Analysis of atresia in bovine follicles using different methods: Flow cytometry, enzyme-linked immunosorbent assay, and classic histology

Abstract

This study was performed in order to validate flow cytometry as an acceptable method for analyzing follicular atresia in bovine granulosa cells by comparing it to two other techniques, histology and ELISA. Ovaries from 35 nontreated cows, all at different times of their estrous cycle, and 12 superovulated cows were collected. Superovulation treatments began between Days 9 and 12 (Day 0 = estrous), and animals were administered 8 doses of FSH-P (32 or 20 mg) at 12-h intervals over 4 days with or without the addition of 1 mg of prostaglandin s.c. on the third day. Animals were slaughtered after the last FSH-P injection. Granulosa cells from 133 follicles from nontreated cows and 85 follicles from superovulated cows were analyzed. Follicular diameters ranged from 2 to 20 and 2 to 16 mm, respectively. Because of the ample amounts of cells collected, it was possible to perform more than one technique for each follicle. Flow cytometry detected in most follicles a subpopulation of cells that possessed less DNA than normal, viable cells (referred to as -G1 cells). Histological classes used (established in previous work) were nonatretic (≤ 5% picnotic nuclei), slightly atretic (> 5 to < 15% picnotic nuclei), and atretic (≥ 15% picnotic nuclei). A strong linear correlation existed between the percentage of picnotic nuclei and the percentage of -G1 cells (R = 0.86; p < 0.001) with granulosa cells from follicles from nontreated cows. In some cases, flow cytometry detected a certain percentage of cells with reduced DNA content while histology revealed very few picnotic nuclei, indicating a higher sensitivity of flow cytometry. Superovulation decreased considerably the percentage of atretic cells seen with both techniques. The linear correlation was not as strong because follicles from superovulated animals represent a very homogenous population (R = 0.54; p < 0.001). The ELISA technique coincided with flow cytometry as seen in the strong correlation between the two techniques (R = 0.91; p < 0.001). Flow cytometry appeared to be very effective and rapid in evaluating the atretic states of follicles from nontreated and superovulated cows. Strong correlations existed between this method and histology and ELISA.