Heat shock during in vitro maturation of bovine oocytes disturbs bta‐miR‐19b and DROSHA transcripts abundance after in vitro fertilization

The objective of the present study was to asses the quality of various cultivation media used for the maturation of bovine oocytes that are prepared for IVF. Upon collection from slaughtered bovine ovaries and after morphological evaluation, a total number of 513 viable oocytes have been selected for cultivation, being divided into 3 batches, 171 oocytes / batch. The oocytes belonging to batch 1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH 20 μl/ml. The oocytes belonging to batch 2 were cultivated in TCM 199 NaHCO3 + 10% FCS + HCG 2.3 x 103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml. The oocytes belonging to batch 3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 1 μl/ml + FSH 20 μl/ml. The cultivation conditions, for all three batches, were: 24 hours at 39°C, 5% CO2. Spermatozoa have been prepared using the Percoll method and IVF of the matured oocytes has been performed. Embryonic development has been assessed 72 hours and then up to 10 days after IVF. The results showed the superior quality of the oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml, as their use for IVF yielded the highest number of viable embryos.

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Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6sup2p4277 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4277

Author(s):

Angelo Bertani Giotto ◽

Daniela Dos Santos Brum ◽

Francielli Weber Santos ◽

Antonio Carlos Galarça Guimarães ◽

Cibele Garcia Moreira Gonçalves ◽

...

Keyword(s):

Antioxidant Activity ◽

In Vitro Fertilization ◽

Oxygen Tension ◽

In Vitro Maturation ◽

Ros Production ◽

Vitro Fertilization ◽

Nuclear Maturation ◽

Cytoplasmic Maturation ◽

Bovine Oocytes

Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the in vitro maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P<0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of in vitro fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).

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