InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Veterinary Medicine and Science » "Insights from Animal Reproduction", book edited by Rita Payan Carreira, ISBN 978-953-51-2268-5, Published: March 23, 2016 under CC BY 3.0 license. © The Author(s).

Chapter 2

Assisted Reproductive Techniques on South American Wild Mammals

By Alexandre R. Silva, Alexsandra F. Pereira, Gabriela L. Lima, Gislayne C. X. Peixoto and Ana Liza P. Souza
DOI: 10.5772/62427

Article top

Assisted Reproductive Techniques on South American Wild Mammals

Alexandre R. Silva1, Alexsandra F. Pereira1, Gabriela L. Lima1, Gislayne C. X. Peixoto1 and Ana Liza P. Souza1

1. Introduction

South America, the fourth largest continent, presents an extreme geographic variation that contributes to its large number of biomes, which includes the marine rainforests, alpine, deserts, savannahs, grasslands, chaparral, desert scrub, fresh water, marine, and deciduous desert. Due to such biome diversity, the South American fauna consists of various unique animals.

In the past decade, a rapid and continuous decline in mammalian species has been documented, indeed affecting South American countries. Therefore, the worry put on the development of conservation strategies, as well as on the rapid expansion of the commercial interest on wild species around the world, requires an equally rapid development and adoption of assisted reproductive techniques (ARTs). These techniques allow the conservation and multiplication of genetic valuable individuals, as well as facilitate the transport of the germplasm among distant regions. However, the application of ARTs is still a challenge, since data related to basic reproductive information is scarce, and it is indispensable for the genetic management of rare species. Because of the high diversity in reproductive mechanisms among mammals, it is not possible to directly apply protocols developed from one species to another, hindering the extrapolation of developed ARTs. In this sense, this chapter highlights the importance of applying ARTs for the South American wild mammals, showing the most recent studies in this area and the perspectives for its use in conservation programs.

2. Manipulation of spermatozoa

Technology of male gamete consists of various forms for sperm processing, since its collection until its storage by cooling or cryopreservation. In wild animals, the gamete recovery is generally conducted by electroejaculation (EEJ) [1]; nevertheless, for animals unable to ejaculate, or immediately postmortem, one possibility is to obtain epididymal sperm or testicular tissue, which can be cultivated and cryopreserved [2]. After collection, samples could be stored in cryobanks, which maintains the sperm quality for an indefinite time, providing valuable genetic material to be used for ARTs destined to the wild mammals’ conservation and multiplication.

2.1. Collection of ejaculated sperm

The EEJ is a safe and effective method for semen collection, especially in wild animals, as it allows handling live animals under anesthesia. It consists of a controlled electrical stimulation of the ejaculatory reflex. A transrectal probe coupled with a specific voltage-producing unit [1] is used to apply the stimulus. The types and disposition of the electrodes, the electric stimulation protocol, and the anatomical characteristics of each species exert important influences on the efficiency of the method [3]. Due to the large species diversity, it is necessary to establish adequate species-specific stimulation protocols using appropriate anesthetic procedures, according to animal responses and well-being [4]. Spermatozoon from different species is presented in Figure 1.


Figure 1.

Sperm images of some animals in Latin America, (A) Spix’s yellow-toothed cavy—Galea spixii, (B) Coati—Nasua nasua, (C) Collared peccary—Pecari tajacu, (D) Six-banded armadillos—Euphractus sexcinctus.

In general, most researchers use adaptations of the original protocol developed for felid collection [5], consisting of a total of 80 electrical stimulations divided into three series: 30 stimuli (10 stimuli, 2 to 4 V, series 01), 30 stimuli (10 stimuli, 3 to 5 V, series 02), and 20 stimuli (10 stimuli, 5 and 6 V, series 03), with a 5-minute interval among series. This serial EEJ protocol was adapted for various South American felids such as the ocelot (Leopardus pardalis), the margay (L. wiedii), the tigrina (L. tigrinus) [6], the jaguar (Panthera onca) [7], and other carnivores such as coatis (Nasua nasua) [8] (Table 1) and maned wolves (Chrysocyon brachyurus) [9]. In addition, this protocol has been adapted for other animals such as sloths [10], giant anteaters [11], six-banded armadillos (Euphractus sexcinctus) [12], tapirs (Tapirus bairdii) [13], and agoutis (Dasyprocta leporina) [14] (Table 1).

A totally different serial protocol was described for primate species. For spider monkey (Ateles geoffroyi), stimulation series begins at 1 V, followed by 1 V increments up to a maximum of 10 V. Each series lasts 5 minutes, with 3-minute rest periods between series [15]. For capuchin monkey (Sapajus apella), stimulation session is comprised of six series, 10, 15, 20, 25, 30, and 35 electrical stimuli (12.5–100 mA), with a 30-second interval between consecutive series [16].

In some species, however, a better response is achieved using continuous electrical stimulation. For collared peccaries (Pecari tajacu) EEJ (Table 1), the stimulatory cycle consists of 10 stimuli in each voltage, starting from 5 V, followed by voltage increases of 1 V up to 12 V, then, the voltage remains being applied during 10 minutes, continuously [17].

Parameters Collared peccary
Ring-tailed coati
Six-banded armadillo
Sperm concentration (x106) 765.0 ± 313.7357.0 ± 61.2197.5 ± 204.9450.0 ± 14.0
Motility (%) 86.7 ± 2.680 ± 29.691.3 ± 9.261.0 ± 7.0
Vigor (0-5) 4.4 ± 0.33.5 ± 1.94.5 ± 0.62.0 ± 0.2
Viability (%) 92.3 ± 1.665.5 ± 26.173.1 ± 14.555.0 ± 7.0
Membrane functional integrity (%) 75.3 ± 2.368.5 ± 17.674.3 ± 12.046.0 ± 6.0
Normal morphology (%) 83.2 ± 2.283.9 ± 8.681.2 ± 11.886.0 ± 2.0

Table 1.

Characteristics of semen obtained by electroejaculation from various South American species.

2.2. Epididymal sperm retrieval

Obtaining viable sperm directly from the epididymis tail is an additional option for the conservation of genetic material of South American wild animals, particularly for those accidentally killed. However, different factors such as the epididymis size and vas deferens diameter [18] as well as the possibility for contamination of samples with blood or epithelial cells are considered as limiting factors [19].

Epididymal sperm can be retrieved by flotation or retrograde flushing. In the flotation method, the epididymis is cut into small pieces in a diluent solution for the removal of sperm; this is one of the preferred techniques for small species due to the reduced size of the epididymis [20]. On the other hand, the retrograde washing is accomplished by injecting a buffered solution at the vas deferens, as it was described for the agouti [21]. In some species, such as collared peccaries [22] and yellow-toothed cavies (Galea spixii) [23], both recovery methods can be conducted with no notorious differences on sperm quality.

2.3. Sperm preservation

The research on gamete preservation needs to focus on the species-specific variations in sperm physiology, as well as the changes that gametes undergo during sperm processing [24]. The factors most often cited as crucial for success include the sperm processing and packaging, type and composition of extender, duration of equilibrium time, freezing rate, storage, and thawing rate [25].

The short-term preservation of sperm on a liquid form is rarely applied for South American species. In capuchin monkey, for instance, dilution of semen in coconut water–based extender allowed its preservation for 24 hours at 33°C [16]. Moreover, the use of a TRIS extender containing egg yolk or Aloe vera extract was efficient in preserving the collared peccary semen for 36 hours at 5°C [26].

Nowadays, intensive research focuses on the development of effective methods for sperm cryopreservation. In general, methods are adapted from the most closely related phylogenetic domestic species; for example, the domestic pig serves as an experiment model for wild pigs; however, such adaptation is not always effective. This fact is verified for six-banded armadillos that present unique semen characteristics, such as high viscosity, large sperm dimensions, and rouleaux formation, which hinders the success of freezing protocols [27].

Extenders based on TRIS plus egg yolk and glycerol were reported for collared peccary semen cryopreservation [17]. Other compounds have been described, such as the INRA® extender for tapir [13], the lactose-egg yolk extender for jaguars [28], and a combination of TES and TRIS extenders for capuchin monkey [16]. Recently, a coconut water–based extender was reported for semen cryopreservation in collared peccaries [29], agoutis [30], and squirrel monkeys (Saimiri collinsi) [31].

Although most researchers use egg yolk and glycerol as main cryoprotectant, a recent study demonstrated that the dimethyl sulfoxide (DMSO) would be more appropriate for the preservation of maned wolf semen than that of glycerol. Moreover, low-density lipoprotein [32] and Aloe vera extract [33] could effectively substitute the egg yolk for the cryopreservation of collared peccary semen.

Regarding preservation of epididymal sperm from South American wild mammals, studies are scarce. For collared peccaries [34] and cavies [35], epididymal sperm was efficiently cryopreserved using TRIS extender added to egg yolk and glycerol. In agoutis, however, epididymal sperm is better cryopreserved in coconut water–based extenders [36].

2.4. Artificial insemination

Artificial insemination (AI) is the single most important technique ever devised for genetic improvement of animals. It refers to the artificial process of sperm deposition into the female genital system, at the appropriate time, seeking the fertilization of the oocyte. The main factors that determine the fertility rates derived from AI include the individual fertility of players, the way in which sperm is collected and manipulated, the ability of the inseminator, the female management (the time of insemination), the type of sperm used (fresh, chilled, or frozen), the insemination dose, and the site for the semen deposition [37].

In spite of AI popularity among domestic animals breeders, its use remains limited in South American wild animals. Initial studies include the successful production of offspring derived from laparoscopic intrauterine AI in ocelots. One female was inseminated with 7.5 × 106 frozen-thawed spermatozoa after receiving 500 IU equine chorionic gonadotropin (eCG) and 225 IU human chorionic gonadotropin (hCG), giving birth to a male offspring after 78 days of gestation [38]. Moreover, deposition of fresh or cryopreserved semen, as well as cryopreserved epididymal sperm, into the cervix of marmoset (Callithrix jacchus) resulted in pregnancy and the production of offspring [39].

2.5. Testicular tissue processing

The preservation of testicular tissue for prolonged periods is one big challenge in the field of cryobiology, because spermatogonia, Sertoli, and Leydig cells contain large amounts of water, therein increasing the risk for intracytoplasmic crystal formation [40]. If the testicular tissue has preserved active spermatogenesis, it can be used for the extraction of sperm or elongated spermatids that could be used for oocyte fertilization using intracytoplasmic sperm injection (ICSI) [31]. Moreover, testicle freezing can be used in cases where the animal dies suddenly [41].

Although this is a promising area, studies on the collection of sperm or even on the preservation of testicular tissues are very scarce. In South American individuals, the unique published results can be found in collared peccaries, in which the first successful xenotransplantation of fresh testicular tissue was also documented [42]. In addition, its testicular tissue was efficiently vitrified using ethylene glycol (EG) as cryoprotectant at 3.0 or 6.0 M [43].

3. Estrous and ovulation control

For wild females exhibiting irregular or regular cycles of sexual activity, estrus synchronization support assisted breeding procedures particularly to maximize the chances of conception and the use of fresh or cryopreserved semen [44]. The administration of exogenous hormones, whether or not in association, can artificially synchronize the estrous and ovulation of wild females, altering its endogenous endocrine environment [45]. Research has dramatically increased the number of synchronization protocols for wild animals, being the prostaglandins, progestins, or gonadotropins the most available, each with specific functions and peculiar mechanism of action.

One of the most ancient methods for estrus synchronization is the use of a luteolytic agent such as the prostaglandin F2α (PGF2α) or its analogues, which provokes the corpus luteum degeneration [46]. Another approach is by using gonadotropin-releasing hormone (GnRH), which represents the final common pathway where by internal and external relevant stimuli converge to control reproduction. GnRH neurons, thus, stimulate pituitary gonadotropin secretion to appropriately regulate gametogenesis and sex steroid secretion [47]. On the other hand, progestins inhibit the synthesis and secretion of GnRH that subsequently inhibits follicular growth and development as well as the ovulation [48]. Additionally, gonadotropin-based protocols include the use of equine and human chorionic gonadotropins (eCG and hCG, respectively), as well as the application of follicle stimulating hormone (FSH) and luteinizing hormone (LH), as a synthetic or extract form [49]. The action of eCG is similar to that of FSH at stimulating the ovary follicles to produce mature oocyte, thus promoting the outward signs of estrus [50]. In contrast, the hCG action is similar to that of LH, causing the release of mature oocyte at ovulation, and promoting corpora lutea formation [51]. Currently, the majority of studies focus on the use of hormonal associations. This is acceptable because the results of studies have shown that such protocols increased rate, occurrence, and ovulation speed, therefore increasing the fertility rate [52].

Various attempts have been made to establish effective protocols for estrous control in South American wild felines, mainly due to the variety of manifestations of reproductive cycles. For example, the margay present spontaneous ovulations during normal estrous cycles, but ocelot and tigrina are induced ovulators. Luteal control in feline is not a feasible option, due to several factors: (1) although some individuals ovulate spontaneously at unpredictable intervals, many felids are obligated to induce ovulations and (2) the diestrus corpus luteum, when present, is refractory to prostaglandins and dopamine agonists up to day 40 postovulation [50].

The use of porcine FSH (pFSH) with minimal LH activity is reported for inducing follicular growth in wild felines [53]. This protocol was effectively applied for pumas (Puma concolor) [54], jaguars (Panthera onca) [55, 56], and jaguarundis (Puma yagouaroundi) [49]. Recently, the ability of an oral progestin, altrenogest (0.192 mg/kg, 14 days), to suppress the ovarian follicular activity in tigrina was demonstrated. This protocol was also effective in reducing hyperstimulation and hyperestrogenism after hCG and eCG administration. However, the authors affirmed that not all females responded uniformly [45]. In fact, studies with both conventional gonadotropins showed diversity of response in different feline species. For example, the ovarian response to a combined treatment with eCG injection followed 80-84 hours later by a hCG administration resulted in a conception rate lower than 30% in ocelots (400 IU eCG and 200 IU hCG) [38] and tigrina (200 IU eCG and 150 IU hCG) [51]. Exogenous gonadotropins are immunologically complex foreign peptides, and thereby, the development of antibodies against gonadotropin is expected sequelae in wild feline submitted to short-term repeated treatments [57]. For this motive, the isolation, characterization, and production of recombinant gonadotropins has been suggested to avoid these complications. On the other hand, it is important to remember that felid species present a large variation in body sizes, a factor that directly influences the synchronization protocols, as well as the individual ovarian response to gonadotropin stimulation [58].

A considerable effort has been made to develop a method to induce estrus and ovulation in the maned wolf, the biggest South American canids. An implant of deslorelin (2.1 mg), a GnRH analogue, was 100% effective for induction of ovary activity, with ovulation occurring between days 9 and 16 (mean: 12.5 ± 1.4 days) after implant when paired with a male, a physiological condition required for the effective ovulation. Alternatively, a single injection of equine recombinant LH (reLH), associated with the deslorelin removal effectively induced ovulation in unpaired females, without apparent adverse impact on fertility in subsequent breeding seasons [59]. Regarding the coati, a carnivore from the Procyonidae family, the unique attempt of estrous control was made by using melengestrol acetate, a progestin, but treatments induced purulent vaginal discharge and uterine adenocarcinoma, and consequently the authors recommend caution when using this drug [60].

In ungulates, as the brown brocket deer (Mazama gouazoubira), a monovulatory nonseasonal breeder, estrous synchronization can be achieved using an intravaginal progesterone device (CIDR®) for 8 days, associated with an injection of 265 µg cloprostenol at the removal of the device [61]. In collared peccaries, on the other hand, the use of two injections of 60 µg cloprostenol administered at a 9-day interval was effective for estrous synchronization [62].

In primates, it is known that PGF2α can act on luteal cells to inhibit the luteotrophic actions of LH or hCG in vitro [63]. In this context, the administration of cloprostenol has a marked and rapid luteolytic action (0.5–0.8 µg) in the marmoset monkey [64]. In this species, the daily use of recombinant human FSH (rehFSH; 50 IU) during the first 6 days of the follicular phase resulted in superovulation [65]. Moreover, when associated, the protocol using rehFSH 25 IU/hCG 500 UI proved to be efficient to collect oocytes [66].

Despite the difficulties in using a great number of individuals for experimentation, there is notably a need for the development of estrous synchronization protocols for South American wild mammals, especially focused on dosage and efficiency of drugs.

4. Female gametes manipulation

As with other ARTs, the main limitation for wild species is the difficulty in obtaining viable oocytes, being the recovery of preovulatory oocytes for in vitro culture (IVC), an available alternative [67]. The possibility to store frozen female gametes as ovary tissue fragments, isolated follicles, or either mature or immature oocytes represents an attractive alternative to cryopreservation [68]; however, the technique is not yet well-established, presenting several challenges that need to be addressed in order to be routinely used.

4.1. Recovery of female gametes

The first step in the manipulation of female gametes is to obtain viable oocytes; however, such procedures are still uncommon in South American wild mammals and are most frequently conducted in Neotropical primates. Oocyte can be collected in vivo, or postmortem.

The recovery of oocytes by mechanical preantral follicle (PF) isolation in ovariectomy specimens [69], the collection of ovarian biopsies by exploratory laparoscopy [70], and the puncture of antral follicles by laparotomy [71] have been described studies in capuchin monkeys (Sapajus apella). In marmoset monkeys, oocyte recovery was achieved through follicle aspiration after laparotomy [72], laparoscopy [65], or uni- or bilateral ovariectomy [73]. The latter was also the method described in squirrel monkeys (Saimiri boliviensis boliviensis) [74] for oocytes collected by ultrasound-guided puncture, after monitorization of the follicular development, in superstimulated females [75]. Finally, in Saimiri sciureus, follicle aspiration was conducted after laparoscopy [76] or laparotomy [77].

Regarding other South American species, in particular, wild felids, ovary collection is often performed immediately after the animal death, as it was reported for Geoffroyi and tigrina cats [78]. Also in puma and jaguar, isolation of small PFs (40-90 µm) was performed after animal death [79]. The oocyte aspiration by laparoscopy was also described for tigrina and ocelot [51], puma [80], and jaguar [55]. In vicunas (Vicugna vicugna), a South American wild camelid, oocytes also were recovered after follicle aspiration [81].

4.2. Characterization of ovarian follicles population

The rescue and manipulation of matured oocytes represent an important genetic source from wild species; nevertheless, it faces many obstacles, particularly the limited knowledge on the species reproductive physiology [82]. Thus, the use of immature oocytes from antral follicles or PFs allows the recovery of gametes from prepubertal, pregnant, old, or even dead animals [83, 84] The characteristics of the ovarian follicles population in various South American wild mammals are described in Table 2 and illustrated in Figure 2.

Specie Preantral follicles populationPreantral follicles category
Right ovaryLeft ovaryTotalTotal number%Diameter (µm)Total number%Diameter (µm)Total number%Diameter
Sapajus apella
56.9 ± 21.949.1 ± 26.9--30.0 ± 4.322.1± 0.5-6.0 ± 1.027.3 ± 0.5-4.0 ± 0.761.2 ± 4.0[85]
Saimiri sciureus
---915.0 ± 78.873.3 ±1.320.5 ± 0.8230.5
± 20.8
18.6 ± 0.733.7 ± 2.4115.9 ± 15.78.1 ± 0.9512.5 ± 67.1[86]
Dasyprocta leporina
4419.8 ± 532.35397.5 ± 574.9--86.618.6 ± 3.4-13.023.8 ± 5.7-0.488.6 ± 17.6[87]
Galea spixii
220.8 ± 175.1195.2 ± 182.8416.0 ± 342.8-32.216.6 ± 0.3-63.728.3 ± 2.1-4.1123.7 ± 18.3[88]
Pecari tajacu --33273.5 ± 579.0 (per ovary)30 466.6 ± 5194.891.631.8 ± 1.12 093.9 ± 595.06.340.9 ± 2.1712.9 ± 95.42.2196.2 ± 17.1[89]

Table 2.

Characterization of ovarian follicles population from different South American wild species


Figure 2.

Photomicrograph of the ovary cortex. (A-C) Preantral follicles from Pecari tajacu and (D-F) Dasyprocta leporina. Primordial (A,D), primary (B,E), and secondary (C,F) follicles.

4.3. In vitro culture and maturation of oocytes

The IVC and in vitro maturation (IVM) of oocytes has highlighted some of the mechanisms underlying the folliculogenesis in particular wild species, particularly among the South American wild mammals, offering new opportunities for the use of many other ARTs [85]. Oocyte IVM is a technique that provides material for the study of the final steps of oocyte meiosis. Nevertheless, the practical application of this procedure remains relatively inefficient, and the embryo production rate is much lower compared with in vivo matured oocytes [73]. Some of the researches that have been developed using IVC and IVM are revised below.

The competence of oocytes retrieved from antral follicles in capuchin monkeys was achieved after 36 hours of IVM, the highest maturation rate occurring in oocytes collected from dominant follicles [71]. It has also demonstrated that the IVC of ovarian cortical strips in TCM199 supplemented of β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) promoted a follicular viability similar to that of controls (89.3%) for this species, while it also increases the rate of secondary follicle formation (44.9%) [70].

The oocyte meiotic competence and the cumulus cell function were positively associated with the follicle size in marmoset monkeys [86], as it was demonstrated in IVM studies. It was shown that chromosome quality is crucial for cytoskeletal organization allowing a correct meiosis in IVC and IVM of the immature oocyte [73]. Moreover, abnormal spindle formation was observed in oocytes derived from small antral follicles failing to complete meiosis, contrasting almost 90% matured oocytes, surrounded by expanded cumulus cells at the time of isolation [66]. For this species, an alternative two-step culture system consisting in the culture of oocytes within stromal tissue fragments for 2 days has been described. Afterwards, follicles were mechanically isolated and transferred to a culture system where they grew for up to further 12 days. This process produces full-sized matured oocytes from primary and early secondary follicles [87].

An immunohistochemical assay conducted in squirrel monkey ovaries during IVC demonstrated that growth differentiation factor 9 (GDF-9) and c-Kit protein were detectable in oocyte cytoplasm from primordial to secondary follicles, whereas the Kit Ligand expression was observed in oocytes and granulosa cells from primordial to secondary follicles. On the other hand, the anti-Müllerian hormone was expressed in primary and secondary follicles, but not in the primordial ones [88]. In a different species from the same gender, the S. boliviensis, IVM of follicles obtained by laparoscopy requested the use of a medium containing high-energy source [89]. Moreover, oocyte IVM was also described for S. scierus [90].

Regarding South American wild felids, the yield of the IMV technique varies greatly among species. Immature oocytes were recovered from Geoffroyi cat, jaguar, and puma after the animal death and further matured in vitro. In Geoffroyi cat, from a total of 45 oocytes retrieved, 23% succeed maturation, similarly to that described for jaguar, from whose oocytes (n=21) only one-third advanced to metaphase II during IVM. In the puma, more than 100 oocytes were recovered from 8 females, from which 43.8% of the oocytes matured in vitro [91]. IVM has been previously described in this species after oocyte recovery using laparoscopy and transabdominal aspiration [92]. In a recent study, 42 mature oocytes were collected by laparoscopic ovum pick up from pumas, after the administration of eCG (750 UI) with hCG (500 UI). In this species, oocytes were also matured in vitro using TCM199 supplemented with LH, FSH, and 17β-estradiol [80].

Studies on ovarian gene expression have been conducted to better understand folliculogenesis in plains viscacha (Lagostomus maximus), an animal showing the highest ovulation rate among all mammals, releasing between 400 and 800 oocytes per estrous cycle [93]. The expression of germ cell–specific VASA protein, apoptotic proteins BCL2 and BAX, as well as DNA fragmentation revealed an unrestricted proliferation of germ cells in this species, without apoptosis-driven elimination, contrary to what is normally found in other mammals [94, 95].

In ungulates, IVC of collared peccaries ovarian tissue reveals that more than 50% follicles remain morphologically intact when the TCM199 medium was used whether supplemented or not with FSH. Moreover, the activation of collared peccaries PFs was stimulated by the addition of FSH to the medium during IVC, the proportion of growing PFs increased from only 31.2 ± 0.7% in the control group to more than 90% after a 7 day in TCM199+FSH [96].

4.4. Female gametes cryopreservation

The conditions required for the complete in vitro development of oocytes remain incompletely established in wild species; therefore, cryopreservation emerges as a promising tool for female germplasm conservation, allowing its future use in ART. It is possible to store ovarian tissue, isolated follicles, and mature or immature oocytes, for which purpose two methods are routinely used: the slow-freezing procedure and vitrification. The value of vitrification has been highlighted to avoid damages caused by conventional freezing protocols, such as the risk of ice crystal formation. It is considered a cheap method that can be performed under field conditions.

Another major factor on the success of cryopreservation of female gametes is the cryoprotectant used. In marmoset monkeys, DMSO was advantageously used as a cryoprotectant for slow freezing of ovarian tissue. It provided a higher percentage of morphologically normal primordial (26.2 ± 2.5%) and primary follicles (28.1 ± 5.4%) compared with propanediol (PROH) (12.2 ± 3.0 and 5.4 ± 2.1%, respectively) [97]. Furthermore, the development up to secondary PFs [98] and the formation of antrum after xenografting cryopreserved tissue in this species were also described [99].

Regarding South American wild felids, short-term IVC of ovarian cortex slices collected from Geoffroyi cat was performed to assess the success of the slow freezing protocol, using EG and sucrose. The results showed an increase in the number of viable PFs in frozen–thawed samples after 7 days of IVC (48%) compared with fresh cultured pieces (31%) obtained in the same sampling day. In tigrina cats, the percentage of primordial follicles after freezing and culture (44%) indicated a normal population of viable follicles [78].

Regarding Neotropical rodents, the conventional freezing was adopted to preserve ovarian fragments from agoutis. After freezing, no differences were found among groups using different cryoprotectants: DMSO (60.6 ± 3.6%), EG (64.0 ± 11.9%), or PROH (62.0 ± 6.9%). However, only follicles cryopreserved with PROH presented a normal ultrastructure, similar to that of the control group [100]. A reduction on the morphologically normal PFs (69.5%) compared with the control group (91.2%) was also observed, after solid surface vitrification of ovarian tissue from yellow-toothed cavies. The preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles were acceptable, and the transmission electronic microscopy showed that the presence of vacuoles in the oocyte and granulosa cells cytoplasm and turgid mitochondria remained the main alteration observed in some vitrified follicles [101].

In collared peccaries, the refrigeration of ovaries for up to 36 hours allowed supporting the morphological integrity and viability of PFs. Further, it was demonstrated that powdered coconut water media (66.7%) was more effective than the phosphate buffered saline (PBS, 49.4%) to preserve the morphological integrity after 36 hours storage, although without statistical differences on respect to the follicular viability [102]. Moreover, the solid surface vitrification of ovarian tissue using EG, DMSO, or dimethylformamide (DMF), at 3.0 or 6.0 M, preserved the morphological integrity of more than 70% PFs using any of these cryoprotectants [103].

5. Embryo technology

ARTs using in vivo embryo production by applying hormonal superovulation protocols, as well as those obtaining in vitro blastocyst through in vitro fertilization (IVF) and ICSI, allowed to establish the adequate conditions for embryonic development in different species. Furthermore, ARTs associated with embryo cryopreservation foster the preservation of genetic material, contributing to conservation of biodiversity. In general, ARTs applied to wild mammals in South America are established in accordance with procedures used in domestic mammals, despite that some wild species may have complex reproductive characteristics, compromising the rapid progress of ARTs in particular species.

5.1. In vivo embryo production

Soon after the recognition of the gametes and embryo physiology, application of ARTs in each new species begins with in vivo embryo production, defined as the selection of female donors, AI or natural mating, embryo collection, cryopreservation, or direct transfer into synchronized recipients. Inevitably, the number of individuals is a crucial point in research, and individual variations nurture the development of in vitro ARTs.

In general, superovulatory protocols consist of a single dose of eCG and/or multiple injections of FSH [104]. The status of follicular development at the moment of superovulation, as well as the stress induced by intense manipulation during ARTs, is the main factor ascribed for the variability on response [105]. In an attempt to provide efficient superovulation protocols for embryo collection, different studies have been developed in various wild mammals. An interesting example corresponds to brown brocket deer [106], in which eCG induced a good response to superovulatory protocol, promoting the formation of functional corpora lutea (7.0 ± 1.8) although 66.7% (4/6) of the females showed premature luteal regression. In comparison, FSH administration resulted in a low formation of corpora lutea (2.6 ± 0.8) and lower proportion premature luteal regression (33.3%; 2/6).

There are few studies on in vivo embryo production protocols in wild camelids. In vicunas, two studies [107, 108] proposed ovarian superstimulatory protocols using eCG with or without CIDR® during 5 days; unfortunately, the results involved only data for follicular development, with no information on embryo recovered or transferred.

5.2. In vitro embryo production

The IVF is still the main in vitro technique for the reproduction of wild mammals, although this technique demands a wide quantity of viable oocytes to be fertilized. Briefly, IVF steps involve the oocyte recovery, the in vivo or IVM and selection of oocytes, the IVF using capacitated spermatozoa, and the preimplantation embryonic development. The aim is to develop high-quality embryos and obtain normal pregnancies, resulting in the birth of healthy offspring. In this sense, Table 3 summarizes the results of in vitro ARTs applied in some South American wild mammals. In general, the protocols used for domestic animals were gradually adjusted to wild species.

Although IVF embryos have been produced from capuchin monkeys [109], marmoset monkeys [66, 110], pumas [92], and tigrinas [51], modifications are needed to increase the efficiency of protocols. Currently, in vitro embryo production depends primarily on two major products: meiotic viable oocytes (meiotic competence) and capacitated viable spermatozoa (sperm competence). Domingues et al. [71] reported the viability of in vitro matured oocytes collected from nonstimulated capuchin monkeys. Lima et al. [109] established 40 hours as the optimal IVM length and showed the positive effect of FSH/LH (0.5 µg/mL/50.0 µg/mL) on IVF of oocytes collected also in nonstimulated females. In addition, spermatozoa obtained from semen diluted in coconut water were able to fertilize oocytes. However, the establishment of experimental conditions is still a requirement for this species.

Another interesting example of the use of IVF in wild felids [111] is that of jaguars; a stimulation protocol using FSH/LH resulted in approximately 25 follicles/female, with more than 80% of high-quality oocytes but the fertilization rates were fairly low (<25%) [55]. Contrastingly, ocelot and tigrina each treated with eCG/hCG produced an average of approximately 10 follicles with ~7–9 high-quality oocytes/female that resulted in 76 ocelot and 52 tigrina embryos [51].

SpeciesOocyte sourceMaturation typeSpermatozoaART appliedEmbryo productionAuthors
Collection/selectionMotility (%)Mediaa Cleavage rateb Blastocyst ratec Stage
Sapajus apela Without stimulation In vitro EEJ, cooled to 4°C/ Swim up~80IVFSOF20---4 cellsCell staining[114]
Callithrix jacchus Hormonal stimulation In vivo FertiCare vibrator, artificial vagina/ Pure gradient"/>70IVF/ICSIG1.2, G2.244.7/94.246.7/0.0d BlastocystCell staining[66]
Callithrix jacchus Hormonal stimulation In vitro FertiCare vibrator, artificial vagina/ Swim upNIIVF/ICSIISM1, ISM293.2/97.639.2/35.4BlastocystEmbryo transfer[115]
Felis concolor Hormonal stimulation In vivo EEJ/ Swim up40-50IVFmKRB------1 cellNI[97]
Pecari Tajacu Without stimulation In vitro Testis
cell suspension xenografts/---
NIICSINI37.5---4 cellsXenograft assay[42]

Table 3.

Results of IVF and ICSI techniques applied in some South American wild mammals.

[i] - Abbreviations: EEJ, electroejaculation; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; ARTs, Assisted reproductive technique; NI, not informed.

[ii] - aMore information about the composition of the media see reference.

[iii] - bCleavage rate represents the number of cleaved oocytes in relation to the number of oocytes entering to maturation in D2.

[iv] - cBlastocyst rate represents the number of blastocyst in relation to the number of zygotes.

[v] - dBlastocyst rate represents the number of blastocyst in relation to the number of cleaved embryos.

Alternatively, ICSI offers a precise control of sperm competence for in vitro procedures. ICSI was first used in marmoset monkeys in 2007, by Grupen et al. [66], using in vivo matured oocytes. Still, no blastocyst was produced by ICSI, despite that 47% of blastocysts were achieved after IVF. In 2014 [110], ICSI embryos derived from in vitro matured marmoset oocytes developed into neonates, through the use of two strategies: the evaluation of the most suitable timing for ICSI and establishing the in vitro and in vivo embryo developmental potential. In this study, the blastocyst rate tended to be low when ICSI was performed 1–2 hours after the extrusion of the first polar body. For in vitro developmental competence, a greater fertilization rate was observed in ICSI embryos (93.2%) compared with IVF embryos (82.2%). However, no differences in developmental rate (blastocyst/fertilized oocytes) were observed between ICSI (35.4%) and IVF (39.2%). In addition, for in vivo development of ICSI embryos, an offspring rate of 28.6% (6/21) and 2.7% (1/37) was obtained after the transference of 6 to 8 cell embryos and blastocysts, respectively.

In collared peccary [42], ICSI was used to evaluate the quality of sperm cells derived from testis cell suspension xenografts. The collared peccary presents an interesting Leydig cell cytoarchitecture, and therefore, it represents an interesting mammalian model for investigating cellular roles in male gonads. In the study, the sperm recovered from the xenografts originated 75.0% (6/8) of two-cell embryos and 37.5% (3/8) of four-cell embryos produced in 24 and 48 after ICSI, respectively.

For South American wild camelids, information on oocyte recovery and maturation are only available for vicuna: using the surgical oocyte aspiration from superstimulated females, a recovery rate of 55.4% (46/83) and an IVM rate of 41% were obtained, characterized by extrusion of the first polar body and cytoplasmic maturation [81], but no information was presented on IVF or ICSI.

5.3. Embryo cryopreservation and transfer

In recent years, advances in the embryo cryopreservation were achieved with studies in slow freezing conventional method or vitrification. Currently, embryo cryopreservation targets the establishment of a consistent vitrification protocol to be applied for the conservation of preimplantation embryos in different stages of development. Protocols developed for other species appear to be adequate for the purpose of embryo cryopreservation in wild felids. Offspring were obtained in ocelot following transfer of cryopreserved embryos [111].

In marmoset monkey, due to its small body size, the technical development of an efficient embryo transfer is being delayed. Different factors are being studied, such as nonsurgical approaches, embryo cryopreservation, use of late-stage embryos, and volume medium [112] to overcome these gaps. Using embryos in different developmental stages (10- to 16-cell, morula, and blastocysts) after vitrification in Cryotop, Ishibashi et al. [112] defended that reducing the transfer volume to 1 µL or less is essential for successful embryo transfer this species. This procedure provides pregnancy and birth rates of 80% (8/10) and 75% (9/12), while the use of larger volumes (2–3 µL) results in pregnancy and birth rates of 50% (5/10) and 27% (3/11), respectively.

6. Somatic cell processing and cloning

Somatic cell nuclear transfer (SCNT, cloning) consists of the transfer of the nucleus of donor cells into enucleated oocytes, resulting in the production of an individual genetically identical to the nucleus donor animal. In general, SCNT is performed according to the following steps: (i) preparing cytoplast receptors from oocyte recovery, selection, and maturation (cytoplast); (ii) isolation, characterization, IVC, and cellular cycle synchronization of the somatic cells to be used as nuclear donors (karyoplast); (iii) embryonic reconstruction by nuclear transfer, fusion of karyoplast–cytoplast complex, cellular activation, and preimplantation embryo development in vitro; and (iv) transfer of embryos for previously synchronized recipients, establishment of pregnancies, fetal development, and birth of offspring. In all these steps, there are determining factors for the success of the technique, including the media composition, the donor cell type and the oocyte source, and the timing of all steps down the success of the reproductive output of viable clones [113].

The use of cloning for conservation is often questionable for the risk of reducing the genetic variability. However, it is now known that SCNT not only provides the production and multiplication of a group of individual species of interest, as well as it allows additional studies in the field of reprogramming and cryopreservation of somatic tissue and cells, providing a larger scene in the reproduction of these species. Up to now, there are no studies on clone embryo production in South American wild mammals.

An important step in cloning is the selection of the cells donating a nucleus. In general, fibroblasts derived from skin biopsies are the cell model used. Numerous reasons justify its use for embryo reconstruction, such as the ease of obtaining and handling for the primary culture or subcultures, freezing patterns, and suitable transfection, in addition to obtaining inexhaustible quantities from a single explant. Moreover, the preservation of cells and tissues of the animal to be cloned allows the preservation of the genetic material of an individual indefinitely.

6.1. Generation and establishment of somatic cell primary cultures

The karyoplast preparation derived from skin biopsies involve multiple steps, harvesting of the biological material, through the isolation, establishment and characterization of cell cultures, and the cryopreservation conditions of cell populations after subculture cycle. Routinely, karyoplasts may be derived from skin biopsies of adult or fetal auricular region, according to established standards of asepsis and sterilization procedures [113]. After processing tissue samples into smaller fragments, these are distributed in Dulbecco Modified Eagles Medium (DMEM) supplemented with sodium pyruvate, sodium bicarbonate, antibiotics, amphotericin B, and fetal bovine serum. The primary cultures are monitored every 24 hours to evaluate the cell growth and total medium replacement. When 70% cell confluence is purchased on the plates, the cultures are subcultured. In general, explants are cultured under these conditions for 7–14 days [114]. To confirm the cells’ characteristics, particular antibodies are used, like vimentin [113].

In native Chilean species, the establishment of donor cell was already observed in Chilean Shrew opossum (Rhyncholestes raphanurus) and chinchilla (Chinchilla lanigera) [115]. In the study, tissue fragments were subjected to culture systems and somatic cells were obtained for future cloning.

6.2. Somatic tissue conservation

The tissue cryopreservation involves some major issues, such as the conservation of cellular functionality, extended storage of the samples, and the easiness of the procedures performed outside the laboratory [115]. Tissue sample cryopreservation of wild species is an interesting step for biodiversity conservation, when there is not an appropriate culture system.

In collared peccary [114, 116], vitrification techniques (conventional or solid-surface vitrification) may be used for cryopreservation of somatic tissue, allowing the isolation of viable cells. In this study, ear fragments (9.0 mm3) were vitrified in a solution containing DMEM plus 3.0 M DMSO, 3.0 M EG, 0.25 M sucrose, and 10% fetal calf serum. The histological analysis and IVC showed that few tissue damages were associated with solid-surface vitrification [116]. Moreover, all cells resulting in vitrified and noncryopreserved fragments showed the characteristics of suitable fibroblasts: fusiform features with oval nucleus in the center. The growth curves showed clear log and lag phases of development, with no difference between treatment and nonvitrified for population doubling time (conventional vitrification directly: 66.1 hours; solid-surface vitrification: 66.3 hours; and nonvitrified: 54.0 hours) [114].

6.3. Cloning and its perspectives

Although some domestic mammals have already been cloned, information on cloning of South American wild animals is scarce. As with other animal species, cloning allows to know the principles of nuclear reprogramming, conservation of cells and tissues for different purposes (reproductive and therapeutic), and clones can also be used as models in several studies. Thus, the efficient production of pluripotent stem cells (iPSC) allows the generation of genetic preservation by different ways, such as gamete production and embryo complementation. Dermal fibroblasts derived from jaguars were transduced using genes encoding the human transcription factors, in an experiment that highlighted the homeobox protein NANOG as crucial to the cell reprogramming in this species, and demonstrated that the technique may represent an efficient method for iPSC production from endangered felids [117].

7. Final considerations

The ARTs have applications in different productive and scientific sectors, whether or not aiming at the conservation of species. As occurred for the domestic species, the use of ART sequence followed the order of complexity and evolution of biotechnologies. Accordingly, the routine use of AI and embryo transfer in different species of mammals is evident. Moreover, in vitro ARTs continue to grow, using a parallel between the protocols applied to domestic species and individual characteristics of each wild ones. With the progress of in vivo or in vitro ARTs, it is expected to know, to manipulate, and to conserve the genetic material, promoting the preservation of biodiversity, especially in South American wild mammals.


1 - Silva AR, Morato RG, Silva LDM. The potential of gamete recovery from non-domestic canids and felids. Animal Reproduction Science. 2004;81:159–175.
2 - Garde JJ, Ortiz N, Garcia AJ, Lopez A, Gallego L. Criopreservación postmortem de material espermático e inceminación artificial en el ciervo iberico. [Postmortem cryopreservation of sperm material and artificial insemination in Iberian deer]. Archivos de Zootecnia.1998;47:351–356.
3 - Castelo TS, Silva AR. Eletroejaculação em mamíferos silvestres: principais fatores que afetam sua eficiência. [Electroejaculation in wild mammals: main factors affecting its efficiency]. Revista Brasileira de Reprodução Animal. 2014;38:208–213.
4 - Durrant BS. Semen collection, evaluation and cryopreservation in exotic animal species: maximizing reproductive potential. Ilar News Journal. 1990;32:2–10.
5 - Wildt DE, Bush M, Howard JG, Obrien SJ, Meltzer D, Van Dyk A, Ebedes H, Brand DJ. Unique seminal quality in the South African cheetah and a comparative evaluation in the domestic cat. Biology of Reproduction. 1983;29:1019–1025.
6 - Morais RN, Muciolo RG, Gomes MLF, Lacerda O, Moraes W, Moreira N, Graham LH, Swanson WF, Brown JL. Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. weidii) and tigrina (L. tigrinus). Theriogenology. 2002; 57:2027–2041.
7 - Morato RG, Verreschi IT, Guimarães MA, Cassaro K, Pessuti C, Barnabe RC. Seasonal variation in the endocrine-testicular function of captive jaguars (Panthera onca). Theriogenology. 2004;61:1273–1281.
8 - Silva HVR, Magalhães FF, Ribeiro LR, Souza ALP, Freitas CIA, Oliveira MF, Silva AR, Silva LDM. Morphometry, morphology and ultrastructure of ring-tailed coati sperm (Nasua nasua Linnaeus, 1766). Reproduction in Domestic Animals. 2015;50:945-951.
9 - Cunha IC, Morato RG, Santos IP, Quirino CR. Biometry of the reproductive system and the ejaculation response of maned wolf (Chrysocyon brachyurus) to electroejaculation procedure. In: Proceedings of the 6th ISCFR & 6th EVSSAR; 2008; Vienna. Austria; 2008, pp. 1–2.
10 - Peres MA, Benetti EJ, Milazzotto MP, Visintin JA, Miglino MA, Assumpção MEOA. Collection and evaluation of semen from the three-toed sloth (Bradypus tridactylus). Tissue Cells Cell. 2008;40:325–331.
11 - Mendonça MAC. Análise descritiva do perfil espermático do tamanduá-bandeira (Myrmecophaga tridactyla Linnaeus, 1758) de cativeiro. [Descriptive analysis of the sperm profile in captive flag anteater (Myrmecophaga tridactyla Linnaeus, 1758) - Master of Sciences Thesis]. São Paulo: Universidade de São Paulo; 2010.
12 - Serafim MKB, Lira RA, Costa LLM, Gadelha ICN, Freitas CIA, Silva AR. Description of semen characteristics from six-banded armadillos (Euphractus sexcinctus) collected by electroejaculation. Animal Reproduction Science. 2010;118:362–365.
13 - Pukazhenthi BS, Della Togna G, Padilla L, Smith D, Sanchez C, Pelican K, et al. Ejaculate traits and sperm cryopreservation in the endangered Baird’s tapir (Tapirus bairdii). Journal of Andrology. 2011;32:260–270.
14 - Castelo TS, Souza AL, Lima GL, Peixoto GC, Campos LB, Oliveira MF, Silva AR. Interactions among different devices and electrical stimulus on the electroejaculation of captive agoutis (Dasyprocta leporina). Reproduction in Domestic Animals. 2015;50:492–496.
15 - Flores-Herrera H, Acuña-Hernández DG, Rivera-Rebolledo JA, González-Jiménez MA, Rodas-Martínez AZ, Swanson WF. Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi). Theriogenology. 2012;78:612–619.
16 - Oliveira KG, Castro PHG, Muniz JAPC, Domingues SFS. Conservação do sêmen e liquefação do coágulo seminal de macaco-prego (Cebus apella) em água de coco em pó (ACP-118®), em diferentes temperaturas. [Spem conservation and seminal clot liquefaction in capuchin monkey (Cebus apella) using powdered coconut water (ACP-118®) at different temperatures]. Ciência Rural. 2010;40:617–621.
17 - Castelo TS, Bezerra FSB, Souza ALP, Moreira MAP, Paula VV, Oliveira MF, Silva AR. Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders. Theriogenology. 2010;74:1060–1065.
18 - Chatdarong K, Thuwanut P, Morrell JM. Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm. Theriogenology. 2010;73:1284–1292.
19 - Rijsselaere T, Van Soom A, Maes D, Verberckmoes S, Kruif A. Effect of blood admixture on in vitro survival of chilled and frozen-thawed canine spermatozoa. Theriogenology. 2004;61:1589–1602.
20 - Morton KM, Evans G, Maxwell WMC. Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm. Theriogenology. 2010;74:311–316.
21 - Ferraz MS, Menezes DJ, Pessoa GT, Cabral RM, Illera MJ, Silva AR, Carvalho MA. Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti). Theriogenology. 2011;75:459–462.
22 - Bezerra JAB, Silva AM, Peixoto GCX, Silva MA, Oliveira MF, Silva AR. Influence of recovery method and centrifugation on epididymal sperm from collared peccaries (Pecari tajacu Linnaeus, 1758). Zoological Science. 2014;31:338–342.
23 - Silva AM, Bezerra JAB, Campos LB, Borges PAC, Lima GL, Castelo TS, Souza ALP, Silva AR. Características gerais de espermatozoides epididimários de préas (Galea spixii Wagler,1831). [General epididymal sperm characteristics of cavies (Galea spixii Wagler, 1831]. In: Proceedings of the XXII Congresso Brasileiro de Zootecnia, 2012, Cuiabá. Mato Grosso; 2012.
24 - Fickel L, Wagener A, Ludwig A. Semen cryopreservation and the conservation of endangered species. European Journal of Wildlife Research. 2007;53:81–89.
25 - Alamo D, Batista M, González F, Rodríguez N, Cruz G, Cabrera F, Gracia A. Cryopreservation of semen in the dog: use of ultra-freezers of −152 °C as a viable alternative to liquid nitrogen. Theriogenology. 2005;63:72–82.
26 - Lago AEA, Nascimento HHL, Souza ALP, Bezerra JAB, Silva AM, Castelo TS, A.R. Silva AR. Aloe vera use on short-term preservation of collared pecari (Pecari tajacu) semen. In: Proceedings of the V International Symposium on Animal Biology of Reproduction, 8-11 October 2014; Campinas. São Paulo: CBRA; 2014.
27 - Sousa PC, Santos EAA, Bezerra JAB, Lima GL, Castelo TS, Fontenele-Neto JD, Silva AR. Morphology, morphometry and ultrastructure of captive six-banded armadillo (Euphractus sexcinctus) sperm. Animal Reproduction Science. 2013;140:279–285.
28 - Paz RCR, Morgado TO,Viana CTR, Arruda FP, Bezerra do Nascimento DO, Guimarães LDA. Semen collection and evaluation of captive coatis (Nasua nasua). Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2012;64:318–322.
29 - Silva MA, Peixoto GCX, Lima GL, Bezerra JAB, Campos LB, Paiva ALC, Paula VV, Silva AR. Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol. Theriogenology. 2012;78:605–611.
30 - Castelo TS, Praxedes ECG, Souza ALP, Peixoto GCX, Lima GL, Sousa PC, Santos EAA, Campos LB, Bezerra JAB, Silva AR. Criopreservação de sêmen de cutias (Dasyprocta aguti) em diluente à base de água de coco em pó – ACP-109c®. [Cryopreservation of agouti (Dasyprocta aguti) semen using powdered coconut water extender – ACP-109®]. In: Proceedings of the Congresso Brasileiro de Reprodução Animal; Maio 2013; Uberlândia. Minas Gerais: CBRA; 2013.
31 - Oliveira KG, Leão DL, Almeida DV, Santos RR, Domingues SF. Seminal characteristics and cryopreservation of sperm from the squirrel monkey, Saimiri collinsi. Theriogenology. 2015;84:743–749.
32 - Souza ALP, Lima GL, Peixoto GCX, Castelo TS, Bezerra JAB, Silva AR. Efeito da Lipoproteína de Baixa Densidade (LDL) na congelação de sêmen de catetos (Pecari tajacu). Acta Veterinaria Brasilica. 2014;8:41–42.
33 - Souza ALP, Lima GL, Peixoto GCX, Bezerra JAB, Silva AM, Oliveira MF, Silva AR. Innovative use of Aloe vera as a cryoprotectant for the collared peccaries (Pecari tajacu) semen freezing. In: Proceedings of the V International Symposium on Animal Biology of Reproduction, 8-11 October 2014; Campinas. São Paulo: CBRA; 2014.
34 - Bezerra JAB, Praxedes ECG, Silva AM, Souza ALP, Castelo TS, Campos LB, Silva AR. Objective assessment of the effect of different extenders on frozen-thawed epididymal sperm from collared peccaries (Pecari tajacu). In: Proceedings of the V International Symposium on Animal Biology of Reproduction, 8-11 October 2014; Campinas. São Paulo: CBRA; 2014.
35 - Silva AM, Apolinario CAC, Almeida LM, Souza ALP, Castelo TS, Lago AEA, Silva AR. Criopreservação de espermatozoides epididimários de preá (Galea spixii Wagler, 1831). [Cryopreservation of cavies (Galea spixii Wagler, 1831) epididimal sperm]. In: Proceedings of the XXI Congresso Brasileiro de Reprodução Animal; 2015; Belo Horizonte. Minas Gerais: CBRA; 2015.
36 - Silva MA, Peixoto GCX, Santos EAA, Castelo TS, Oliveira MF, Silva AR. Recovery and cryopreservation of epididymal sperm from agouti (Dasyprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders. Theriogenology. 2011;76:1084–1089.
37 - Leão KM. Técnicas de inseminação artificial [Techniques for artificial insemination – Graduate monography]. Botucatu: Faculdade de Medicina Veterinária e Zootecnia da Universidade Estadual Paulista; 2003.
38 - Swanson WF, Horohov DW, Godke RA. Production of exogenous gonadotropin-neutralizing immunoglobulins in cats following repeated eCG/hCG treatment and relevance for assisted reproduction in felids. Journal of Reproduction and Fertility. 1995;105:35–41.
39 - Morrell JM, Nubbemeyer R, Heistermann M, Rosenbusch J, Küderling I, Holt W, Hodges JK. Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm. Animal Reproduction Science. 1998;52:165–174.
40 - Woelders H, Chaveiro A. Theoretical prediction of optimal freezing programmes. Cryobiology. 2004;49:258–271.
41 - Abrishami M, Anzar M, Yang Y, Honaramooz A. Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice. Theriogenology. 2010;73:86–96.
42 - Campos-Junior PHA, Costa GMJ, Avelar GF, Lacerda SMSN, Costa NN, Orashi OM, Miranda MS, Barcelos LS, Jorge EC, Guimarães DA, França LR. Derivation of sperm from xenografted testis cells and tissues of the peccary (Tayassu tajacu). Reproduction. 2014;147:291–299.
43 - Borges PAC, Bezerra JAB, Praxedes ECG, Lima GL, Souza ALP, Peixoto GCX, Silva AR. Cryopreservation of testicular tissue: a potential tool for the conservation of male genetic material from collared peccaries (Pecari tajacu). In: Proceedings of the V International Symposium on Animal Biology of Reproduction, 8-11 October 2014; Campinas. São Paulo: CBRA; 2014.
44 - Durrant BS. The importance and potential of artificial insemination in CANDES (companion animals, non-domestic, endangered species). Theriogenology. 2009;71:113–122.
45 - Micheletti T, Brown JL, Walker SL, Cubas ZS, Furtado PV, Putman SB, Moraes W, Liveira MJ, Oliveira CA, Moreira N. The use of altrenogest to ovoid hyperestrogenism after eCG-hCG ovulation induction in southern tigrina (Leopardus guttulus). Theriogenology. 2015;84:575–582.
46 - Pfeifer LFM, Leonardi CEP, Castro NA, Viana JHM, Siqueira LGB, Castilho EM, Singh J, Krusser RH, Rubin MIB. The use of PGF2a as ovulatory stimulus for timed artificial insemination in cattle. Theriogenology. 2014;81:689–695.
47 - Kriegsfeld LJ, Ubuka T, Bentley GE, Tsutsui K. Seasonal control of gonadotropin-inhibitory hormone (GnIH) in birds and mammals. Frontiers in Neuroendocrinology. 2015;37:65–75.
48 - Ahmed HMM, Yeh JY, Lin WJ, Forsberg NE, Cheng WTK, Ou BR. Validation of a luciferase bioassay to detect the progestative activity in gilts whose estrus was induced by an uterotonic herb (Ligusticum chuanxiong). Livestock Science. 2014;163:159–164.
49 - Howard J, Wildt DE. Approaches and efficacy of artificial insemination in felids and mustelids. Theriogenology. 2009;71:130–148.
50 - Pelican KM, Wildt DE, Pukazhenthi B, Howard J. Ovarian control for assisted reproduction in the domestic cat and wild felids. Theriogenology. 2006;66:37–48.
51 - Swanson WF, Paz RCR, Morais RN, Gomes MLF, Moraes W, Adania CH. Influence of species and diet on efficiency of in vitro fertilization in two endangered Brazilian felids—the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus). Theriogenology. 2002;57:593.
52 - Silva ME, Colazo MG, Ratto MH. GnRH dose reduction decreases pituitary LH release and ovulatory response but does not affect corpus luteum (CL) development and function in llamas. Theriogenology. 2012;77:1802–1810.
53 - Crichton EG, Bedows E, Miller-Lindholm AK, Baldwin DM, Armstrong DL, Graham LH, et al. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biology of Reproduction. 2003;68:105–113.
54 - Phillips LG, Simmons LG, Bush M, Howard JG, Wildt DE. Gonadotropin regimen for inducing ovarian activity in captive wild felids. JAVMA. 1982;181:1246–1250.
55 - Morato RG, Crichton EG, Paz RCR, Zuge RM, Moura CA, Nunos AVL, Teixeira RH, Porto L, Priscila M, Guimaraes MABV, Correa SHR, Barnabe RC, Armstrong DL, Loskutoff NM. Ovarian stimulation and successful in vitro fertilization in the jaguar (Panthera onca). Theriogenology. 2000;53:339.
56 - Morato RG, Bueno MG, Malmheister P, Verreschi ITN, Barnabe RC. Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress. Brazilian Journal of Medical and Biological Research. 2004;37:1903–1907.
57 - Swanson WF. Application of assisted reproduction for population management in felids: the potential and reality for conservation of small cats. Theriogenology. 2006;66:49–58.
58 - Brown JL. Female reproductive cycle of wild female felids. Animal Reproduction Science. 2011;124:155–162.
59 - Johnson AEM, Freeman EW, Colgin M, McDonough C, Songsasen N. Induction of ovarian activity and ovulation in an induced ovulator, the maned wolf (Chrysocyon brachyurus), using GnRH agonist and recombinant LH. Theriogenology. 2014;82:71–79.
60 - Chittick E, Rotstein D, Brown T, Wolfe B. Pyometra and uterine adenocarcinoma in a melengestrol acetate-implanted captive coati (Nasua nasua). Journal of Zoo and Wildlife Medicine. 2001;32:245–251.
61 - Zanetti ES, Polegato BF, Duarte JMB. Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira). Animal Reproduction Science. 2010;117:266–274.
62 - Maia KM, Peixoto GC, Campos LB, Silva AM, Castelo TS, Ricarte ARF, Silva AR. Estrous synchronization in captive collared peccaries (Pecari tajacu) using a prostaglandin F2α analog. Zoological Society. 2014;31:836–839.
63 - Michael AE, Abayasekara DRE, Webley GE. The luteotrophic actions of prostaglandins E2 and F2α on dispersed marmoset luteal cells are differentially mediated via cyclic AMP and protein kinase C. Journal of Endocrinology. 1993;138:291–298.
64 - Webley GE, Michael AE, Abayasekara DRE. The relationship between the production and the anti-gonadotrophic action of prostaglandin F2a in luteal cells from the marmoset monkey (Callithrix jacchus) in the early and mid-luteal phase. General and Comparative Endocrinology. 2010;166:436–442.
65 - Marshall VS, Browne MA, Knowles L, Golos TG, Thomson JA. Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone. Journal of Medical Primatology. 2003;32;57–66.
66 - Grupen CG, Gilchrist RB, Nayudu PL, Barry MF, Schulz SJ, Ritter LJ, Armstrong DT. Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus). Theriogenology. 2007;68:861–872.
67 - Pukazhenthi B, Comizzoli P, Travis AJ, Wildt DE. Applications of emerging technologies to the study and conservation of threatened and endangered species. Reproduction, Fertility and Development. 2006;18:77–90.
68 - Lermen D, Blömeke B, Browne R, et al. Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes. Molecular Ecology. 2009;18:1030–1033.
69 - Domingues SFS, Ferreira HS, Muniz JAPC, Lima AKF, Ohashi OM, Figueiredo JR, Silva LDM. Mechanical isolation of capuchin monkey (Cebus apella) preantral ovarian follicles. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2003;55:301–308.
70 - Brito AB, Santos RR, van den Hurk R, Lima JS, Miranda MS, Ohashi OM, Domingues SF. Short-term culture of ovarian cortical strips from capuchin monkeys (Sapajus apella): a morphological, viability, and molecular study of preantral follicular development in vitro. Reproductive Sciences. 2013;20:990–997.
71 - Domingues SF, Caldas-Bussiere MC, Petretski MD, Ohashi OM, Lima JS, Santos RR, Cordeiro MS, Gomes de Castro PH. Effects of follicular phase and oocyte–cumulus complexes quality on the protein profile and in vitro oocyte meiosis competence in Cebus apella. Fertility and Sterility. 2010;93:1662–1667.
72 - Marshall VS, Browne MA, Knowles L, Golos TG, Thomson JA. Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone. Journal of Medical Primatology. 2003;32:57–66.
73 - Delimitreva S, Tkachenko OY, Berenson A, Nayudu PL. Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization—what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects? Theriogenology. 2012;77:1297–1311.
74 - Yeoman RR, Helvacioglu A, Williams LE, Aksel S, Abee CR. Restoration of oocyte maturational competency during the nonbreeding season with follicle-stimulating hormone stimulation in squirrel monkeys (Saimiri boliviensis boliviensis). Biology of Reproduction. 1994;50:329–335.
75 - Schuler AM, Westberry JM, Scammell JG, Abee CR, Kuehl TJ, Gordon JW. Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) by using pregnant mare serum gonadotropin. Comparative Medicine. 2006;56:13–17.
76 - Dukelow WR, Jarosz SJ, Jewett DA, Harrison RM. Laparoscopic examination of the ovaries in goats and primates. Journal of Laboratory Animal Science. 1971;21:594–597.
77 - Kuehl TJ, Dukelow WR. Maturation and in vitro fertilization of follicular oocytes of the squirrel monkey (Saimiri sciureus). Biology of Reproduction. 1979;21:545–556.
78 - Wiedemann C, Zahmel J, Jewgenow. Short-term culture of ovarian cortex pieces to assess the cryopreservation outcome in wild felids for genome conservation. BMC Veterinary Research. 2013;9:37.
79 - Jewgenow K, Stolte M. Isolation of preantral follicles from nondomestic cats–viability and ultrastructural investigations. Animal Reproduction Science. 1996;44:183–193.
80 - Baldassare H, Carelli JB, Requena LA, Rodrigues MG, Ferreira S, Salomão J, Jorge Neto PN. Efficient recovery of oocytes from onça parda (Puma concolor) by laparoscopic ovum pick-up of gonadotropin-stimulated females. Animal Reproduction. 2015;12:717.
81 - Chaves MG, Miragaya MH, Capdevielle EF, Rutter B, Giuliano SM, Agüero A. In vitro maturation of vicuna oocytes recovered by surgical aspiration of follicles from superstimulated ovaries. Biocell. 2004;28:545.
82 - Luvoni GC. Cryosurvival of ex situ and in situ feline oocytes. Reproduction in Domestic Animals. 2012;47:266–268.
83 - Tingen C, Kim A, Woodruff TK. The primordial pool of follicles and nest breakdown in mammalian ovaries. Molecular Human Reproduction. 2009;15:795–803.
84 - Comizzoli P, Songsasen N, Wildt DE. Protecting and extending fertility for females of wild and endangered mammals. Cancer Treatment and Research. 2010;156:87–100.
85 - Matos MH, Lima-Verde IB, Luque MC, Maia JE Jr, Silva JR, Celestino JJ, Martins FS, Báo SN, Lucci CM, Figueiredo JR. Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro. Zygote. 2007;15:173–182.
86 - Gilchrist RB, Nayudu PL, Hodges JK. Maturation, fertilization, and development of marmoset monkey oocytes in vitro. Biology of Reproduction. 1997;56:238–246.
87 - Nayudu PL, Wu J, Michelmann HW. In vitro development of marmoset monkey oocytes by pre-antral follicle culture. Reproduction in Domestic Animals. 2003;38:90–96.
88 - Scalercio SRRA, Brito AB, Domingues SFS, Santos RR, Amorim CA. Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi). Reproductive Sciences. 2015;22:68–74.
89 - Yeoman RR, Williams LE, Abee CR. Low oxygen inhibits but complex high-glucose medium facilitates in vitro maturation of squirrel monkey oocyte-granulosa cell complexes. Journal of Assisted Reproduction and Genetics. 1999;16:102–107.
90 - Asakawa T, Chan PJ, Dukelow WR. Time sequence of in vitro maturation and chromosomal normality in metaphase I and metaphase II of the squirrel monkey (Saimiri sciureus) oocyte. Biology of Reproduction. 1982;27:118–124.
91 - Johnston LA, Donoghue AM, Obrien SJ, Wildt DE. Rescue and maturation in vitro of follicular oocytes collected from nondomestic Felid Species. Biology of Reproduction. 1991;45:898–906.
92 - Miller AM, Roelke ME, Goodrowe KL, Howard JG, Wildt DE. Oocyte recovery, maturation and fertilization in vitro in the puma (Felis concolor). Journal of Reproduction and Fertility. 1990;88:249–258.
93 - Weir BJ. The reproductive physiology of the plains viscacha, Lagostomus maximus. Journal of Reproduction and Fertility. 1971;25:355–363.
94 - Jensen FC, Willis MA, Leopardo NP, Espinosa MB, Vitullo AD. The ovary of the gestating South American plain vizcacha (Lagostomus maximus): suppressed apoptosis and corpora lutea persistence. Biology of Reproduction. 2008;79:240–246.
95 - Leopardo NP, Jensen F, Willis MA, Espinosa MB, Vitullo AD. The developing ovary of the South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia): massive proliferation with no sign of apoptosis-mediated germ cell attrition. Reproduction. 2011;141:633–641.
96 - Silva AR, Lima GL, Lima LF, Rocha RMP, Oliveira MF, Rodrigues APR, Figueiredo JR. Effect of FSH supplementation in the TCM 199 medium for in vitro culture of ovarian preantral follicles from collared peccaries (Pecari tajacu). In: Proceedings of the Systems Biology of Reproduction Fertility A global challenge; 2014; Grand Rapids. EUA: SSR; 2014. p. 442.
97 - Schönfeldt V, Chandolia R, Kiesel L, Nieschlag E, Schlatt S, Sonntag B. Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant. Reproduction. 2011;141:481–490.
98 - Schönfeldt V, Chandolia R, Kiesel L, Nieschlag E, Schlatt S, Sonntag B. Advanced follicle development in xenografted prepubertal ovarian tissue: the common marmoset as a nonhuman primate model for ovarian tissue transplantation. Fertility and Sterility. 2011;95:1428–1434.
99 - Candy CJ, Wood MJ, Whittingham DG. Ovary and ovulation: follicular development in cryopreserved marmoset ovarian tissue after transplantation. Human Reproduction. 1995;10:2334–2338.
100 - Wanderley LS, Luz HK, Faustino LR, Lima IM, Lopes CA, Silva AR, Báo SN, Campello CC, Rodrigues AP, Figueiredo JR. Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol. Theriogenology. 2012;77:260–267.
101 - Praxedes ECG, Lima GL, Silva AM, Bezerra JAB, Souza ALP, Campos LB, Silva AR. Vitrificação de tecido ovariano de preá (Galea spixi). [Vitrification of cavies (Galea spixi) ovarian tissue]. In: Proceedings of the XXI Congresso Brasileiro de Reprodução Animal, 2015, Belo Horizonte. Minas Gerais: CBRA; 2015.
102 - Lima GL, Santos EAA, Lima LF, Luz VB, Rodrigues APR, Silva AR. Short-term preservation of Pecari tajacu ovarian preantral follicles using phosphate buffered saline (PBS) or powdered coconut water (ACP®) media. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2014;66:1623–1630.
103 - Lima GL, Luz VB, Alves AMCV, Lunardi FO, Souza ALP, Peixoto GCX, Rodrigues APR, Oliveira MF, Silva AR. Vitrification of collared peccaries (Tayassu tajacu) ovarian tissue using various cryoprotectants preliminary results. In: Proceedings of the IV International Symposium on Animal Biology of Reproduction. 2012; Campinas. São Paulo: ISABR; 2012.
104 - Asher GW, Jabbour HN, Thompson JG, Tervit HR, Morrow CJ. Superovulation of farmed red deer (Cervus elaphus) and fallow deer (Dama dama): incidence of ovulation and changes in plasma hormone concentrations during the pré-ovulatory period in relation to ova recovery and fertilization. Animal Reproduction Science. 1995;38:137–154.
105 - González R, Berlinguer F, Espeso G, Ariu F, del Olmo A, Garde JJ, Gomendio M, Ledda S, Rolda ERS. Use of a neuroleptic in assisted reproduction of the critically endangered Mohor gazelle (Gazella dama mhorr). Theriogenology. 2008;70:909–922.
106 - Zanetti ES, Munerato MS, Cursino MS, Duarte JMB. Comparing two different superovulation protocols on ovarian activity and fecal glucocorticoid levels in the brown brocket deer (Mazama gouazoubira). Reproductive Biology and Endocrinology. 2014;12:24.
107 - Aller JF, Rebuffi GE, Cancino AK. Superovulation response to progesterone-eCG treatment in vicuna (Vicugna vicugna) in semicaptive conditions. Theriogenology. 2002;57:576.
108 - Aba MA, Miragaya MH, Chaves MG, Capdevielle EF, Rutter B, Agüero A. Effect of exogenous progesterone and eCG treatment on ovarian follicular dynamics in vicunas (Vicugna vicugna). Animal Reproduction Science. 2005;86:153–161.
109 - Lima JS, Leão DL, Sampaio RV, Brito AB, Santos RR, Miranda MS, Orashi OM, Domingues SFS. Embryo production by parthenogenetic activation and fertilization on in vitro matures oocytes from Cebus apella. Zygote. 2012;21:162–166.
110 - Takahashi T, Hanazawa K, Inoue T, Sato K, Sedohara A, Okahara J, Suemizu H, Yagihashi C, Yamamoto M, Eto T, Konno Y, Okano H, Suematsu M, Sasaki E. Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes. Plos One. 2014;9:e95560.
111 - Swanson WF, Brown JL. International training programs in reproductive sciences for conservation of Latin American felids. Animal Reproduction Science. 2004;82–83:21–34.
112 - Ishibashi H, Motohashi HH, Kumon M, Yamamoto K, Okada H, Okada T, Seki Kazuhiko. Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos. Biology of Reproduction. 2013;88:1–5.
113 - Pereira AF, Santos MLT, Borges AA, Queiroz Neta LB, Santos MVO, Feitosa AKN. Isolation and characterization of skin-derived donor cells for nuclear transfer. Acta Veterinaria Brasilica. 2014;8:311–316.
114 - Borges AA, Queiroz Neta LB, Lima GL, Santos MVO, Santos MLT, Oliveira MF, Silva AR, Pereira AF. Atividade proliferativa de células provenientes de tecido auricular de catetos (Pecari tajacu) após distintas técnicas de vitrificação. [Proliferative activity of cells derived from the ear tissue of collared peccaries (Pecari tajacu) after diferente vitrification techniques]. In: Proceeding of the XXV Congresso Brasileiro de Zootecnia; Fortaleza, Ceará; 2015.
115 - Tovar H, Navarrete F, Rodríguez L, Skewes O, Castro FO. Cold storage of biopsies from wild endangered native Chilean species in field conditions and subsequent isolation of primary culture cell lines. In Vitro Cellular & Developmental Biology – Animal. 2008;44:309–320.
116 - Borges AA, Lima GL, Santos MLT, Queiroz Neta LB, Praxedes ECG, Santos MVO, Oliveira MF, Silva AR, Pereira AF. Histological evaluation of ear tissue of collared peccary (Pecari tajacu) after different vitrification techniques. Animal Reproduction. 2015;12:230.
117 - Verma R, Liu J, Holland MK, Temple-Smith P, Williamson M, Verma PJ. Nanog is an essential factor for induction of pluripotency in somatic cells from endangered felids. BioResearch Open Access. 2013;2:72–76.