Chronological Changes of Bovine Follicular Oocyte Maturation in Vitro

Abstract

Chronological changes of bovine follicular cumulus-oocyte-complexesi were studied after in vitro maturation over a period of 48 h. According to their thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enriched Ham’s F-10 medium with or without human chorionic gonadotrophin (hCG) and estradiolbenzoate (EB) for 0, 6, 12, 18, 21, 24, 27, 30 and 48 h. Representative samples were taken at each time interval for evaluation of nuclear maturation stages, ooplasm quality and size of the peri vitelline space (PVS). The results showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture, and the peak of the first polar abstriction occurred at 24 h. The culture period required for ONBD and abstraction of the first polar body were related to the thickness and compactness of cumulus investments with and approximately 6 h delay in heavily compacted complexes. Ooplasm quality evaluation failed to show a clear trend, but the PVS increased in size from 0 h to 30 h and then, retracted again from 30 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substantial proportion (68.8 %) of nude or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophase I was observed. In the absence of hCG and EB the percentage of oocytes reaching metaphase II was much lower (48.6%) in comparison with oocytes matured in the presence of these hormones (79.1 %). It was concluded a very high proportion of slaughterhouse oocytes could be matured in vitro and that the cumulus investments and addition of certain hormones affected the maturation rate.

Sammendrag

Oocytmodningens kronologiske forløb blev studeret efter in vitro modning af slagtehusoocyter. Deres cumulus oophorus, perivitelline rum størrelse samt cytogenetiske karakteristika blev undersøgt umiddelbart efter aspiration (kontroloocyter) og efter dyrkning i henholdsvis 6, 12, 18, 21, 24, 27, 30 og 48 timer i Ham’s F-10 medium eller Ham’s F-10 medium tilsat humant chorion-gonadotropin (hCG) og østradiolbenzoat (EB). Resultaterne viste, at oocytkernen blev nedbrudt (ONBD) inden for de første 6–12 timer, og at hovedparten af oocyteme afsnørede deres første pollegeme (PB1) efter 24 timers dyrkning in vitro. Begge disse processors tidsmæssige forløb var på-virket af cumuluscellelagenes antal og tæthed, således at oocyter omgivet af mange lag, taetsluttende cumulusceller brugte længere tid både til ONBD og til udstødelse af PB1.

Oocytmodningen var ligeledes under indflydelse af dyrknings-mediets hormonindhold, idet Ham’s F-10 med hCG og EB resulterede i en højere modningsrate (79,1 %) and Ham’s F-10 uden hormoner (48,6 %), bl. a. fordi en stor procentdel af oocyterne tilhørende denne sidste gruppe ikke nåede metafase II stadiet, men standsede ved metafase I og anafase-telofase I.

Nærvaerende undersøgelse har vist, at en meget høj procentdel af slagtehusoocyter kan modnes in vitro, at tilsætning af hormoner samt cumuluscellelagenes antal og tæthed påvirker det kronologiske forløb af denne proces.