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You can request this article ( The effect of sperm-oocyte incubation time on in vitro embryo development using sperm from a tetraparental chimeric bull )

C. Sumantricorrespondenceemail, A. Boediono, M. Ooe, M. Murakami, S. Saha, T. Suzuki United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi 753, Japan May 27, 1997

Abstract For this study :

The present study was designed as 5 × 4 factorial to investigate the effects of using sperm from 5 bulls, and varied sperm-oocyte incubation times (5, 10, 15 and 20 h) on the fertilization, cleavage rates and blastocyst formation on an in vitro bovine embryo production system. The bulls included a tetraparental Chimera, its sires (Japanese Black and Limousin), its maternal grand-sires (Japanese Brown and Holstein). The proportion of polyspermy, 2-pronuclei formation, fertilization, cleavage and development to blastocyst were affected (p < 0.01) by the duration of sperm-oocyte incubation, as well as by the interaction between bulls and their corresponding sperm-oocyte incubation time. Blastocyst rate observed after 5 h in oocytes inseminated with Chimera, Japanese Black and Limousin were higher (p < 0.05) than those observed at 20 h incubation. The proportion of blastocysts from oocytes inseminated with Japanese Black observed at 10 h of incubation did not differ from that of Chimera, but both were higher (p < 0.05) than those observed for the Limousin, Japanese Brown and Holstein sires. The present study showed that there was an effect by the duration of sperm-oocyte incubation on in vitro embryo development. The optimal time of sperm-oocyte incubation for the Chimera was similar to that of its sires (Japanese Black and Limousin) but differed from its maternal grand-sires (Japanese Brown and Holstein). The fertilization rates for the sperm from the Holstein bull increased up to 15 h suggesting that this might be the only bull that would benefit from a long incubation period for insemination.

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Dear sir, Thank you a lot for your response.

The problem is, after 24 hours of incubation of bovine oocytes there isq a film between oocytes that does not allow me to observe them properly, before coming to co incubation with spermatozoa

Hi Hamza, I don't know if you found a response to your inquiry inside the article proposed by Saleh, but I think that if you cultured oocytes inside their cumulus oophorus, the film you see is the product of hyaluronic acid secreted by cumulus cells. Therefore, if you incubate the complexes in hyaluronidase for five minutes you should get rid of the film.

Please, let us know more details about your procedure and good luck.

Thank you very mutch sir, your response is very satisfactory. 

we are just using now folliculr flude with some hormons to do some trials. 

Hi. Firstly you can separte the OCCs mecanically from each other by cutting the "film" by a needle.The best, as was advised by  Arturo Bevilacqua, is to use hyaluronidase. However, according to your description, regarding the morpology of the complex,I am not sure  it will affect the cumulus cell mass. In such a case you will have to work carefully with 2 needles. Do it one by one (not more than 1 in a dish as they might stuck to each other) and try to flatten the cumulus on the dish. In such a case if the corona radiata cells are not densed, it might expose the Oocyte . Good Luck

Professor Brackett's Lab published numerous articles on bovine and other species in vitro fertilization, embryo development and cryopreservation.  The oocytes source was slaughter house ovaries.  You may like to review those publications.

In human ICSI procedure, removal of cumuls cells is necessary to confirm that oocytes are mature (Metaphase II).  For removal of cumulus cells, hyaluronidase is commonly used.  A brief exposure of oocytes in hyaluronidsae solution and gentle pipetting through Pasteur pipette removes these cells without any harmful effect on the oocytes or further development.   

Hi, it is same in bovine, prof Murid,  the removal of cumulus cells is the signe of maturation as well as the emission of the first  "polar globule" . that why I need to remove the film to verify the maturation and to select oocyte to be used in fertilization and remove degenerative ones.

I am very thankful for your contributons professors.

Hi, I quote Arturo's answer, then you can remove HA and cumulus cells just by vortex or by pipetting, depending what is your endpoint. If you need just meiotic assessment, just choose one of them, that's fine. If you have to keep oocyte viability, or fix for IF or doing IVF then you should develop other protocols. Hope this helps.

Thanks a lot Alberto, Murid Yona, Saleh and Arturu, ur responses are very very helpful. I ll tell u about the results when I try them.

Hope u happy new year

Nothing to add.  Good luck

Hi dr. Hamza I think all answers give you the ways for seperating oocyte film in dish ; I used to use pasture pippite and another needle like pasture ir insuline syringe to separate cumulus cells and also to keep ut to do ordinary ivf after that without any effect on it but to avoid it completely after that and to avoidavoid loss if oocyte after mechanical pippiting you must work in minimum range of maturation hours ; I mean in if maturation hours 22-24 hours we can take oocyte to wash them and complete your experiment at 22hours and this is from my observations during my work; I wish it could help you , good luck