Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species

Abstract

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Number and density of equine preantral follicles in different ovarian histological section thicknesses

“…Furthermore, as expected, oocyte nucleus diameter and the number of granulosa cells in a cross-section plane also increased as the follicular classes advanced; these were novel results for the equine species. These findings together indicate the normal growth and development of preantral follicles and the gradual expansion of the follicular compartment, which have been associated with the increase of granulosa cell number in other species [26,27]. Another new finding was the percentage increase among the follicular classes (primordial to primary) for the diameter of follicles (31%), oocytes (22%), and oocyte nuclei (18%), and the number of granulosa cells (148%).…”

Morphologic characterization of isolated bovine early preantral follicles during short-term individual in vitro culture

“…First, the abundant stromal tissue of the ovaries hampers follicle isolation. Different mechanical [33] and enzymatic (collagenase [34] or liberase [35]) isolation methods have been suggested. Although optimal retrieval and minimal follicle damage are aimed for, follicle viability is often negatively influenced [35].…”

The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests

“…Dissection and culture methods developed for young or prime reproductive age domestic species need to be adapted because a considerable portion of the ovary may be devoid of follicles. Follicle isolation by mechanical 41 or enzymatic digestion 42 of the tissue can be considered although these protocols can damage follicles and decrease viability 14 , 43 . Another possibility is to develop a way to scale up the capacity of ovarian tissue culture methods to allow bulk processing of ovarian tissue.…”