Immunolocalization of angiotensin-(1-7), angiotensin II and angiotensin- converting enzyme 2 in goat ovary / Imunolocalização de angiotensina-(1-7), angiotensina II e enzima-conversora de angiotensina 2 em ovário de cabras

There is increasing evidence as to the participation of the ovarian renin-angiotensin system in important reproductive processes. The inhibition of the angiotensin-converting enzyme (ACE) caused an increase in the rate of ovulation and pregnancy in the artificial insemination protocol has fixed time (TFIA). This study aimed to investigate the presence and location of Ang II, Ang- (1-7) and ACE2 in goat ovaries and the possibility of the involvement of these peptides in previous results. Ten ovaries from goats were collected in a slaughterhouse, washed in buffered PBS, perfused with protease inhibitor solution and processed for immunohistochemistry protocol. The search for peptides was performed using the avidin–biotin–peroxidase method. A strong immunoreactivity for Ang II in theca cells of antral follicles and corpus luteum was observed. Antral follicles (theca cells), corpus luteum and oocyte cytoplasm in early antral follicles exhibited strong immunoreactivity for Ang-(1-7). There was strong immunoreactivity for ACE2 in the cytoplasm of luteal cells and theca cells of antral follicles. In this study, for the first time, the presence and location of Ang II, Ang-(1-7) and ACE2 are reported in goat ovary, suggesting that there is participation in follicular development, oocyte maturation and corpus luteum development.

Há evidências crescentes quanto à participação do sistema renina-angiotensina ovariano em processos reprodutivos importantes. A inibição da enzima conversora de angiotensina (ECA) ocasionou aumento na taxa de ovulação e gravidez no protocolo de inseminação artificial por tempo fixo (TFIA). Este estudo teve como objetivo investigar a presença e localização de Ang II, Ang-(1-7) e ECA2 em ovários de cabras e a possibilidade do envolvimento desses peptídeos em resultados anterio-res. Dez ovários de cabras foram coletados em abatedouro, lavados em PBS tamponado, perfundidos com solução inibidora de protease e processados para protocolo de imunohistoquímica. A busca por peptídeos foi realizada usando o método avidina-bio-tina-peroxidase. Foi observada uma forte imunorreatividade para Ang II em células da teca de folículos antrais e corpo lúteo. Os folículos antrais (células da teca), corpo lúteo e citoplasma do oócito nos folículos antrais iniciais exibiram forte imunor-reatividade para Ang-(1-7). Houve forte imunorreatividade para ECA2 no citoplasma das células luteais e células da teca dos folículos antrais. Neste estudo, pela primeira vez, a presença e localização de Ang II, Ang- (1-7) e ECA2 são relatadas em ovário caprino, sugerindo que há participação no desenvolvimento folicular, maturação oocitária e desenvolvimento do corpo lúteo.

Immunolocalization of angiotensin-(1-7), angiotensin II and angiotensin- converting enzyme 2 in goat ovary / Imunolocalização de angiotensina-(1-7), angiotensina II e enzima-conversora de angiotensina 2 em ovário de cabras

There is increasing evidence as to the participation of the ovarian renin-angiotensin system in important reproductive processes. The inhibition of the angiotensin-converting enzyme (ACE) caused an increase in the rate of ovulation and pregnancy in the artificial insemination protocol has fixed time (TFIA). This study aimed to investigate the presence and location of Ang II, Ang- (1-7) and ACE2 in goat ovaries and the possibility of the involvement of these peptides in previous results. Ten ovaries from goats were collected in a slaughterhouse, washed in buffered PBS, perfused with protease inhibitor solution and processed for immunohistochemistry protocol. The search for peptides was performed using the avidin–biotin–peroxidase method. A strong immunoreactivity for Ang II in theca cells of antral follicles and corpus luteum was observed. Antral follicles (theca cells), corpus luteum and oocyte cytoplasm in early antral follicles exhibited strong immunoreactivity for Ang-(1-7). There was strong immunoreactivity for ACE2 in the cytoplasm of luteal cells and theca cells of antral follicles. In this study, for the first time, the presence and location of Ang II, Ang-(1-7) and ACE2 are reported in goat ovary, suggesting that there is participation in follicular development, oocyte maturation and corpus luteum development.(AU)

Há evidências crescentes quanto à participação do sistema renina-angiotensina ovariano em processos reprodutivos importantes. A inibição da enzima conversora de angiotensina (ECA) ocasionou aumento na taxa de ovulação e gravidez no protocolo de inseminação artificial por tempo fixo (TFIA). Este estudo teve como objetivo investigar a presença e localização de Ang II, Ang-(1-7) e ECA2 em ovários de cabras e a possibilidade do envolvimento desses peptídeos em resultados anterio-res. Dez ovários de cabras foram coletados em abatedouro, lavados em PBS tamponado, perfundidos com solução inibidora de protease e processados para protocolo de imunohistoquímica. A busca por peptídeos foi realizada usando o método avidina-bio-tina-peroxidase. Foi observada uma forte imunorreatividade para Ang II em células da teca de folículos antrais e corpo lúteo. Os folículos antrais (células da teca), corpo lúteo e citoplasma do oócito nos folículos antrais iniciais exibiram forte imunor-reatividade para Ang-(1-7). Houve forte imunorreatividade para ECA2 no citoplasma das células luteais e células da teca dos folículos antrais. Neste estudo, pela primeira vez, a presença e localização de Ang II, Ang- (1-7) e ECA2 são relatadas em ovário caprino, sugerindo que há participação no desenvolvimento folicular, maturação oocitária e desenvolvimento do corpo lúteo.(AU)

Tissue thickness may influence the outcome of vitrification of goat ovarian cortex

Background: The main advantage of the cryopreservation of ovarian fragments is a thinner tissue, which facilitates the penetration of cryoprotective agents, but the size of tissue may not be a limiting factor in achieving a successful cryopreservation of the ovarian tissue. This information is highly significant considering that the cryopreservation of hemi-ovary or whole ovary may preserve the entire or major part of the contingent of primordial follicles of ovarian fragments. Therefore, the aim of this study was to evaluate the vitrification of different dimensions goat ovarian tissue on the follicular morphology, viability, diameter, and the stromal cell density. Materials, Methods & Results: The ovarian tissue was vitrified as fragment, hemi-ovary, or whole ovary, and after warming, the preantral follicles were examined by trypan blue dye exclusion test and histological analysis. Preantral follicles incubated with trypan blue were considered viable if the oocyte and granulosa cells remained unstained. Preantral follicles were classified as morphologically normal only when they contained intact oocyte and granulosa cells. The follicular diameter was measured considering the major and minor axes of each follicle; the average of these 2 measurements was used to determine the diameter of each follicle. Ovarian stroma cells density was evaluated by calculating the number of stromal cell in an area of 100 × 100 µm. There was no difference in the percentage of morphologically normal and viable follicles after vitrification compared to the control (fresh tissue), regardless of the dimension of the vitrified ovarian tissue (P > 0.05). In addition, there were no differences in the follicular diameter after ovarian tissue vitrification, independent of the dimension (P > 0.05). However, after vitrifi cation, a decrease in the ovarian stromal cells density was observed (P < 0.05). This reduction was more intense after the vitrification of the hemi-ovary and whole ovary, compared to the ovarian fragment vitrification (P < 0.05). Discussion: No differences were observed in the percentages of morphologically normal and viable follicles from fresh or vitrified ovarian tissue (fragment, hemi-ovary, and whole ovary). These results are in agreement with other reports which no showed morphological changes after cryopreservation of the whole ovary, and the ovarian fragments. With respect to follicular diameter, only the diameter of the preantral follicles in ovarian tissue vitrified as hemi-ovary was similar to that observed in the fresh control, in the present study. The results demonstrate that fragments and whole ovary vitrification had greater cell dehydration (exposure to VS) and/or less cell rehydration (VS removal), showing that minor adjustments are needed in the protocols of cryoprotectants addition or removal from the fragments and the whole ovary. However, this reduction in follicular diameter did not appear to have affected the follicular architecture or cellular viability, which were maintained in all dimensions of ovarian tissue undergoing vitrifi cation. A reduction in the stromal cell density was observed, especially in the hemi-ovary and whole ovary as compared to the ovarian fragment. Previous reports have shown that ovarian stromal cells are responsible for the production of essential substances for follicular development and these substances are fundamental for follicles development and these cells tend to be more sensitive to cryopreservation procedure than ovarian follicles. In conclusion, the maintenance of follicular morphology and viability demonstrated that vitrification of goat ovarian tissue under the conditions applied in this study can be performed in any dimension of ovarian tissue (fragment, hemi-ovary, and whole ovary).

Caracterização dos danos celulares em gametas femininos e embriões após criopreservação / Characterization of cellular damage in female gametes and embryos after cryopreservation

Background: Cryopreservation is a biotech successfully employed in female gametes and embryos. This technique is of great importance for propagation of genetic material from animals with high-value livestock as well as to preserve the fertility of women undergoing cancer treatments. However, low temperatures can result in damage to different cellular compartments and organelles. This damage culminates in reduced viability, since they affect cell metabolism. Review: Cryopreservation consists of maintenance of biological material at low temperatures, in which chemical reactions are ceased, however, allowing the cells to preserve their viability. However, the decrease of temperature and subsequent warming may result in cellular damage. These damages occur in the cell membrane, cytoplasmic organelles and the cell nucleus. It is believed that the first cell structure undergoing cryoinjury is the plasma membrane, responsible for maintaining homeostasis within the cell, and the loss of plasma membrane during the reduction temperature reported the main damage. The membrane damage due to cryopreservation appears to correlate with the reduction of thermal energy at low temperatures, thus limiting the movement of molecules through the phospholipids of lipid bilayer. Cryopreservation also alters the morphology, structure and cellular distribution of lipid droplets, reducing the survival of oocytes and embryos. The physical state changes, besides changing physicochemical properties of intracellular lipid may also result in damage to lipids associated with the cellular cytoskeleton. The interaction between cell lipid phase and components of cytoskeleton is complex and hardening of these lipids can cause deformation and disruption of cytoskeleton with consequent negative effect on cell survival and development. The disruption of cytoskeleton can also be intrinsic to change in dehydration and cellular form that follow the cryopreservation process. Among the cellular organelles, mitochondria are sensitive to cryopreservation procedures, since the formation of intracellular ice crystals considered one of the most relevant cryoinjury, leading to damage to the mitochondrial cristae and matrix. Another important organelle undergoes damage as result of cryopreservation is the endoplasmic reticulum, which change in morphology has been described after vitrification of embryos. It is also known that cryopreservation may result in fragmentation of DNA even in the absence of deformation of the cellular morphology, and that these changes can lead to delay in the development or cell death. In addition to the direct damage to double-stranded DNA, cryopreservation may trigger chromosomal abnormalities, these the aneuploidy is the most frequent and seems to compromise the developmental competence of oocytes in addition to being indicated as the main factor for the low achieve of live births. Conclusion: Cryopreservation is an important alternative for the preservation of gametes and embryos, however, the cells are subjected to unfavorable conditions that can compromise cell recovery after thawing/warming. The main cause of reduction in survival oocytes and embryos after cryopreservation appear to be related to damage in different cellular compartments and structures, which are essential for maintaining cell metabolism. Thus, it is observed the need to achieve cryopreservation protocols capable of maintaining the integrity of different cellular components.

Criopreservação de tecido ovariano: limitações e perspectivas para a preservação da fertilidade de fêmeas / Ovarian tissue cryopreservation: limitations and perspectives for female pertility preservation

Background: The ovarian tissue cryopreservation has been achieved a great notoriety in the Reproductive Biology area, due to its potential in preserving female fertility through the protection of exocrine and endocrine functions of the ovary. The association of this technique with in vitro culture and/or transplant in adult or young individuals who has not initiated its reproductive activities represents not only the conservation and perpetuation of the genetic material of economic valuable animals, but also the preservation of female gametes from endangered species, or even from young women who may have ovarian dysfunctions caused by gonadotoxic treatments. Studies with some species (human, mice and ovine) have demonstrated the recovery of the ovarian function and the birth of healthy offspring after transplant of ovarian tissue which has been previously cryopreserved. However, most studies have shown that ovarian cryopreservation process offer risks to different structures (follicle and stroma ) as well as to the different cell types (oocyte, granulosa, thecal and stromal cells), which constitute this tissue. Review: Extreme cold, intracellular ice crystallization, osmotic shock and the toxicity of the cryoprotectant agents are factors that are usually associated with the injuries caused by the cryopreservation process. As a direct or indirect consequence, those factors limit the success of the cryopreservation of ovarian tissue, since they affect the survival or alter the tissue functionality or cellular structure, like the ovarian follicles, for example, after the thawing/warming procedure. Among the injuries that may take place as a consequence of those factors, we can mention: cell death by the necrotic or apoptotic pathways; alterations in normal levels of genic expression; ischemia and changes of communication and interaction between the oocyte and follicular cells. As a result, many authors have studied and developed protocols of cryopreservation that may prevent or minimize the cryoinjuries, since the cryopreservation per si or combined to other techniques (in vitro culture and/or transplant) can compromise the ovarian integrity, leading consequently to a significant loss of follicles. In this regard, the present review seeks to app roach the advantages of the cryopreservation of ovarian tissue; indicating the difficulties and challenges that encompass this procedure, with purpose of pointing out solutions to overcome the damages of ovarian tissue cryopreservation, through of convenient cryopreservation protocols that avoid those follicular losses. For this purpose, it is necessary the preservation of the follicular viability as well as the preservation of the tissue integrity and the contact between reproductive (oocytes) or somatic cells, which are essentials to the follicle development, and, consequently, to the embryo production. Conclusion: The use of cryopreserved ovarian tissue is an important strategy to the preservation of female fertility. This tool has being pointed as an alternative way to the cryopreservation of mature oocytes and embryos. However, additional studies are necessary to diminishing the cellular damages inherent to this procedure, especially those related to the comprehension of the obstacles and mechanisms associated to the exposition to extreme cold.

Vitrificação: uma alternativa para a preservação de embriões e material genético de fêmeas mamíferas em criobancos / Vitrification: an alternative for preserving embryos and genetic material mammalian females in cryobanking

A vitrificação é um método de criopreservação barato, rápido e fácil de ser realizado e tem sido usado com relativo sucesso para a preservação de embriões e oócitos obtidos a partir de folículos antrais e pré-antrais. A presente revisão descreve os diferentes métodos de vitrificação, os principais resultados obtidos, bem como sua importância para a tecnologia de reprodução assistida em humanos, animais de genética superior e espécies mamíferas ameaçadas de extinção.

Vitrification is a cryopreservation method's cheap, fast and easy to perform and has been used, with relatively success, for the preservation of embryos and oocytes from preantral and antral follicles. The present review describes the different methods of vitrification, the main results obtained so far as well as its important for the assisted reproduction technologies in human, genetic superior animal and endangered mammalian species.

Criopreservação de tecido ovariano: limitações e perspectivas para a preservação da fertilidade de fêmeas / Ovarian tissue cryopreservation: limitations and perspectives for female pertility preservation

Background: The ovarian tissue cryopreservation has been achieved a great notoriety in the Reproductive Biology area, due to its potential in preserving female fertility through the protection of exocrine and endocrine functions of the ovary. The association of this technique with in vitro culture and/or transplant in adult or young individuals who has not initiated its reproductive activities represents not only the conservation and perpetuation of the genetic material of economic valuable animals, but also the preservation of female gametes from endangered species, or even from young women who may have ovarian dysfunctions caused by gonadotoxic treatments. Studies with some species (human, mice and ovine) have demonstrated the recovery of the ovarian function and the birth of healthy offspring after transplant of ovarian tissue which has been previously cryopreserved. However, most studies have shown that ovarian cryopreservation process offer risks to different structures (follicle and stroma ) as well as to the different cell types (oocyte, granulosa, thecal and stromal cells), which constitute this tissue. Review: Extreme cold, intracellular ice crystallization, osmotic shock and the toxicity of the cryoprotectant agents are factors that are usually associated with the injuries caused by the cryopreservation process. As a direct or indirect consequence, those factors limit the success of the cryopreservation of ovarian tissue, since they affect the survival or alter the tissue functionality or cellular structure, like the ovarian follicles, for example, after the thawing/warming procedure. Among the injuries that may take place as a consequence of those factors, we can mention: cell death by the necrotic or apoptotic pathways; alterations in normal levels of genic expression; ischemia and changes of communication and interaction between the oocyte and follicular cells. As a result, many authors have studied and developed protocols of cryopreservation that may prevent or minimize the cryoinjuries, since the cryopreservation per si or combined to other techniques (in vitro culture and/or transplant) can compromise the ovarian integrity, leading consequently to a significant loss of follicles. In this regard, the present review seeks to app roach the advantages of the cryopreservation of ovarian tissue; indicating the difficulties and challenges that encompass this procedure, with purpose of pointing out solutions to overcome the damages of ovarian tissue cryopreservation, through of convenient cryopreservation protocols that avoid those follicular losses. For this purpose, it is necessary the preservation of the follicular viability as well as the preservation of the tissue integrity and the contact between reproductive (oocytes) or somatic cells, which are essentials to the follicle development, and, consequently, to the embryo production. Conclusion: The use of cryopreserved ovarian tissue is an important strategy to the preservation of female fertility. This tool has being pointed as an alternative way to the cryopreservation of mature oocytes and embryos. However, additional studies are necessary to diminishing the cellular damages inherent to this procedure, especially those related to the comprehension of the obstacles and mechanisms associated to the exposition to extreme cold.(AU)

Ativina intraovariana: uma atualização sobre estrutura, regulação e função / Intraovarian activin: an update on structure, regulation and function

Background: Important advances have been made recently that clarify our understanding of the structural basis, signaling and regulation, as well as the biological role of activin in ovaries. During folliculogenesis various growth factors are produced locally in the mammalian ovary. Among these factors, activin has been a focal point in research as it has emerged as a crucial substance capable of inducing follicular development. The important actions indicate that activin has many relevant homeostatic functions in the reproduction of several species. Therefore, this review discusses the ligand protein structure, activin receptors, mechanisms of action and regulation, as well as the importance of activin on in vitro culture of preantral follicles. Review: Activin belongs to the transforming growth factor β (TGF - β) super family. It is a homodimer or heterodimer of two similar but distinct subunits (βA and βB). The dimerisation of activin subunits gives rise to three forms of activin, which are classified as, activin A (βA - βA), activin B (βB - βB) and activin AB (βA - βB). The biological activity of activin occurs through its connection with two types of cell surface receptors designated type I and type II. These receptors are represented by two isoforms, activin receptor types IA (ActR - IA), IB (ActR - IB), IIA (ActR - IIA) and IIB (ActR - IIB). Activin receptors are transmembrane proteins, composed of a ligand-binding extracellular domain, a transmembrane domain and a cytoplasmic domain with serine/threonine kinase activity. The transient activation of the receptor induces phosphorylation of protein mediators called Smads. Activation of Smad 2/3 by phosphorylation causes trimerization and hetero-oligomerization with the common Smad, Smad 4. This complex translocates to the nucleus to activating and regulating transcription of target genes. Members of another class of Smads act as negative regulators of the signal transduction pathway. Inhibitory Smad 7 can bind to type I receptors, preventing receptor–Smad 2/3 association, or by competitively binding of Smad 4, which blocks Smad intracellular translocation. In addition, within the extracellular environment, binding proteins such as follistatin and inhibin can modulate the biological activity of activin. In the ovaries of mammals specifically, activin participates in several cellular events, including cellular proliferation, differentiation, and survival, as well as assisting steroidal hormones during follicular development. Activin has been localized in the oocytes and granulosa cells of rodent, porcine, caprine and bovine follicles. Activin is also within the granulosa cells of human follicles and in the thecal layers of porcine and human. In addition, activin stimulates follicle growth in-vitro, is used in pre-antral ovine and caprine follicles and enhances growth and survival of human pre-antral follicles in vitro. Conclusion: Activin is controlled by competitive substances and a dynamic interaction between the various regulatory proteins responsible for coordinating several signaling pathways. The balance between the actions of these proteins is critical for regulation of gene expression in different structures, including pre-antral follicles. However, the nature of physiological effects of activin in the ovary is still equivocal and awaits clarification.(AU)

Agentes crioprotetores intracelulares: características e utilização na criopreservação de tecido ovariano e oócitos / Intracellular Cryoprotectant Agents: Characteristics and Use of Ovarian Tissue and Oocyte Cryopreservation

Background: The application of cryopreservation in human and animal reproductive medicine has stimulated several studies about the effects of low temperatures and freezing processes on cells and tissues, in order to develop efficient protocols for gamete and embryo preservation. Moreover, cryopreservation is a fundamental tool for the establishment of animal germplasm banks, allowing the preservation of genetic material from several species and breeds or for further study and/or recovery of desirable characteristics. For the success of cryopreservation, the addition of an intracellular cryoprotectant agent in the freezing solution is indispensable. However, issues related to intracellular cryoprotectant agents used, e.g., their metabolism and potential toxicity, must be examined carefully so we can choose the cryoprotectant most suitable for a specific structure. Review: In this regard, this review introduces several aspects of cryopreservation, such as basic principles and methods used (slow freezing and vitrification), describing the fundamental steps of cryoprotectant agent’s exposure, cooling, storage, thawing or warming and removal of the cryoprotectant agent. The addition of an intracellular cryoprotectant to the freezing solution is essential, but does not guarantee the success of the cryopreservation protocol, due to its toxic effect, which requires a perfect balance between cryoprotectant concentration, temperature and exposure time to the structure which will be cryopreserved. Some studies attribute the toxicity of these agents mainly to the secondary metabolites formed when the cell resumes its activi ty and gradually begins to metabolize the cryoprotectant agent.[...]

Ativina intraovariana: uma atualização sobre estrutura, regulação e função / Intraovarian activin: an update on structure, regulation and function

Background: Important advances have been made recently that clarify our understanding of the structural basis, signaling and regulation, as well as the biological role of activin in ovaries. During folliculogenesis various growth factors are produced locally in the mammalian ovary. Among these factors, activin has been a focal point in research as it has emerged as a crucial substance capable of inducing follicular development. The important actions indicate that activin has many relevant homeostatic functions in the reproduction of several species. Therefore, this review discusses the ligand protein structure, activin receptors, mechanisms of action and regulation, as well as the importance of activin on in vitro culture of preantral follicles. Review: Activin belongs to the transforming growth factor β (TGF - β) super family. It is a homodimer or heterodimer of two similar but distinct subunits (βA and βB). The dimerisation of activin subunits gives rise to three forms of activin, which are classified as, activin A (βA - βA), activin B (βB - βB) and activin AB (βA - βB). The biological activity of activin occurs through its connection with two types of cell surface receptors designated type I and type II. These receptors are represented by two isoforms, activin receptor types IA (ActR - IA), IB (ActR - IB), IIA (ActR - IIA) and IIB (ActR - IIB). Activin receptors are transmembrane proteins, composed of a ligand-binding extracellular domain, a transmembrane domain and a cytoplasmic domain with serine/threonine kinase activity. The transient activation of the receptor induces phosphorylation of protein mediators called Smads. Activation of Smad 2/3 by phosphorylation causes trimerization and hetero-oligomerization with the common Smad, Smad 4. This complex translocates to the nucleus to activating and regulating transcription of target genes. Members of another class of Smads act as negative regulators of the signal transduction pathway. Inhibitory Smad 7 can bind to type I receptors, preventing receptor–Smad 2/3 association, or by competitively binding of Smad 4, which blocks Smad intracellular translocation. In addition, within the extracellular environment, binding proteins such as follistatin and inhibin can modulate the biological activity of activin. In the ovaries of mammals specifically, activin participates in several cellular events, including cellular proliferation, differentiation, and survival, as well as assisting steroidal hormones during follicular development. Activin has been localized in the oocytes and granulosa cells of rodent, porcine, caprine and bovine follicles. Activin is also within the granulosa cells of human follicles and in the thecal layers of porcine and human. In addition, activin stimulates follicle growth in-vitro, is used in pre-antral ovine and caprine follicles and enhances growth and survival of human pre-antral follicles in vitro. Conclusion: Activin is controlled by competitive substances and a dynamic interaction between the various regulatory proteins responsible for coordinating several signaling pathways. The balance between the actions of these proteins is critical for regulation of gene expression in different structures, including pre-antral follicles. However, the nature of physiological effects of activin in the ovary is still equivocal and awaits clarification.

Agentes crioprotetores intracelulares: características e utilização na criopreservação de tecido ovariano e oócitos / Intracellular Cryoprotectant Agents: Characteristics and Use of Ovarian Tissue and Oocyte Cryopreservation

Background: The application of cryopreservation in human and animal reproductive medicine has stimulated several studies about the effects of low temperatures and freezing processes on cells and tissues, in order to develop efficient protocols for gamete and embryo preservation. Moreover, cryopreservation is a fundamental tool for the establishment of animal germplasm banks, allowing the preservation of genetic material from several species and breeds or for further study and/or recovery of desirable characteristics. For the success of cryopreservation, the addition of an intracellular cryoprotectant agent in the freezing solution is indispensable. However, issues related to intracellular cryoprotectant agents used, e.g., their metabolism and potential toxicity, must be examined carefully so we can choose the cryoprotectant most suitable for a specific structure. Review: In this regard, this review introduces several aspects of cryopreservation, such as basic principles and methods used (slow freezing and vitrification), describing the fundamental steps of cryoprotectant agents exposure, cooling, storage, thawing or warming and removal of the cryoprotectant agent. The addition of an intracellular cryoprotectant to the freezing solution is essential, but does not guarantee the success of the cryopreservation protocol, due to its toxic effect, which requires a perfect balance between cryoprotectant concentration, temperature and exposure time to the structure which will be cryopreserved. Some studies attribute the toxicity of these agents mainly to the secondary metabolites formed when the cell resumes its activi ty and gradually begins to metabolize the cryoprotectant agent.[...](AU)