Elsevier

Theriogenology

Volume 78, Issue 4, 1 September 2012, Pages 731-736
Theriogenology

Research article
Effect of holding medium, temperature and time on structural integrity of equine ovarian follicles during the non-breeding season

https://doi.org/10.1016/j.theriogenology.2012.03.019Get rights and content

Abstract

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h.

Introduction

In the last few years, there has been increased interest in assisted reproductive biotechnologies to produce foals from mares that died unexpectedly or were euthanized. In these cases, the ovaries can be collected after death and processed to recover viable oocytes [1], [2], [3] for in vitro maturation and fertilization. Preantral follicles represent 90% of the follicular population and constitute a potential source of gametes, with the possibility of yielding thousands of oocytes for assisted reproductive biotechnologies [4]. However, a limiting factor for the successful use of these oocytes is the condition of the follicles after removal from the ovaries and transportation of the ovaries to the laboratory. Another important consideration is the non-breeding season, when reproductive activity is limited [5], [6].

Many researchers have analyzed ovarian tissue preservation protocols for short intervals to compare combinations of temperatures and media for several species [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. The type of medium previously used for in vitro studies has an important influence on survival and growth of follicles. For example, Minimal Essential Medium (MEM) has been used for culturing bovine ovarian tissue [20] and isolating preantral follicles [21]. Furthermore, phosphate-buffered saline (PBS) is often used for transportation of embryos and oocytes in several species [22], [23], [24], [25], [26], [27], [28], [29].

Equine preantral follicles were isolated from ovaries using collagenase [30] or collagenase and desoxyribonucleases [31]. After isolation, individual follicles can be submitted to in vitro culture. Another biological model is preantral follicles within ovarian tissue, but there are no reports of the viability of these follicles after various intervals of transport, temperatures, and media. Therefore, development of protocols for preservation of equine ovarian tissue is crucial to ensure oocyte quality and, consequently, the success of reproductive biotechnologies.

The purpose of this study was to investigate the effect of time, temperature and holding medium on the structural integrity of equine follicles from ovaries obtained during the non-breeding season.

Section snippets

Collection of ovaries

Ovaries (n = 10) without any CL or dominant follicle (derived from 10 mares in seasonal anestrus) were collected at an abattoir in Parana State, Brazil. This abattoir was approximately 150 km (90 min) away from the Laboratory of Animal Reproduction at Londrina State University. The ovaries were dissected with a scalpel blade and all adipose and connective tissues were removed. The ovaries were then cut sagittally and the cortical portion (more internal) was divided into small fragments

Results

From 3000 slides, 2567 follicles were evaluated, comprised of 1752 primordial follicles (68.2%), 808 developing follicles (31.5%), 774 primary follicles (30.1%), 34 secondary follicles (1.3%), and 7 antral follicles (0.3%). The presence of pycnosis was not detected in granulosa cells in any group. Combined for all treatments, 355 (13.8%) follicles were viable, and 2212 (86.2%) follicles were degenerated. Based on histologic evaluation immediately after collection, only 2% of follicles in the

Discussion

To our knowledge, this is the first study regarding the transport of equine preantral follicles within ovarian tissue, emphasizing the importance of optimizing the use of equine genetic material, because of the small number of abattoirs for this species.

Regarding the protocols tested, equine preantral follicles from ovaries during the non-breeding season were best preserved in PBS at 4 °C for 4 h. The low temperature contributed to the maintenance of follicular morphology. In that regard, it

References (33)

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