The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.
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