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