Transport of Domestic and Wild Animal Ovaries: A Review of the Effects of Medium,Temperature,and Periods of Storage on Follicular Viability

Abstract

Short-term storage of ovaries during their transport from the collection sites to the specialized laboratories allows the recovery of thousands of oocytes from females of high genetic value, endangered species, and companion or transgenic animals, which sometimes die unexpectedly in the field, or are ovariectomized for medical reasons. Therefore, several studies have been performed to find ideal protocols to preserve oocyte viability during ovarian tissue transport, thus ensuring the success of techniques that are performed after the storage, such as cryopreservation and/or in vitro follicle culture. To achieve this goal, some factors are essential to maintain oocyte quality, such as medium, temperature, and storage time. Currently, techniques for short-term storage of ovaries have been developed for several animal species. This review aims to present the state of the art with respect to the transport of domestic and wild animal ovaries, highlighting the advantages, limitations, and prospects.

Introduction

In vitro-produced mammalian embryos are usually derived from oocytes collected from ovaries obtained at abattoirs or places far from specialized laboratories.¹ Therefore, it usually takes a long time after the collection of the ovaries until their handling in the laboratory.² In addition, improper transport and preservation conditions might result in the failure of oocyte maturation and fertilization.¹ Therefore, the establishment of an ideal protocol (medium, time, and temperature) for temporary storage of ovarian tissue that maintains follicular and oocyte quality is essential for cryopreservation and/or in vitro culture (IVC).^3,4 This will allow the conservation and further recovery of oocytes from females of high genetic value, endangered species, and companion or transgenic animals,⁵ increasing the availability of fertilizable oocytes.⁶ Therefore, temporary storage of ovaries will not only increase tissue accessibility for research purposes,⁷ but it also has important implications for the preservation of animal germplasm to be used for the conservation of biodiversity.⁸

Current techniques for short-term storage of ovaries have been developed for human⁹ (see ref. Duncan et al.⁹ for review), nonhuman primate,⁷ murine,¹⁰ domestic ruminant (bovine,¹¹ caprine,¹² and ovine¹³), nonruminant (swine² and equine¹⁴), carnivore (canine¹⁵ and feline¹⁶), and wild mammals (Cervus elaphus hispanicus,¹⁷ Cervus nippon yesoensis,¹⁸ and Pecari tajacu¹⁹). The current status of human ovarian tissue transport and its safe and efficient use for fertility preservation in women has been reviewed elsewhere⁹ and, therefore, will not be covered in this review. Thus, this article reviews the major findings that emerged from research on the transport of domestic and wild mammalian ovaries, highlighting the advantages, limitations, and prospects.

General Aspects of Ovarian Tissue Transport and Preservation

Medium composition, transport temperature, duration of storage, and/or the species studied are among the factors affecting follicular viability and the success of techniques that come after the storage, such as cryopreservation and/or IVC.

Media used for ovarian tissue transport

One of the main challenges is to find the optimum composition for a storage solution, which might have less toxicity or damage to the ovarian tissue. In fact, it is difficult to identify the components lacking in the medium, or which compounds may be detrimental. Moreover, the choice of an ideal preservation medium depends on its cost, availability, and time necessary for the transport of ovaries to the laboratory. In this context, many studies have evaluated the efficiency of different solutions on the follicle health during ovarian tissue transport.

Saline is isosmotic, low cost, and, although poor in nutrients (composed of water and sodium chloride), has been widely used in ovarian tissue transport, generally associated to low temperatures (4°C) (equine,²⁰ caprine,²¹ ovine,²² and swine²³). Phosphate buffered saline (PBS) has also been commonly used for short-term ovarian tissue preservation (canine,¹⁵ feline,²⁴ and equine²⁵). However, the use of a richer media has been suggested for female genetic material preservation such as Braun-Collins and coconut water solutions, minimum essential medium (MEM), tissue culture medium 199 (TCM-199), among others.

The hyperosmotic Braun-Collins solution has been used for ovarian tissue transport because it promotes low cellular dehydration, avoiding cell swelling.²¹ The coconut water solution has proved to be efficient for maintaining well-preserved follicular morphology and/or ultrastructure after the transport of ruminant ovarian tissue (bovine,²⁶ caprine,²⁷ and ovine²²), which could be attributed to its rich contents of amino acids, sugars, vitamins, and minerals.²⁸ In addition, 3-indol-acetic acid (IAA) is an auxin present in coconut water that has beneficial effects on the preservation of caprine preantral follicles when added to TCM-199. It is possible that the IAA interacts with growth factors present in the ovarian tissue, which in turn improves the action of these growth factors in animal cells and maintains the cell permeability and respiratory patterns.²⁹ Since coconuts are not universally available, powdered coconut water (ACP^®) has been developed. This powder can be easily stored and transported, and after reconstitution, its biochemical characteristics are very similar to those of fresh coconut water, being a suitable alternative for preserving canine ovarian tissue.¹⁵

TCM-199 is rich in nutrients, such as glucose, vitamins, amino acids, and adenine sulfate, and has been used to preserve bovine ovaries³⁰ and cumulus oocyte complexes (COCs)³¹ during the transport to the laboratory. Other nutritionally complex and rich media commonly used in cell culture and sometimes used to ovarian tissue transport are MEM,¹² alpha-MEM (α-MEM),¹⁴ and Dulbecco's modified Eagle's medium (DMEM).¹⁰ These media can be used alone¹² or enriched with other supplements such as insulin, transferrin, selenium (ITS), glutamine, hypoxanthine, and bovine serum albumin (BSA)^4,10,14 for maintenance of ovarian follicle morphology and/or prevention of DNA damage.

A promising strategy to avoid the high costs of the commercial medium (TCM-199, MEM, α-MEM, or DMEM) is the use of medicinal plant extracts as a storage medium. Amburana cearensis and Morus nigra ethanolic extracts have been successfully used to preserve caprine³² and ovine³³ ovarian tissue, respectively, at 4°C for 6 hours, maintaining the percentages of normal preantral follicles (54%–64%) and apoptosis rates (33%–37%) similar to those observed in the control medium (32%–57% and 16%–65% in MEM, respectively). These results could be attributed to the natural antioxidants present in these plants (flavonoids, stilbenes, and coumarins),^32,33 which may reduce oxidative stress and DNA damage in different cell types.^34,35

Temperature and duration of ovarian tissue transport

The temperature and the duration of the ovarian tissue transport to the laboratory are important components in the maintenance of oocyte viability that may ultimately affect subsequent development. The temperatures range from 4°C to 39°C.^1,13,18,36 Some researchers choose 4°C based on their frequent use and practical convenience because it can be achieved in a refrigerator and is the typical cryogenic preservation temperature. The preservation temperature of 18°C can be achieved in an incubator, the room temperature of 20°C–25°C is easily achieved by air conditioning,¹ and 35°C may also be achieved using an incubator or oocyte transporter.² In addition, it is suggested that low temperatures (4°C) decrease cell metabolism, minimizing the metabolic need and increasing follicular resistance to reduced nutrients and oxygen conditions, allowing the efficient storage for up to 24 or 48 hours.^11,36 However, the increase in cell metabolism at physiological (39°C) or near-room (20°C) temperatures may cause depletion of intracellular energy sources, followed by the consumption of nutrients and oxygen available in the preservation medium, resulting in higher rates of follicular atresia even during short transport periods (e.g., only 2 or 4 hours).^22,30 Therefore, in general, when the storage temperature increases, it may be necessary to reduce the preservation period.

It is important to highlight that species and follicular category (preantral or antral) respond differently to storage temperature. Overall, the low temperature (4°C) contributed to the maintenance of preantral follicle morphology (bovine³⁰ and caprine¹²) and did not affect COCs competence (feline³⁷ and canine³⁸) compared with higher temperatures (20°C–39°C). However, in swine² and equine³⁹ species, higher temperatures (35°C and 22–25°C, respectively) for transport of ovaries, even for longer periods (from 4 to 25 hours), are the most optimal to maintain oocyte quality from antral follicles as well as meiotic and cytoplasmic developmental competence. Studies have indicated that lower temperatures lead to reduced oxidative activity, which seems to be related to a reduction in oocyte viability (from 29% to 92.3% of degeneration depending on the storage time), especially in pigs.^1,2,40

Other factors that affect follicular quality during ovarian tissue transport

Ovarian tissue transport as small fragments of ∼3 × 3 ×1 mm^4,12 may allow the perfusion of the medium through the tissue, but the long period of manipulation during the collection procedure may lead to possible contamination. Nevertheless, the transport of whole ovaries or larger fragments is more practicable and needs less time of manipulation; however, the larger volume of tissue may constrain the availability of nutrients and oxygen to the follicles preserved in vitro.^27,41

In the few studies that evaluate the effect of the form of preservation (whole ovary, half of the ovary, or smaller fragments of the organ) on the follicular survival, the results are contradictory^14,27,41,42 and it is believed that these differences may be related to the species and/or preservation media used. Using only histological analysis, two studies showed that caprine (coconut water solution)²⁷ or ovine (0.9% saline)⁴² preantral follicles can be equally preserved into the whole, half, or fragments of ovary at 4°C, 20°C, or 39°C, for 24, 12, or 4 hours, respectively. Nevertheless, compared with the transport of half or whole ovary, Barberino et al.⁴¹ have shown that storage of one-eighth of the ovine ovary in MEM at 4°C for 24 hours maintained the health of the follicles (apoptosis rates similar to fresh control: ∼10%) without affecting their ability to grow and activate in vitro. Similarly, the transport of equine ovarian fragments (biopsy size: 2 × 2 × 12 mm) at 4°C in α-MEM enriched with BSA and sodium pyruvate was best to preserve preantral follicle morphology for as long as 24 hours compared with the whole ovary.¹⁴

The temporary storage of the ovaries may also be influenced by the methods of transport, and the choice will depend on the size of the organ or tissue fragment, temperature, and availability. Fragmented ovarian tissue or whole ovary can be transported in glass or polystyrene tubes, maintained in thermo boxes in different temperatures.^12,32 Other authors used methods capable of maintaining the temperature near to physiological limits, such as a water bath,⁴³ portable incubator,⁴⁴ or oocyte transporter.⁴

Factors such as the phase of reproductive cycle at the moment of the collection of the ovaries and the presence or absence of part of the reproductive tract during preservation also influence the transport and further oocyte quality. In canines, the highest maturation rate was obtained from oocytes harvested from anestrous ovaries preserved at 4°C, whereas the lowest was at 37°C.³⁷ Moreover, transport temperature (4°C or 37°C) had no effect on the maturation rate of oocytes collected from ovaries in luteal or follicular phases. Furthermore, the transport of canine ovaries surrounded by an intact bursa and adjacent uterine horn at 37°C maintained more viable oocytes (61%) than excised ovaries (19%).⁴⁵ However, further investigations to evaluate the influence of different factors (stage of the estrous cycle, body condition, age of the female, etc.) on the transport of ovarian tissue are still needed.

Main Results of Ovarian Tissue Transport in Different Mammalian Species

Many efforts have been made to optimize transport conditions by simple and effective procedures of storage. Some results seem to be related to the different species used. It is also important to highlight the importance of using specific laboratory techniques to determine if the oocytes and their surrounding cells are of acceptable quality after ovarian tissue transport. Most of the studies used classical histological evaluation and others used more sophisticated techniques, such as transmission electron microscopy, immunohistochemistry, or quantitative real-time polymerase chain reaction. Table 1 summarizes the main results obtained from the transport of mammalian ovarian tissue.

Table 1.

Main Results Obtained After In Vitro Transport of Mammalian Ovarian Tissue in Different Incubation Media, Temperatures, and Periods

Animal model	Ref. no.	Brief description of preservation protocol	Medium	Temperature (°C)	Time (h)	Evaluated follicular category	Findings
Goat	¹²	Ovarian fragments (3 × 3 × 1 mm) and further in vitro culture	MEM	4	4	Preantral follicles	Maintain follicular viability and ultrastructure similar to fresh control ovaries and promote in vitro development (follicular activation and increase in diameter)
Sheep	³⁶	Ovarian fragments (3 × 3 × 1 mm)	0.9% saline	4	24	Preantral follicles	Follicular morphology similar to fresh control ovaries (>70% normal follicles) and maintenance of the ultrastructure
Sheep	¹³	Whole ovary and subsequent IVM	PBS	4	24	Antral follicles: COCs collection	Oocytes with ability to mature and fertilize in vitro
Cow	³⁰	Ovarian fragments (3 × 3 × 1 mm)	TCM-199	4	24	Preantral follicles	Maintain follicular morphology similar to fresh control ovaries (65% normal follicles)
Cow	¹¹	Whole ovary and subsequent IVM	0.9% saline	4	48	Antral follicles: COCs collection	Low DNA fragmentation rate and oocytes with ability to mature in vitro
Pig	²³	Ovarian fragments (5 × 3 × 1 mm) and further in vitro culture of isolated follicles	0.9% saline	4	18	Secondary follicles (220–320 μm)	Maintain follicular viability and promote antrum formation and growth similar to fresh follicles
Pig	²	Whole ovary and subsequent IVM	0.9% saline	35	4	Antral follicles: COC collection	Maintain oocyte cytoplasmic developmental competence
Horse	¹⁴	Biopsy size fragment (2 × 2 × 12 mm)	α-MEM	4	24	Preantral follicles	Maintain follicular morphology
Horse	³⁹	Whole ovary and subsequent IVM	TCM-199 with Hank's salts	22–25	18–25	Antral follicles: COCs collection	Improve the rates of MII oocytes
Bitches	¹⁵	Ovarian fragments (3 × 3 × 1 mm)	ACP^®-based medium	3–9	24	Preantral follicles	Maintain follicular viability similar to fresh control
Bitches	³⁸	Whole ovary and subsequent IVM	DPBS	4	4	Antral follicles: COCs collection	Increased rates of COC maturation (58%)
Cat	¹⁶	Ovarian fragments (3 × 3 × 1 mm)	TCM 199	4	24	Preantral follicles	Maintain follicular morphology similar to fresh control ovaries (59%–63%)
Cat	²⁴	Whole ovary and subsequent IVM	PBS	4	72	Antral follicles: COCs collection	Oocytes with ability to mature in vitro similar to fresh COC
Collared peccary	¹⁹	Ovarian fragments (2 × 2 × 2 mm)	Coconut water solution	4–8	4	Preantral follicles	Maintain follicular morphology and viability similar to fresh control
Dromedary camel	⁴⁸	Whole ovary and subsequent IVM	0.9% saline	4 or 20	24	Antral follicles: COCs collection	Maintain the viability and showed high maturation rates (78.3%)
Iberian red deer	¹⁷	Whole ovary and subsequent IVM and FIV	0.9% saline	5–8 or 20–25	12	Antral follicles: COCs collection	In vitro embryo production similar to fresh control
Nippon deer	¹⁸	Whole ovary and subsequent IVM	0.9% saline	10–15	24	Antral follicles: COCs collection	High maturation rates (55%)

PBS, phosphate buffered saline; TCM 199, tissue culture medium 199; COC, cumulus oocyte complex; α-MEM, alpha-MEM; MEM, minimum essential medium.

Effect of ovarian tissue transport on the morphology of preantral follicles

Several studies have analyzed the effects of the transport conditions specifically on the population of preantral follicles, showing that ovarian tissue storage is possible without harming the ovarian follicular reserve.^15,19,25 Overall, increasing the time and temperature of ovarian tissue transport reduced the percentage of morphologically normal preantral follicles in different species.^25,36,46 In domestic ruminants, the percentage of normal preantral follicles was similar to the fresh control (70%–95%) when ovarian fragments were transported at 4°C for up to 24 hours (bovine 65%,³⁰ caprine 90%,²¹ and ovine 79%³⁶), at 20°C for 12 hours (bovine 62%³⁰ and ovine 71%³⁶), at near-physiological temperature (35°C) for 6 hours (caprine 54%⁴⁷ and ovine 71%⁴), and at 39°C for only 2 hours (ovine 58%³⁶). One of the most used ovarian tissue transport protocols in domestic ruminant was reported by Chaves et al.¹² who had shown the efficiency of preserving caprine ovarian fragments in MEM at 4°C for up to 4 hours, keeping viability of preantral follicles similar to fresh control even after IVC for 7 days (71.9% normal follicles).

Experimental data from the equine model showed that transport of ovarian fragments (3 × 3 × 1 mm) at 4°C for 4 hours in PBS²⁵ or fragments of biopsy size (2 × 2 × 12 mm) at 4°C for 24 hours in α-MEM enriched with BSA and sodium pyruvate¹⁴ did not compromise the health of the follicles (52.3%–65% of normal follicles). However, more studies are still necessary to evaluate the ability of those follicles to grow in vitro after the storage of the ovarian tissue. Nevertheless, in swine species, after ovarian tissue transport in 0.9% saline at 4°C for up to 18 hours or at 20°C for up to 6 hours, Lucci et al.²³ isolated and cultured the preantral follicles (220–320 μm in diameter) for 3 days, showing their viability and their ability to grow and form antrum in vitro in the same pattern as fresh follicles.

For canine species, ovarian tissue transport in MEM in hypothermic (4°C or 20°C, for 12 hours) or near-physiological (38°C, for 6 hours) temperatures maintained preantral follicle viability and ultrastructural integrity similar to controls.⁴⁶ Morphological integrity of canine preantral follicles (>75% of normal primordial follicles) was also maintained after transport of ovarian fragments in low temperatures (3°C–9°C) for 12 hours (in PBS) or for up to 24 hours (in ACP-based medium).¹⁵ These results are consistent with work done on feline ovaries in which the transport of ovaries at 4°C for 24 hours in TCM 199 maintained the percentage of normal preantral follicles similar to that observed in the control group (59% vs. 63%, respectively).¹⁶ After transport, these authors also observed that primordial follicles have a higher viability than the developing follicles (primary and secondary follicles).¹⁶ It is possible that primordial follicles may be less affected because they are presumably quiescent in the ovary and have a smaller oocyte volume that may contribute to the reduced cellular metabolism rate at cold temperatures.^9,25

Using collared peccaries (Pecari tajacu) as a model, ovarian tissue transport in ACP-based medium in low temperatures (4°C–8°C) maintained preantral follicle integrity similar to the control group after 4 hours of preservation (87.8% vs. 94.4%, respectively).¹⁹

Effect of ovarian tissue transport on COCs competence from antral follicles

Ovarian tissue also contains small and large antral follicles that can be a source of immature oocytes destined to in vitro maturation (IVM) and further in vitro fertilization (IVF) to obtain mammalian embryos.⁹ Thus, studies about ovarian tissue transport conditions for the preservation of oocytes enclosed in antral follicles deserve attention. The data regarding how well COCs withstand transport vary between animal species. For example, in bovine, whole ovaries can be preserved in low temperatures (4°C or 10°C) for 24–48 hours without affecting the DNA integrity (only 5%–20% of oocytes with DNA damages)¹¹ and maturation of oocytes from antral follicles (47.7%–67.5% mature oocytes)^6,11 and further blastocyst development after IVF (25% vs. 27% on control), parthenogenetic activation (19% vs. 25% on control), or somatic cell nucleus transfer (27% vs. 32% on control).⁶ In the ovine model, preservation of ovaries for up to 24 hours at 4°C maintained oocyte competence, whereas a higher temperature (20°C) had a detrimental effect on oocyte viability, maturation, fertilization, and subsequent development after IVF.¹³ Similarly, preservation of canine (transport for 4 hours)³⁸ or feline (transport for 24 hours)³⁷ ovaries at 4°C showed better rates of oocyte maturation (58.1% and 53.4%, respectively) than higher temperatures (variation of 23–38°C; in some cases, only 2.4%–20% of metaphase II were observed). In addition, Wolfe and Wildt (1996) have shown that preservation of cat ovaries for a long period (up to 72 hours) in PBS at low temperatures (4°C) did not affect the in vitro nuclear maturation of oocytes from antral follicles (55.4% vs. 61% on control).

In relation to temperature, opposite results were observed in swine and equine compared with other species. Regardless of the time, meiotic competence of swine oocytes from antral follicles can be maintained when ovaries are preserved at 25°C for 2 hours (60%) or at 35°C for up to 4 hours (52%) in comparison with 15°C (40%).² Adversely, during the transport of swine ovaries in lower temperatures (4°C or 15°C), oocytes from large antral follicles showed a deficiency in maturation due to the reduced oxidative activity, which can lead to the disruption in the meiotic spindle or loss of membrane integrity.¹¹ Moreover, despite maintaining the pH of follicular fluid and decreasing the occurrence of DNA fragmentation in the oocytes, Wongsrikeao et al.⁴⁰ observed that lower temperature (4°C) during the storage of swine ovaries for 6 hours has a detrimental effect on oocyte maturation, fertilization, and subsequent development. Similarly, equine COCs appear to be highly sensitive to low temperature of ovarian tissue transport. Love et al.³⁹ observed that the rates of oocyte maturation from ovaries preserved between 22°C and 25°C for 18–25 hours were higher than those found in ovaries preserved at 4°C for the same period (27% and 10%, respectively). Moreover, maturation rates can reach 37.5% after transport of COCs enclosed in equine ovaries in 0.9% saline in temperatures ranging from 27°C to 37°C for up to 8 hours.²⁰

Nevertheless, experimental data from wild animal models show that it is possible to obtain acceptable results in different temperatures. For example, Abd-Allah⁴⁸ reported high maturation rates of COCs (average of 78.3%) in dromedary camels (Camelus dromedaries) after storage of their ovaries in 0.9% saline at 4°C or 20°C for 24 hours. Similarly, Iberian red deer embryos (Cervus elaphus hispanicus) were obtained in equal numbers after recovery of COCs that were transported in whole ovaries at low (5°C–8°C; 20.61%) or at room (20°C–25°C; 13.68%) temperatures for up to 12 hours.¹⁷ For Japanese deer (Cervus nippon yesoensis), when ovaries were kept at 10°C–15°C (for 24 hours) or at 20°C–25°C (for 12 hours), maturation rates of oocytes were 55% and 71%, respectively.¹⁸

Final Considerations

Several protocols for storage and transport of ovarian tissue in different species are available. However, the preservation conditions of ovaries significantly affect the in vitro follicular quality (Fig. 1). It can be highlighted that (i) the effectiveness of each transport solution is dependent on the temperature and incubation time and (ii) species and follicular category (preantral or antral) respond differently to the transport conditions and, in this case, the transport temperature is an important consideration. Overall, the low temperature (especially 4°C) contributed to preantral follicle health and developmental competence of oocytes from antral follicles in most species regardless of the transport media. In swine and equine species, however, the meiotic competence of oocytes derived from antral follicles is maintained when the ovaries are stored at higher temperatures (22°C–35°C). The use of ovarian tissue transport protocols can optimize animal production, and can be used in the recuperation of threatened animals, representing an important tool in economic, medical, and scientific applications.

FIG. 1.

Establishment of an ideal protocol for storage and transport of ovarian tissue is dependent on several factors such as medium composition, transport temperature, duration of storage, and the species studied.

Footnotes

Acknowledgments

R.S.B. received a scholarship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE, Brazil). J.R.V.S., J.R.F., and M.H.T.M. were supported by a grant from CNPq. The authors thank Luciana da Paz dos Santos and Rodrigo José de Sousa Gonçalves who contributed with the first draft of the article.

Author Disclosure Statement

No conflicting financial interests exist.

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