Effect of culture medium replacement protocol on the in vitro development of isolated caprine secondary follicles

Abstract

The aim of this study was to verify the influence of three different protocols for medium refreshing on the in vitro culture of isolated caprine preantral follicles. Independently of the protocol, preantral follicles were individually cultured for 18 days, the initial volume of medium was 25 μl, and the interval of medium refreshing was every two days. The protocols tested were: T1 (Control) – refreshing of 15 μl (removal of 15 μl of culture medium followed by the addition of the same volume of fresh medium), maintaining a final volume of 25 μl, T2 – only the addition of 5 μl of fresh medium every two days (the medium volume increases 5 μl for each change up to a final volume of 65 μl at day 18), and T3 – initial removal of 15 μl of medium in the first change, with addition of 20 μl of fresh medium (net increase of 5 μl in the final volume at each change). In the subsequent changes for T3, the amount of medium added in the previous change was removed, followed by the addition of the same volume plus 5 μl fresh medium (as occurred for T2 the final volume at day 18 is also 65 μl). Analyses of survival, diameter and antrum formation, as well as the rate of daily follicular growth were performed every 6 days. At the end of the culture period, normal oocytes ≥110 μm were destined for in vitro maturation (IVM). The results showed that only T2 (addition without removal of medium) maintained follicular survival until the end of the culture period. In day 18, both follicular diameter and the rate of daily growth was similar in T2 and T3 (Removal + Addition of medium), which were both higher than in T1 (Partial change). Moreover, T2 obtained a greater percentage of oocytes >110 μm destined for IVM and was the only treatment that achieved an oocyte in the telophase-I stage. In conclusion, periodic addition of medium is recommended because it is more practical, maintains survival and promotes the development of caprine preantral follicles in vitro.