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Theriogenology - Recent Advances in the Field [Working Title]
Open access peer-reviewed chapter - ONLINE FIRST
Strategies for the Establishment of Fibroblastic Lines for the Conservation of Wild Mammals
Written By
Alexsandra Fernandes Pereira, Lhara Ricarliany Medeiros de Oliveira, Leonardo Vitorino Costa de Aquino, João Vitor da Silva Viana and Luanna Lorenna Vieira Rodrigues
Submitted: 27 April 2023Reviewed: 30 November 2023Published: 21 December 2023
The loss of wild biodiversity has encouraged the development of fibroblastic lines, mainly fibroblasts derived from skin, which can be interesting tools for the conservation of wild mammals. These biological samples, when properly well-established, are essential elements for the reproduction of species through their use in cloning by somatic cell nuclear transfer and induction of cells to pluripotency. In general, the establishment of fibroblastic lines involves the following strategies: (i) cell isolation techniques and identification of fibroblasts; (ii) conditions for in vitro culture of fibroblasts; (iii) conditions for cryopreservation of fibroblasts; and (iv) nuclear reprogramming studies. At each stage, species-specific factors are involved, and determining these lines in the species of interest represents the first step toward its successful use for animal conservation. Therefore, this chapter discusses the stages and parameters involved in the strategies for establishing fibroblastic lines, delving into the main technical aspects and results obtained from the use of these cells in recent years in wild mammals.
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil
Lhara Ricarliany Medeiros de Oliveira
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil
Leonardo Vitorino Costa de Aquino
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil
João Vitor da Silva Viana
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil
Luanna Lorenna Vieira Rodrigues
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil
*Address all correspondence to: alexsandra.pereira@ufersa.edu.br
1. Introduction
Fibroblastic lines are defined as populations of fibroblasts that proliferate in vitro over an extended period when given an appropriate medium and space, and therefore can be maintained by serial subculturing while maintaining their morphological and physiological characteristics [1]. Although they represent part of an animal’s tissue, they can be used for extensive in vitro experimentations to understand cell behavior through analyzes such as morphology evaluation, population doubling time, metabolism performance, and tolerance to oxidative stress [2].
For wild mammals, studies with cell lines are focused on promoting the conservation and multiplication of such species, which are often endangered. With only one sample collected, different strategies can be developed to propagate the genetic material of this population, ensuring its future use in improved techniques over the years. For example, for different species, these cells can be cultured in vitro and maintained as finite lines for more than 10 passages, since a prior establishment of the ideal culture conditions for the cell type is established, in addition to the development of ideal temperature, gaseous atmosphere, culture medium, and supplements [3].
Moreover, these fibroblastic cells can be stored by cryopreservation protocols both to guarantee their viability in the following stages of experiments that require some time to perform and be used many years later in the search to recover representatives of those populations through advanced assisted reproduction techniques, such as cloning by somatic cell nuclear transfer (SCNT) or generation of gametes by induced pluripotent stem cells (iPSC). Therefore, this chapter presents the steps involved in the establishment and improvement of all these protocols with fibroblasts in wild mammals and describes the difficulties and advances that have already been made, with the goal of reproducing such specimens in the near future.
2. Isolation techniques and identification of fibroblasts
The skin has been the chosen organ for fibroblast obtaining, due to it being the largest organ, with distribution over the individual’s body. Nevertheless, fibroblasts have already been obtained from other regions of individuals [4]. Initially, when establishing a cell line, it is necessary to conduct a study of the skin regions where the fragments can be obtained, permitting the knowledge of its morphology and types of cells that are present and their distribution among the skin layers [5]. In fishing cat (Prionailurus viverrinus), the primary culture showed to be influenced by the skin region, resulting in a major number of keratinocytes and presence of lipid droplets obtained in abdominal and ear skin samples [6]. However, in red-rumped agouti (Dasyrocta leporina), no difference was observed between the cell recovery in inguinal, abdominal, and ear regions [7]. Through this base knowledge, the adequate method of cell isolation and the possible cell type to be utilized can be established.
After the determination of skin region, tissue fragments are recovered by in-vivo or post-mortem biopsies (Figure 1) via the use of scalpel, surgical scissors, and punch. Subsequently, the sample transport is conducted, which should be performed in a short time in animal corporal temperature (37–38°C). For longer periods, the transport should be performed under refrigerated temperature (4–6°C) in nutritive medium or saline solution, avoiding tissue damage. In collared peccary (Pecari tajacu), the presence of nutritive medium was a determinant factor of cellular quality, promoting the maintenance of cell membrane integrity after 10 days of refrigeration (4–6°C) and obtaining cells after 50 days [8].
Figure 1.
Steps for the establishment of cell lines in wild mammals.
In laboratory settings, the first step to be performed is the cleaning of the fragment by removing hair and adipose tissue. This process is fundamental, as this stage is conducted with the aim to prevent bacterial and fungus contamination during cell culture [6]. After the tissue-cleaning step, tissue fragmentation is conducted, aiming to obtain a larger cell detachment. Similar to cell isolation methods, this step can be conducted by mechanical dissolution with scissors, sieves, and meshes, enzymatic dissolution by tissue matrix dissolution with enzymes, or putting the explant directly on a dish (Figure 1).
Specifically, regarding the methods of cell isolation, the three cited methods can be conducted alone or combined. The mechanical dissolution in combination with dissolution enzymatic was conducted in Cheetah (Acinonyx jubatus) [9], Asian elephant (Elephas maximus) [10], Iberian hare (Lepus granatensis), and European rabbit (Oryctolagus cuniculus) [11] with cells recovered after 4–7 days. However, enzymatic, and mechanical methods can result in damage, reducing the cell viability if inconsistently utilized. Thus, the explant isolation method is the most utilized in wild mammals due to its lower cost and lesser capacity to damage the fragment. The isolation by explants showed success in several wild felids, such as jaguar (Panthera onca) [12], and wild rodents, such as red-rumped agouti (Dasyprocta leporina) [13], and large Japanese field mice (Apodemus speciosus) [14] with cells recovered after 4 or 11 days.
During primary culture, several cell types become present that can be isolated, such as fibroblasts, mesenchymal cells, epithelial, and adipocytes. However, fibroblasts have been the cell of choice, due to its morphology with large nucleus and lower quantities of cytoplasm, several distributions on tissues, and the facility of isolation [2]. For obtaining these cells, several methodologies have been efficiently utilized such as the addition of medium factors that improve the multiplication of such cells (for example: fibroblast growth factors [FGF]) or maintaining the major quantitative cells that overlap the cells of the lower quantitative among the passages [6].
After obtaining the cells, one essential step is the cell type identification, which determines the success of their future applications. In this identification, the first characteristic is cell morphology identification by optical microscope or scanning electron microscopy. However, molecular analysis has been shown to be more reliable for this purpose [15]. Some methods have been applied, such as the florescence in immunocytochemistry by an antibody specific for cell surface protein conjugate with fluorophore [13]. The quantitative real-time polymerase chain reaction (qRT-PCR) by gene expression has shown great results in several wild mammals [16].
To mimic a physiological environment when using cell cultures, careful control over environmental factors is typically needed, as even small deviations of environmental parameters from physiological levels may impair cellular function, and variable conditions in cell culture can substantially affect the reproducibility of experiments [3]. Certain parameters are directly involved in the success of cell culture, the main one among which is the culture medium, which has important effects on cellular functions and that maintains physiologically relevant conditions is necessary to maintain in vivo behaviors [17]. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with fetal bovine serum (FBS) to stimulate replicative growth and media exchange every several days has become a standard protocol and this compound is possibly the most used medium for mammalian fibroblast cell culture [17].
Another important aspect of cell culture is the levels of oxygen and carbon dioxide, which are known to be vital for cell growth [2]. Variations in the levels of these gasses can have significant effects on cell cultures. For many cell cultures, the commonly used atmosphere is 5% of carbon dioxide in the air, but the optimum condition will depend primarily on the buffering system of the culture medium and the desired features of the cell culture [18]. Also, it is critical to ensure that O2 levels in cell culture are both physiological and relatively constant, and these aspects are typically controlled by the incubator [3, 17].
After the establishment of primary conditions, the specific needs were observed for some species cells, requiring the addition of more FBS around 20%, glucose supplements, promoting a high level of cell duplication [19]. In fishing cat (P. viverrinus), the supplementation with glutamax and FGF promotes cell viability [6]. However, the epidermal growth factor (EGF) does not change the cell parameters of collared peccary (Pecari tajacu), but the addition of 20% of FBS increases the cell viability and metabolic activity [20]. This characteristic demonstrates the necessity to understand the species-specific medium requirements.
Furthermore, the effect of cell passage is one of the reasons to implement cell supplements, as some species are more affected by stress, causing telomeres shortening and cell senescence [21]. The cells in senescence decrease the cell application for the conservation of wild animals, where the cells cannot divide and may undergo DNA damage, resulting in less success with SCNT [14]. The time of cell culture was determined by the number of passages—a passage consisting of cell detachment from the dish. In some felid species such jaguar (Panthera onca) and puma (Puma concolor) [22], the 10th passage affected the morphology, metabolic activity, and growth curve. However, in red-rumped agouti (D. leporina), the eighth passage did not affect cells parameters. The effect of cell passage as the subject of several studies has been variable in wild mammals, as shown in Table 1.
Morphology altered after the 13th passage and changes in cell proliferation
After 7th passage, the cells become flat
No chromosome damage was notified
No difference occurred after a long cell culture
Morphological damage after 10th passage
Duplication time
25 h
68.9 h
24 h
—
17 h
30 h
Viability
~97%
~95%
~93%
—
~80%
~75%
Cell passages
7 passages
13 passages
7 passages
8 passages
8 passages
10 passages
Culture conditions
37°C 5% CO2
37°C 5% CO2
38°C 5% CO2
37°C 5% CO2
38.5°C 5% CO2
38.5°C 5% CO2
Culture medium
DMEM +10% FBS + 1× ATB/ATM
DMEM + HAM’S F12 + 15% FBSe
MEM-Alpha+10% FBS + 1% ATB/ATM
DMEM + 10% FBS
DMEM + 10% FBS + 2% ATB/ATM
DMEM + 10% FBS + 2% ATB/ATM
Species
Elephas maximus
Feresa attenuata
Vulpes corsac
Artibeus planirostris
Dasyprocta leporina
Puma concolor
Table 1.
In vitro culture conditions of fibroblastic lines on the passage. DMEM: Dulbecco modified eagle medium; MEM: Modified eagle medium; FBS: Fetal bovine serum; ATB/ATM: Antibiotic/antimycotic solution.
Regarding cell detachment, enzymatic detachment has been applied with a dissolution of the cellular matrix by an enzyme [26]. The enzymatic method is the most applied, but in longer periods cell culture can cause morphological changes in the shape of fibroblasts and even in the senescence state [27]. Thus, the knowledge of the effect of the number of passages and the right cell culture supplementation in each specific species is necessary for maintaining the applicability of these cells.
Cryopreservation consists of the use of low temperatures to inhibit metabolism and retard biochemical processes responsible for the degradation of living cells [28]. This methodology permits the formation of biobanks, increasing the possibility of developing assisted reproduction technologies (ARTs) by enabling the use of biological materials with their genetic variability preserved regardless of time and space, since cryopreserved cell lines provide a viable and expandable source of material genetic, facilitating the protection of endangered species in the future [29, 30]. Therefore, for somatic cell-based technologies, it is crucial to monitor the quality of the original cell lines and ensure the integrity of the genome, including chromosomal stability and the absence of relevant epigenetic changes during prolonged culture and differentiation, which occur randomly and unpredictably [31]. Thus, it is fundamental to define an efficient method of cryopreservation, aiming to maintain the efficiency and reliability of fibroblastic lines.
Slow freezing (Figure 2) is the most used technique for fibroblastic lines cryopreservation, in which the temperature is reduced in a gradual and controlled manner, and the cell viability and function are preserved due to slow cooling rates and cell dehydration [32, 33]. The success of any cryopreservation protocol is dictated by cryoprotectant agent type, including permeating and non-permeating agents or a combination of both, as well as appropriate cooling and thawing rates [34]. Cryoprotectant agents (CPAs) are used to prevent such damages. The ideal CPA should be able to protect cell membrane integrity, avoiding damage to membrane lipids and consequently other cellular components, including cellular proteins and nucleic acids [28]. They are classified into nonpenetrating (NPAs) and penetrating agents (PAs) that protect biological samples through different mechanisms. NPAs are small molecules organized into polymers that increase extracellular osmolality to cause cell dehydration as a stabilization mechanism [35], such as sucrose and FBS [34, 36]. On the other hand, PAs exist currently as dimethyl sulfoxide (DMSO) and ethylene glycol (EG). These agents must be highly water soluble at low temperatures, able to easily cross biological membranes, and be minimally toxic [34, 37].
Figure 2.
Steps of slow freezing for fibroblast cryopreservation. (A) Addition of cell concentration in cryovials with cryoprotectant solution. (B) Cryovials being allocated on the freezing device Mr. Frosty® to the slow freezing. (C) Transfer of cryovials to liquid nitrogen after slow freezing.
Due to DMSO’s well-proven high efficacy in cryoprotection, it remains the most used CPA in research on wild mammals. This compound is thought to increase cellular permeability by affecting membrane dynamics in a concentration-dependent manner, and at low concentrations (5%) it decreases membrane thickness and, in turn, increases membrane permeability. However, at commonly used concentrations (10%), water pore formation in biological membranes is induced and the formation of pores can be advantageous, as intracellular water can be more readily replaced by cryoprotectants that promote vitrification [28, 34]. This compound has strong molecular stability, a high dielectric constant, properties of basicity, solvation of salts (particularly anions), and a propensity to act as the acceptor atom in hydrogen bonding [38].
Cryoprotectant solutions should not only benefit from the cryoprotective effect of DMSO but also diminish its toxicity by using adequate dilutions; thus, the equilibrium is believed to be satisfactory at 10% [39, 40]. Several studies have focused on evaluating the efficiency of different combinations of NPAs and PAs in fibroblasts of different species, such as in red-rumped agouti (D. leporina) [36], which evaluated the interactions among sucrose and concentrations of FBS on the cryopreservation of fibroblasts and verified that DMSO plus 90% FBS and 0.2 M sucrose promote greater ability of these cells after thawing. Other authors have employed this concentration of DMSO plus FBS and/or sucrose to preserve fibroblasts of different wild mammals around the world for different purposes (Table 2).
Cryoprotective solutions used in the cryopreservation of fibroblasts in different wild mammals. NI: Non-informed.
Therefore, slow freezing has established itself as the technique of choice for fibroblast cryopreservation, and, when there are variations between the protocols, it occurs in terms of the combination of cryoprotectants employed.
The highest priority of animal conservation programs is to guarantee the reproduction and propagation of species that are threatened or in danger of extinction. Although promising in maintaining the genetic diversity of wild populations, this current model of animal conservation depends on some factors that have not yet been overcome, such as the broad knowledge of the reproductive biology of the species and the mechanisms that govern this process [44]. Moreover, the limited space observed in scientific breeding sites makes it difficult or restricts the application of research on this topic [45].
As a result of the previously mentioned problems, associated with the alarming situation to maintain wild biodiversity, ARTs are constantly incorporated and optimized to guarantee the best reproductive performance of these individuals [46]. Among these technologies, the establishment of genetic banks, mainly in the form of fibroblast cell lines, has been suggested [47]. This is because it is believed that these cells are the most suitable for restoring or expanding populations of individuals through cloning and using the SCNT technique [29]. For this reason, the formation of wildlife genetic resource banks is constantly investigated, since it allows not only conservation [33] but also the application of these samples in different periods [48], contributing to reproductive optimization and biotechnological purposes.
Theoretically, SCNT can be defined as a biological, embryological, and genetic study tool that allows the epigenetic reprogramming of the transcriptomic signatures of a fibroblast (karyoplast) inside a single cell capable of redefining its nuclear organization for the generation of an individual, known as an oocyte (cytoplast) [49]. This process results in obtaining a reconstructed embryo that is artificially activated through electrical pulses or chemical stimuli, inducing it after embryonic development [50]. With this, it is possible to produce replicas that are genetically identical to the nucleus donor individual [51].
In wild mammals, SCNT is used to clone endangered species for their conservation, in the rescue of extinct populations, and for the evolutionary understanding of the species [52]. However, to meet this goal, it is necessary to have the availability of female gametes, which are not always possible to access [53]. In this sense, an alternative technique known as interspecific cloning (iSCNT) is used when sufficient biological resources are not available for the study of the species of interest [45]. In some wild mammals, iSCNT has been efficiently performed, and some of these results are shown in Table 3.
Successfully performed studies using fibroblastic lines in the reproductive cloning of wild mammals. SCNT: Somatic cell nuclear transfer; iSCNT: Interspecific somatic cell nuclear transfer.
However, even though intraspecific and interspecific cloning has already efficiently allowed the generation of more than 25 species [49], including domestic and wild ones, since the first-born clone [69], this technique remains with considerably low efficiency, ranging from 0.1 to 16% [70, 71]. Among the main causes for this, aberrant reprogramming and epigenetic memories inherited by the donor cell [53, 70] are one of the main barriers that prevent the proper development of clone embryos. Because of this failure, some abnormalities are reported, and in wild species, chromosomal alterations [63], incompatibility between the mitochondrial and nuclear genome [61], and disorders related to placental dysfunction of embryos, conceptuses, and offspring generated [56, 68] are continually observed problems.
As a result, efforts are being made to improve cloning efficiency that would enable a substantial and positive change for the best performance of the technique, ensuring a greater number of applications. Therefore, considering that in recent years the biggest challenge in relation to obtaining cloned offspring is related to the donor nucleus, studies on cellular and molecular knowledge involving this cell proved to be a decisive step toward the further improvement of SCNT and iSCNT. This is because, after insertion into the oocyte cytoplasm, the donor nucleus is expected to return to its state of totipotency through undifferentiation [71]. However, when this process is performed in a disorganized manner, it generates disorders that, in addition to causing the death of the embryo, reduce the efficiency of the technique.
Thus, among the orchestrated efforts to increase the success of cloning between wild species, the phylogenetic and reproductive relationship can guarantee not only a higher rate of success but also help in the gestational period of the embryo and fetus [72]. Moreover, cell type selection, where fibroblasts are the chosen cell (Table 3), and synchronization of their cell cycle phase in G0/G1 [45] are other factors that may contribute to the efficiency of this technique. This is because, after insertion of the donor DNA, its nucleus rapidly breaks down its membrane to form condensed metaphase-like chromosomes [73], triggering the process of premature chromosomal condensation, which seems to be necessary for adequate embryonic reprogramming, thus enabling greater efficiency in the generation of cloned embryos [74]. Additionally, this process facilitates compatibility between the cell-cycle stage and the recipient oocyte, allowing accessibility of chromatin transcriptional factors [53].
However, it has been clearly demonstrated that in addition to the choice of karyoplasts and synchronization of the cell cycle phase contributing to the success of cloning in wild mammals, other barriers may be related to this technical application. Studies associated with compression and correction of mRNA expression patterns through methylation and acetylation [62], modifications of histone variants [63], X chromosome inactivation, and accessibility of the structure of chromatin have begun to be extensively investigated [47, 73].
Lastly, the knowledge of the previously mentioned characteristics can make a difference in the efficiency of nuclear reprogramming in wild mammals, since for the generation of a clone, the process is governed by the remodeling of transcriptional factors, and any scientific advance in this regard would provide not only unquestionable benefits to the success of the technique but also contribute positively to the conservation and genetic multiplication of wild biodiversity.
In the last decade, great strides have been made in the development of advanced ARTs, not only in the areas of animal conservation but also for animal reproduction. Among these new technologies that use fibroblastic cell lines, induced pluripotent stem cells (iPSC) are generated by reprogramming adult or differentiated somatic cells to a pluripotent stem cell-like state using defined transcription factors. The whole process consists of the insertion of factors that are only expressed in multi and totipotent cells in fibroblasts through different viral or molecular carriers, enabling the expression of these genes that comprise the characteristics of stem cells [75].
In 2006, the first papers on fibroblast reprogramming were published, disclosing the list of some factors considered essential because they are associated with cellular pluripotency: OCT4 (octamer-binding transcription factor 4), SOX2 (SRY-box transcription factor 2), KLF4 (kruppel-like factor 4), and MYC (MYC proto-oncogene). Since then, many important improvements have been made to this iPSC technology, where researchers have started screening for factor combinations that could direct line reprogramming for wild mammalian cells (Table 4). Thus, we can highlight the potential of NANOG (homeobox protein), which helps stem cells maintain pluripotency by suppressing cell determination factors, and LIN28 (lin-28 homolog A) that regulate the self-renewal of stem cells, both of which are extremely important functions to guarantee the reprogramming of these fibroblasts.
Induction of pluripotent stem cells (iPSC) from fibroblast cell lines of wild mammals. ND: No data. AP: Alkaline phosphatase.
Verma et al. [86] made the first attempt to produce iPSC in three different wild felids (Panthera tigris, Panthera onca, and Leptailurus serval), comparing the efficiency of NANOG as a reprogramming factor in these species. After the tests, it was possible to conclude that the insertion of NANOG doubled the efficiency of reprogramming and colony formation, in addition to having remained present in these colonies until the 14th passage. In contrast, colonies that were not transfected with NANOG did not remain beyond seven passages. The iPSCs reprogrammed with NANOG from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers, confirming the success of the cell reprogramming.
However, the success of this technique is still low and extremely variable. While the infection efficiency of the retrovirus ranged from 91.3 to 98.3%, the reprogramming efficiency ranged from 0.00052 to 0.00064% in wild felids [86]. Similar results were observed in the Tasmanian devil (Sarcophilus harrisii), where in an attempt to produce the iPSC in this mammal, the authors observed a transduction rate of 80%, but the reprogramming efficiency dropped to 0.00050 to 0.00075% [79].
Another gap that influences the results is the source of the factors that are inserted into the fibroblasts, since these can be from the same researched animal or even from humans. In the literature, we can observe authors who were successful with factors of the same kind, such as Katayama et al. [78], who used mouse OCT3/4, SOX2, KLF4, CMYC, LIN28, and NANOG to successfully generate iPSC in a wild rodent (Microtus ochrogaster) and Shimozawa et al. [83] using monkey KLF4, SOX2, POU5F1 and CMYC factors to reprogram Macaca fascicularis cell lines. In contrast, authors have also been successful in Ornithorhynchus anatinus [80], Neovison vison [77], Pongo pygmaeus [76], and Prionailurus viverrinus [6] using human factors.
Based on these results compiled over the years, it is clear that, despite the technique’s importance, improvements are still needed. The production of iPSC in wild mammals is far from being a common and low-cost method, but we cannot fail to highlight the future glimpses of success that this technology could provide in animal reproduction. In the future, it may serve as a blueprint for harnessing the potential of in-vitro-generated gametes, where, theoretically, iPSCs from male donors can be used to generate oocytes to make the best use of available genetic diversity. However, to date, viable offspring have only been generated from iPSC-derived gametes in mice (Mus musculus), but efforts are underway to extend this approach to rescue highly endangered wild mammals.
Technological advances based on cell culture in recent decades have offered an alternative for the development of methods that can preserve and disseminate the valuable genetics of wild mammals, whether threatened with extinction or not. Due to the benefit of this technique, many wild mammals mentioned in this study had their genetics conserved through the establishment of cell lines.
This is because, from the establishment of in vitro culture conditions through fibroblastic lines, multiple applications can be performed, as observed in the species C. pyrenaica pyrenaica, when these cells were used in interspecific cloning for the birth of a wild species that was previously considered defunct. Moreover, the in vitro culture of fibroblasts allows the production of iPSC, which despite its low efficiency in optimizing protocols, represents yet another application for these cells, as it allows subsequent differentiation into male or female gametes, which, although not having been reported so far, still represents an innovative application for the culture of this cell type.
For this reason, the primary culture of fibroblasts aiming at the formation of a somatic bank is already a widely used technique in the development of protocols aimed at the conservation of wild mammals. We reviewed in this chapter the development of protocols, methods, and technical optimizations used efficiently in the in vitro culture and cryopreservation of fibroblasts from different species of wild mammals. Therefore, a better understanding of the subject is generated, allowing the development of new reproductive strategies that can not only improve the application of in vitro culture of fibroblast lines in wild mammals but also guarantee the development of strategies that can help overcome the challenges associated with this technique, since any advance or further knowledge provides unquestionable benefits for animal conservation and the genetic reintroduction of endangered species.
This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES, Financial Code 001) and National Council for Scientific Development (CNPq, Financial Code 309078/2021-0).
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Written By
Alexsandra Fernandes Pereira, Lhara Ricarliany Medeiros de Oliveira, Leonardo Vitorino Costa de Aquino, João Vitor da Silva Viana and Luanna Lorenna Vieira Rodrigues
Submitted: 27 April 2023Reviewed: 30 November 2023Published: 21 December 2023
Open access peer-reviewed chapter - ONLINE FIRST
