Growth of bovine oocyte-granulosa cell complexes cultured individually in microdrops of various sizes

In mammalian embryo culture, the embryo:medium volume ratio can substantially affect embryo developmental performance. In the present study, we tested the possibility of improving the growth of bovine oocytes by reducing the medium volume, from a typical volume used in mouse follicle culture to a minimum possible level. A total of 282 complexes, each containing a growing oocyte 87-100 mum in diameter, were individually placed in microdrops of 2, 5, 10 or 20 microl and cultured for 13 days in a modified TCM-199 supplemented with 4% polyvinylpyrrolidone (molecular weight: 360 kDa). Oocyte diameter was measured every other day to trace the growth of each oocyte. Half the medium was replaced every other day or every day, and comparison revealed that daily replacement was more favorable for culture of these microdrops. The highest survival rate, 95%, occurred in the 20-microl microdrops, where most oocytes continued to grow throughout the culture period. In comparison, in the 5- and 10-microl microdrops, more oocytes died, and growth slowed towards the end of culture. In the 2-microl microdrops, which had the highest death rate, growth virtually ceased after 9 days. The surviving oocytes were usually accompanied by a characteristic dome-like structure of the granulosa cell mass, except in the 2-microl microdrops. In conclusion, the 20-microl microdrops allowed oocyte growth at an acceptable level, and any further reduction of the volume only had a negative impact on oocytes.