Pawlak, P., E. Warzych, A. Chabowska, and D. Lechniak. 2014. Differences in Cytoplasmic Maturation Between the BCB+ and Control Porcine Oocytes Do Not Justify Application of the BCB Test for a Standard IVM Protocol. Journal of Reproduction and Development. 60(1):28–36.
9. Kuzmina, T. I., T. I. Stanislavovich, D. N. Tatarskaya, H. M. Mutieva, and I. Ya. Shahtamirov. 2015. ВСВ-diagnostics of donor's oocytes of bos taurus and sus scrofa domesticus - prospects in cell reproductive technologies. Questions regulatory veterinary medicine. 2:212–214.
10. Kuzmina, T. I., H. Alm, and J. Torner. 2008. Methods for producing of porcine embryos in vitro. St.-Petersburg–Pushkin, 36.
11. Tarkowski, A. 1966. An air-drying method for chromosomal preparation from mouse eggs. Cytogenetic. 1:394–400.
12. Luca, X., E. A. Martіnez, J. Roca , J. M. Vіzquez, M. A. Gil, L. M. Pastor, and J.L. Alabart. 2002. Relationship between antral follicle size, oocyte diameters and nuclear maturation of immature oocytes in pigs. Theriogenology. 58(5):871–85.
13. Dode, M.A.N., and C. N. Graves. Role of estradiol-17_ on nuclear and cytoplasmic maturation of pig oocytes. Animal Reproduction Science. 78 (2003):99–110
14. Kuzmina, T. V. Kravtsov, H. Alm, H. Torner, and K.-P. Brіssow. 2015. Developmental competence of porcine oocytes that have finished growth phase from follicles of different diameter. Anim. Reprod. 12(3):604.
15. Algriany, O., M. Bevers, E. Schoevers, B. Colenbrander, S. Dieleman. 2004. Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes. Theriogenology. 62(8):1483–97.
Published
2018-03-28
How to Cite
Kuzmina, T. I., Kovtun, S. I., Usenbekov, E. C., Epishko, O. A., & Stefanova, V. N. (2018). ANALYSIS OF INDICATORS OF FERTILITY OF PORCINE OOCYTES THAT HAVE FINISHED GROWTH PHASE IN VIVO ASPIRATED FROM THE FOLLICLES OF DIFFERENT DIAMETERS. Animal Breeding and Genetics, 51, 240-247. https://doi.org/10.31073/abg.51.32

Most read articles by the same author(s)