Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation

Abstract

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

Introduction

Many species are currently endangered, which could result in a major loss of biological diversity in the next decades. To prevent extinction, various efforts have been conducted, from preservation of natural habitats to creation of germplasm banks.^1,2 Most biobanking efforts have been focused on the development of effective protocols for preservation of sperm cells,³ oocytes,⁴ and embryos.⁵ Preservation of other germplasms including somatic⁶ and gonadal^7,8 tissues has recently emerged, including the cryopreservation of testicular tissue that recently led to the birth of a live offspring in Rhesus monkey.⁹

Testes contain a large number of germ cells, especially spermatogonia that can provide an unlimited source of male gametes if appropriately preserved and cultured.¹⁰ In domestic species and also in some wild mammals, several studies have shown collection and preservation of testes from sexually immature individuals^11,12 and adult animals,¹³ alive¹⁴ or postmortem.¹⁵ In general, a first strategy consists of conserving testicular tissue fragments appropriately using cryopreservation systems, such as slow freezing, fast freezing, and vitrification.^16,17 Nevertheless, the use of these systems depends on the choice of cryoprotectants¹¹ and procedures.¹⁸ Following the tissue's immediate recovery or cryopreservation, fragments can be cultured in vivo or in vitro to obtain viable spermatozoa destined to other reproductive techniques.^10,19

Despite the progress, adequate conditions for direct application of that technology in wild animals remain a challenge, especially due to species-specific differences that lead to the need for improvement and adaptations of current protocols. Therefore, this review aims to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility.

Collecting and Processing Testicular Tissues Before Cryopreservation

Various factors could affect the success of testicular tissue storage and culture. Those include the temperature of the environment where the animal's death occurred, the interval between animal death and sample collection, and the transportation conditions of the samples (temperatures and media).

Collection of testicular samples from wild species generally occurs in places where handling of individuals is possible (in farms, laboratories, breeding centers, zoos, or animal parks), and where tissues can be collected immediately postmortem or after castration. An initial study conducted by Schlatt et al.²⁰ showed the possibility of recovering testes from newborn marmoset monkeys (Callithrix jacchus). Monkey testes were xenografted to castrated immunodeficient mice and were able to produce viable spermatocytes. Later, the possibility of recovering and culturing fresh testicular tissue from wild species was demonstrated for various species as in Indian black bucks (Antilope cervicapra L.),²¹ bisons (Bison bison),²² and collared peccaries (Pecari tajacu).²³

One study reported the use of testicular tissues from a wild species for biobanking.² Testes and epididymis were recovered from seven Iberian lynx (Lynx pardinus) from 5 weeks to 5 years of age. Collections were conducted between 24 and 60 hours postmortem. However, only epididymal and testicular sperm cells were collected for cryopreservation, but no testicular tissues were processed. This missed opportunity also has been observed in a recent rhinoceros report.²⁴

In 2012, Poels et al.²⁵ reported a successful trial of recovery and cryopreservation of testicular tissues from an adult nonhuman primate, the Rhesus monkey (Macaca mulatta), opening the possibility of using this germplasm for biobank formation. An impressive study in this area was conducted in Thailand.¹⁶ The authors demonstrated the possibility of recovering and cryopreserving testes from adult felids such as jungle cats (Felis chaus), lions (Panthera leo), and leopards (Panthera pardus). It also included adult ungulates such as Rusa deer (Rusa timorensis), Fea's muntjacs (Muntiacus feae), and Sumatran serows (Capricornis sumatraensis). Samples were obtained during necropsy, which was conducted within 56 hours postmortem and transported to the laboratory in sterile saline supplemented with 1% penicillin–streptomycin at 4°C. However, the authors highlighted that timing of testis removal postmortem was an important factor affecting testicular morphology (detachment of the epithelium of seminiferous tubules for instance). Moreover, they found that high ambient temperatures experienced by animals before death could also affect the quality of testicular sperm and testicular morphology.¹⁶

Environmental temperatures, however, seem to act in different ways on distinct species since Pothana et al.¹⁵ reported the recovery of testes from immature Indian spotted mouse deer (Moschiola indica) that were found 1 hour postmortem due to heat stress during summer in India. The authors reported that testes were cryopreserved, xenografted to immunodeficient mice, and resumed spermatogenesis.¹⁵

Moreover, testicular tissue cryopreservation and culture would also be an alternative for germplasm preservation in wild species in which procedures for semen collection is not efficient, such as in agoutis³ or when spermatogenesis has not started yet. However, to the best of our knowledge, no other procedures for testicular tissue recovery in living individuals have been reported, except for full castration. One possibility would be testicular biopsies, which is currently used as an elective methodology for the collection of testicular samples destined to studies related to reproductive physiology and pathologies in wild animals.^26,27 Although in humans, testicular biopsy is a widely used technique to recover tissue before being submitted to cryopreservation, especially for men who will undergo chemotherapy.²⁸

Besides testicular tissue recovery, adequate media for washing and transport also are essential. Various media can be effectively used for this purpose, such as the sterile saline supplemented with 1% penicillin–streptomycin used in the Thai study¹⁶ or phosphate-buffered saline (PBS) used for Indian spotted mouse deer¹⁵ and chimpanzee (Pan troglodytes).²⁹ As an alternative, Dulbecco's modified Eagle medium (Gibco) was used for transport of ice-cooled testes of domestic species, such as pigs³⁰ and dogs.³¹

Another important factor related to the cryopreservation is the size of testicular tissue fragments used for the procedure. It can vary from 0.5 to 1 mm³ as reported for Capuchin monkeys (Cebus sp.),²⁰ 1 to 2 mm³ in Indian spotted mouse deer (Mo. indica),¹⁵ and 3 mm³ for wild felids and cervids.¹⁶ However, fragments as large as 9–20 mm³ were also reported for prepubertal rhesus macaques.⁹

Cryopreservation Methods

After recovery, tissues are then destined for cryopreservation. Different methods have been reported for this purpose, considering different cooling rates, as well as cryoprotectant types and concentrations. In general, methods used for wild species are adapted from previous experiments conducted in laboratory and domestic models.

Slow- and fast-freezing methods

During slow freezing, tissue fragments are submitted to gradual reduction of temperature using a cooling rate of −1°C/min between 2°C and −80°C, following −259°C/min between −80°C and −196°C, at low cryoprotectant concentrations to reduce possible cellular toxicity.¹⁸ However, fast-freezing or two-step freezing methods use three cooling rates of −10.8°C/min between 4°C and −50°C, −18°C/min between −50°C and −90°C, and −252°C/min between −90°C and −196°C.^16,32

Among these methods, slow freezing has been used for the conservation of testicular tissue from laboratory and domestic mammals (Table 1). In ovine species, slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ∼10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa.⁴² At this time, the most promising results obtained from the use of slow freezing of testicular tissue are related to the production of viable sperm by in vitro culture, resulting in the birth of viable offspring in immature mice.¹⁷

Table 1.

Cryopreservation and Culture of Testicular Tissues from Laboratory and Domestic Species

Species	Technical approach	Objectives	Main outcomes	References
Mouse	Cryopreservation and in vivo culture	Evaluation of spermatogenesis in immature testicular pieces in xenogeneic recipients after slow-freezing cryopreservation	Birth of offspring following transplantation of cryopreserved immature testicular pieces	Shinohara et al.³³
	Cryopreservation and in vivo culture	Slow-freezing cryopreservation and transplantation of stem cells into the seminiferous tubules	The combination of cryopreservation and transplantation of stem cells produced viable sperm resulting in the birth of viable offspring	Kanatsu-Shinohara³⁴
	Cryopreservation and in vivo culture	Comparison of slow-freezing and vitrification techniques of immature mouse testes	Production of viable sperm by in vitro culture of testicular tissue preserved using both methods, resulting in the birth of viable offspring	Yokonishi et al.¹⁷
	Cryopreservation and in vitro culture	Comparison of slow freezing with vitrification techniques	The vitrification protocol resulted in success rates better than slow freezing in maintaining testicular tissue structure, tubular morphology, tissue functions, and production of flagellated spermatozoa	Dumont et al.³⁵
	Cryopreservation and in vivo culture	Comparison of slow-freezing and vitrification techniques of neonatal mouse testes	The complete spermatogenesis was obtained after testicular tissue controlled-rate freezing and vitrification	Yildiz et al.³⁶
Rat	Cryopreservation	Evaluation of several protocols for cryopreservation of rat immature testicular tissue	A testicular tissue piece of 7.5 mg cryopreserved in cryovial using 1.5 M DMSO, an equilibration time of 30 minutes at 4°C, showed fewer morphological alterations than the other protocols tested	Travers et al.¹⁸
Rat	Cryopreservation	Evaluation of the cryoprotectants toxicity in the vitrification of testicular tissues from immature and adult rats	DMSO would be preferable for the preservation of testes from immature individuals, whereas EG would be more indicated for adults	Unni et al.³⁷
Pig	Cryopreservation and in vivo culture	Evaluation of cryopreservation protocols, cryoprotectant, and cryoprotectant exposure times in immature testis	Obtaining elongated spermatids after programmed slow freezing using glycerol, as well as after vitrification using glycerol with 5 or 15 minutes of exposures or using DMSO for a 5-minute exposure	Abrishami et al.¹⁴
		Evaluation of vitrification and culture on spermatogenesis in immature testis	Obtaining spermatozoa fertile for production of live piglets	Kaneko et al.¹⁹
		Evaluation of complete spermatogenic activity after vitrification and grafting into nude mice	Obtaining spermatozoa fertile for production of embryo by intracytoplasmic injection	Kaneko et al.³⁸
Bull	Cryopreservation	Comparison of different cryoprotectants in adult testis	Cell viability rates were better for DMSO (30%–80%) when compared with propanediol (30%–60%) and glycerol (20%–40%)	Wu et al.³⁹
Bull		Investigation of the effects of concentration of various cryoprotectants during slow freezing on the cell viability as well as expression of spermatogenesis-related genes	10% DMSO exhibited the highest cell viability and mRNA expression level of the spermatogenesis-related genes, CREM, Stra8, and HSP70–2	Zhang et al.⁴⁰
		Evaluation at the molecular level in post-thaw bovine calf testicular tissue, the effects of different solutions at slow-freezing cryopreservation	30 mg/mL BSA combined with 5% DMSO may best protection against cryodamage for the spermatogenesis of bovine calf testicular tissue	Li et al.⁴¹
Ram	Cryopreservation and in vivo culture	Comparison between slow-freezing and vitrification techniques	Slow freezing of neonatal lamb testes was superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ∼10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa	Pukazhenthi et al.⁴²
Ram	Cryopreservation and in vivo culture	Evaluation of the influence of S1P on explants of frozen-thawed testis	S1P promotes germ cell proliferation during first week of culture and may exert an antiapoptotic influence on the seminiferous cord in sheep testicular explants in vitro	Singh et al.⁴³
Dog	Cryopreservation and in vivo or in vitro culture	Evaluation of conditions of slow freezing and culture of spermatogonial stem cells	StemPro^®-34 SFM as culture medium and DMSO as cryoprotectant were adequate for testis freezing, providing cells viable to develop during both in vivo or in vitro culture	Lee et al.³¹
Cat	Cryopreservation	Comparison of different cryoprotectants in the vitrification of prepubertal testes	DMSO associated with glycerol resulted in a better preservation of proliferative potential of testicular cells when compared with EG plus glycerol and DMSO plus EG	Lima et al.⁴⁴
Cat		Comparison of different warming conditions for the vitrification of prepubertal testes.	Warming at 50°C for 5 seconds can be successfully used to ensure reanimation for vitrified testicular tissue from immature cats	Lima et al.¹²
Stallion	Cryopreservation	Comparison of cryoprotectant solutions and controlled cooling rates	PBS +1.5 M DMSO in a programmable freezer preserved structural integrity	Gómez et al.⁴⁵

BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EG, ethylene glycol; PBS, phosphate-buffered saline; S1P, sphingosine-1-phosphate.

Among wild species (Table 2), promising results with slow-freezing methods have been reached in nonhuman primates, such as adult white-headed marmosets (Callithrix geoffroyi), mandrills (Mandrillus sphinx), and chimpanzees, where tissues derived from these three species were able to express sperm- and spermatid-specific proteins (PRM2 and TNP1, respectively), proving maintenance of spermiogenesis after cryopreservation.²⁹ Studies have also highlighted differences between species since mandrill and marmoset testicular tissues can be effectively cryopreserved in media containing 10% dimethyl sulfoxide (DMSO) and 80% fetal bovine serum (FBS), whereas chimpanzee requires only 20% DMSO without FBS. The most promising result obtained in primates, however, was recently reported by Fayomi et al.,⁹ who demonstrated the birth of a healthy female derived from intracytoplasmic injection of a sperm produced by the autologous graft of cryopreserved testicular tissue.

Table 2.

Cryopreservation of Testicular Tissues Followed or not by Culture in Wild Mammalian Species

Species	Technical approach	Objectives	Main outcomes	References
Antilope cervicapra L.	Cryopreservation and in vivo culture	Evaluation of vitrification, slow freezing, and in vivo culture of spermatogonial stem cells in mature testis	Recovery of spermatocytes and round spermatids post-grafting	Goel et al.²¹
Rhesus monkey	Cryopreservation and in vivo culture	Evaluation of vitrification and culture of spermatogonial stem cells in immature testis	Proliferative activity was maintained after vitrification and in vivo culture	Poels et al.²⁵
Rhesus monkey	Cryopreservation and in vivo culture	Evaluation of slow freezing and autologously grafted in immature testis	Graft-derived sperm cells fertilized rhesus oocytes, resulting embryo led to birth of a healthy female offspring	Fayomi et al.⁹
Felis chaus, Panthera leo, Panthera pardus, Rusa timorensis, Muntiacus feae, Capricornis sumatraensis	Cryopreservation	Comparison of the slow-freezing and fast-freezing rate	Both methods preserved testicular tissues from wild animals	Thuwanut et al.¹⁶
Lynx pardinus, Gazella cuvieri, Nange dama mhorr	In vivo culture of mature testis	Evaluation of the effect of donor adult and slow freezing on testicular survival after grafting was also assessed	All samples degenerated	Arregui and Dobrinski⁴⁶
Moschiola indica	Cryopreservation and in vivo culture	Slow-freezing cryopreservation of testis tissue from immature individuals and evaluation of spermatogenesis after xenografting	The use of DMEM/12 HEPES supplemented with 10% DMSO and 80% FBS was the most advanced for pachytene spermatocytes	Pothana et al.¹⁵
Mandrillus sphinx, Pan troglodytes, Callithrix geoffroyi	Cryopreservation	Slow-freezing cryopreservation of testicular tissue of three adult primates	Mandrill testis (∼15% viability) and marmoset testis (∼40% viability) can be cryopreserved in 10% DMSO with 80% FBS, whereas chimpanzee testis (∼15% viability) in 20% DMSO	Pothana et al.²⁹
Hyelaphus porcinus, Muntiacus muntjak, Rusa unicolor	Cryopreservation	Comparison of different cryoprotectants during slow freezing	20% DMSO in combination with 20% FBS is acceptable for cryopreservation of testis of three species of cervids	Pothana et al.⁴⁷
Collared peccary	Cryopreservation	Comparison of different cryoprotectants for the vitrification of testes from mature individuals	The combination 1.5 M EG and 1.5 M DMSO provided preservation of the ultrastructure and the proliferative capacity of spermatogonia and Sertoli cells	Silva et al.⁴⁸
Galea spixii	Cryopreservation	Comparison of different cryoprotectants on seminiferous tubule morphology during vitrification	3 M EG maintained histological architecture of testicular tissues	Silva et al.⁴⁹
Dasyprocta leporine	Cryopreservation	Comparison of different cryoprotectants using slow freezing	1.5 M EG preserved mitochondrial activity in testicular cells	Silva et al.⁵⁰
Dasyprocta leporine	Cryopreservation	Comparison between slow-freezing and vitrification methods	Both methods were adequate for the conservation of the testicular histological architecture	Silva et al.⁵¹

DMEM, Dulbecco's modified Eagle medium.

For wild ungulates (Table 2), Thuwanut et al.¹⁶ demonstrated that fast freezing tended to cause less damage to sperm cells recovered from testicular tissues of Rusa deer, Fea's muntjac, and Sumatran serows than slow freezing. However, the protective effect of fast freezing on testicular intratubular cells was likely compromised, evidence for the incidence of apoptosis. In addition, Pothana et al.¹⁵ reported the slow freezing of testes from immature Indian spotted mouse deer and highlighted that the increase of DMSO concentration impairs spermatogenesis development during the xenograft of cryopreserved testicular fragments. Subsequently, Pothana et al.⁴⁷ used slow freezing of testes from hog deer (Hyelaphus porcinus), barking deer (Muntiacus muntjak), and sambar deer (Rusa unicolor), showing that it was possible to detect the expression of TNP1 and PRM2 proteins, which prove the maintenance of spermiogenesis after cryopreservation. However, the authors highlighted that even among cervids, different species require different freezing protocols since the presence of FBS in cryopreservation media is necessary for cryopreservation of sambar and hog deer testes but not for the barking deer.

In carnivore species (Table 2), using both slow- and fast-freezing methods for the cryopreservation of testicular tissue from jungle cats, lions, and leopards, viable sperm cells with intact DNA were only obtained from jungle cats. Moreover, fast freezing provoked more apoptotic changes in testicular tissue from all three felids than slow freezing.¹⁶

Finally, a preliminary study conducted in a wild hystricognath rodent (Table 1), the red-rumped agouti (Dasyprocta leporina), revealed that ethylene glycol (EG) would be a cryoprotectant more appropriate for the preservation of the mitochondrial activity of testicular cells rather than the DMSO or the combination of these cryoprotectants.⁵⁰

Vitrification methods

Vitrification consists of a rapid decrease of temperature using a cooling rate of −20,000 to −40,000°C/min and high cryoprotectant concentrations that can cause toxicity to cells and tissue.⁵² It is a practical system that is more cost-effective than slow freezing.

This technique has been applied for the testicular conservation in some laboratory and domestic mammals (Table 2). The most promising results were reported in swine, in which successfully generated porcine offspring were produced utilizing sperm from immature testicular tissues after vitrification and transplantation into nude mice.¹⁹ Also, an interesting study showed that both slow freezing and vitrification were equally efficient for the preservation of immature mice testicular tissue, which resulted in obtaining viable sperm after in vitro culture, and then in the production of live offspring.¹⁷

For wild species, however, the use of vitrification was applied to a few species (Table 1). Promising results were reported for nonhuman primates, in which the preservation of testicular tissue integrity, maintenance of proliferating spermatogonia, and Leydig cell functionality were demonstrated for the Rhesus monkey.²⁵

In ungulate species (Table 1), Silva et al.⁴⁸ recently reported the use of solid surface vitrification for testicular tissues of adult collared peccary. The authors highlighted that 1.5 M EG and 1.5 M DMSO combination instead of isolate use of these cryoprotectants provided a better preservation of testicular structure (tubular lumen, cells junctions, and cell membrane integrity) as well as of proliferative capacity of spermatogonia and Sertoli cells.

Preliminary studies conducted in wild hystricognath rodents also highlighted the use of a solid surface vitrification protocol (Table 1). For the red-rumped agouti (D. leporina), Silva et al.⁵¹ verified that both slow-freezing and vitrification methods were adequate for conservation of the testicular histological architecture. However, EG at a 3 M concentration was more effective than DMSO for the vitrification of histological architecture of testicular tissues from the Spix’ yellow-toothed cavy (Galea spixii⁴⁹).

Grafting and In Vitro Culture of Testicular Tissues

After cryopreservation, the great challenge is to provide adequate conditions for tissues to resume spermatogenesis. Currently, the most promising results have been achieved through an association between cryopreservation techniques and in vivo culture using different graft methods. This procedure has been conducted by the transplant of small fragments (1–9 mm³) of testicular parenchyma both for the same donor individual (autografting)⁹ or for different interest species as the immunodeficient orchiectomized mouse (xenografting).¹⁹

As previously cited, an important novelty for the science related to the autologous grafting of cryopreserved testicular tissues from prepubertal Rhesus monkey, which resulted on the production of sperm and offspring, was recently reported.⁹ Despite the importance of this work, its applicability for wildlife is limited since there is no interest in autografting.

However, current literature reports the successful use of xenografting to support complete spermatogenesis, thus resulting in spermatozoa production after cryopreservation of testicular tissues from immature individuals in some domestic models such as ovine⁴² or swine, which resulted in the birth of piglets.¹⁹

Regarding wild species (Table 3), despite that sperm cells were obtained after xenografting of fresh samples from immature ferrets (Mustela putorius furo),⁵⁵ white-tailed deer (Odocoileus virginianus),⁵⁶ and collared peccaries,²³ this was not achieved when frozen samples were used. At this point, xenografting of cryopreserved samples provided the maintenance of proliferating spermatogonial cells in Rhesus monkey,²⁵ while the development of spermatogenesis until spermatocytes was observed in marmoset,²⁰ and the establishment of spermatogenesis with initiation of meiosis was verified in Indian spotted mouse deer (Mo. indica).¹⁵

Table 3.

Culture of Fresh Testicular Tissues in Wild Mammals

Species	Technical approach	Objectives	Main outcomes	References
Marmoset monkeys (Callithrix jacchus)	In vivo culture of immature testis	Evaluation of germ cell differentiation	Recovery of spermatozoa post-grafting	Schlatt et al.²⁰
Rhesus monkey	In vivo culture of mature testis	Evaluation of germ cell differentiation	All cells degenerated	Arregui et al.⁵³
Rhesus monkey	In vitro culture of mature testis	Evaluation of kisspeptin on hormonal secretion	Kisspeptin has no role in testicular regulation related to testosterone and inhibin release	Tariq and Shabab⁵⁴
Mustela putorius furo	In vivo culture of immature testis	Evaluation of germ cell differentiation	Recovery of spermatozoa post-grafting	Gourdon and Travis⁵⁵
Bison bison	In vivo culture of immature testis	Evaluation of germ cell differentiation	Resulted in testicular maturation and complete development of spermatogenesis in the grafts	Abbasi and Honaramooz²²
Odocoileus virginianus	In vivo culture of immature testis	Evaluation of germ cell differentiation	Recovery of spermatozoa post-grafting	Abbasi and Honaramooz⁵⁶
Collared peccary	In vivo culture of immature testis	Evaluation of germ cell differentiation	Sperm recovery from the xenografts resulted in diploid embryos after intracytoplasmic injection	Campos-Junior et al.²³

For mature individuals from both domestic⁵³ or wild species, the complete spermatogenesis after testicular tissue cryopreservation and xenografting has not been achieved, once cell degeneration was verified for Iberian lynx (L. pardinus), Cuvier's gazelle (Gazella cuvieri), and mohr gazelle (Nanger dama mhorr).⁴⁶ Possibly this is due to the delay in angiogenesis since adult animals have a diverse cell range in their testes, without a blood supply to physiological needs.⁵³ Then, from xenografting of fragments derived from mature mammals, some hypotheses have been generated.⁵³ The timing and the progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting, as observed in any animals tested until now.⁵³ At this point, the improvement of protocols for sperm production from the xenotransplantation of testicular tissue of mature individuals remains as scientific challenge.

To overcome the obstacle for reestablishing complete spermatogenesis in adult individuals, a recent study demonstrated the possibility of inducing infertility in mice and then injecting spermatogonia cells derived from dogs into mouse testes, where it was possible to verify the repopulation capacity of germ cells.⁵⁷ This study shows an alternative for the use of samples from domestic and wild adult animals that have not yet succeeded in the xenotransplantation of testicular fragments.

In addition, the in vitro culture of male gonadal tissue has been reported as an alternative for preserving the mammals' fertility.⁵⁸ Its success depends on the optimization of culture medium and supplements, such as specific growth factors, hormones, vitamins, and lipids,⁵⁹ besides the development of adequate in vitro systems, such as classical or 2D and three-dimensional or 3D methods.¹⁰ Using this approach, the main outcome was reached in both the prepubertal¹⁷ and mature mouse,⁶⁰ in which viable spermatozoa was obtained. Overall, in vitro culture has been used for the conservation of testicular tissue in various domestic species (Table 1), but in wild mammals, research remains limited to primates.⁶¹

Final Considerations

Considering the large potential for testicular tissues be used for the formation of biobanks, it is necessary that zoos and reserves have staff people able to recover samples immediately postmortem and conduct adequate procedures for lavage and transport of samples.

Additionally, it is necessary to consider that although conventional freezing is the method chosen for a couple of studies, it has a disadvantage of needing a long time-consuming process, besides needing programmable freezers. At this point, vitrification provides an alternative more appropriate to be conducted under field conditions. Moreover, there is a lot to learn from domestic models, once results currently applied for the vitrification of testes from domestic cats,¹² for instance, could be extrapolated to the wild felids, in which vitrification is not yet reported. Anyway, we may consider that there is probably no ideal cryopreservation protocol for all species, but each species has its own characteristics and sensitivity to cell preservation. The study of different cryopreservation techniques and cryoprotectants concentrations should be tested for each species to determine what it is the most adequate protocol.

Despite the possibilities of using xenografts for sperm development, in vitro culture has become an interesting tool since it may help in understanding the complete communication of spermatogenesis in adult individuals. Nevertheless, more studies are needed regarding the culture and cryopreservation conditions, culture media, and supplementation to ensure the success in obtaining spermatozoa for the species of interest.

Although testicular tissue cryopreservation and culture have been reported in some domestic and wild mammals, limitations, especially regarding species-specific peculiarities and the age of the animal, have been important factors for the success of these techniques. Nevertheless, the age of the animal is still the most representative factor for the conservation of testicular fragments since testes present morphological differences between individuals of different sexual maturity. Thus, adult-derived tissues, regardless of organ source, are generally vulnerable to ex vivo conditions and difficult to maintain under these conditions due to the cell diversity they exhibit.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was financed in part by the CAPES (Financial Code 001) and CNPq (No 406931/2016-0). Dr. Alexandre R. Silva and Dr. Alexsandra F. Pereira are recipients of grants by CNPq.

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