Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers

Abstract The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.

Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers

The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.(AU)

Biobanking and use of gonadal tissues - a promising strategy for conserving wildlife from the Caatinga biome

Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.(AU)

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina) / Morfometria, estimativa e ultraestrutura da população folicular ovariana preantral de cutia (Dasyprocta leporina)

The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40µm, 12.28±2.37µm and 6.10±0.93µm for primordial, 23.75±5.70µm, 14.22±3.00µm and 6.70±1.24µm for primary and 88.55±17.61µm, 52.85±17.56µm and 22.33±17.61µm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.(AU)

O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40µm, 12,28±2.37µm e 6,10±0,93µm para primordial, 23,75±5,70µm, 14,22±3,00µm e 6,70±1,24µm para primário e 88,55±17,61µm, 52,85±17,56µm e 22,33±17,61µm de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.(AU)

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina) / Morfometria, estimativa e ultraestrutura da população folicular ovariana preantral de cutia (Dasyprocta leporina)

The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40µm, 12.28±2.37µm and 6.10±0.93µm for primordial, 23.75±5.70µm, 14.22±3.00µm and 6.70±1.24µm for primary and 88.55±17.61µm, 52.85±17.56µm and 22.33±17.61µm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.(AU)

O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40µm, 12,28±2.37µm e 6,10±0,93µm para primordial, 23,75±5,70µm, 14,22±3,00µm e 6,70±1,24µm para primário e 88,55±17,61µm, 52,85±17,56µm e 22,33±17,61µm de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.(AU)

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina)

ABSTRACT: The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40m, 12.28±2.37m and 6.10±0.93m for primordial, 23.75±5.70m, 14.22±3.00m and 6.70±1.24m for primary and 88.55±17.61m, 52.85±17.56m and 22.33±17.61m for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.

RESUMO: O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40m, 12,28±2.37m e 6,10±0,93m para primordial, 23,75±5,70m, 14,22±3,00m e 6,70±1,24m para primário e 88,55±17,61m, 52,85±17,56m e 22,33±17,61m de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.

Vitrification of ovarian tissue from non-human primates

Background: One of the strategies to preserve genetic material from nonhuman primates (NHP) consists in the implementation of germplasm banks, for future application in reproductive biotechniques, as well as for biomedical research. Based on the success rates achieved in human, there is a prominent possibility to succeed also with NHP. However, studies with NHP are still scarce, especially regarding the cryopreservation of ovarian tissue.Review: Neotropical non-human primates, especially males, have been used in research related to reproductive biotechniques in Brazil. Regarding research on female reproduction and ovarian tissue preservation, most studies were performed using domestic animals as models. Current concepts and controversies in the restoration of gametes in adult females does not exclude the needs to preserve ovarian tissue. Importantly, ovarian tissue can be collected and preserved even after the death of the donors, being applied when finding dead females. Furthermore, collection of ovarian biopsies is also feasible and will not affect reproductive function. Among the cryopreservation methods, the vitrification has been indicated due to practical logistic, as well as because it will avoid the formation of large intracellular ice crystals, and it is claimed that ovarian stromal damage will be decreased under vitrification. Considering the number of threatened primate species and the needs to preserve their habitat, but also their gametes, development of preservation protocols are needed. Among the procedures, vitrification appears as a practical method to be applied in the near future. Although a low number of studies is reported, most of them were performed in the recent years. In this context, this article reviews recent information on the vitrification of ovarian tissue of non-human primates. Due to the limited number of studies in these species, observed data are compared with the literature in domestic and human mammals.[...]

Vitrification of Ovarian Tissue from Non-Human Primates

Background: One of the strategies to preserve genetic material from nonhuman primates (NHP) consists in the implementation of germplasm banks, for future application in reproductive biotechniques, as well as for biomedical research. Based on the success rates achieved in human, there is a prominent possibility to succeed also with NHP. However, studies with NHP are still scarce, especially regarding the cryopreservation of ovarian tissue.Review: Neotropical non-human primates, especially males, have been used in research related to reproductive biotechniques in Brazil. Regarding research on female reproduction and ovarian tissue preservation, most studies were performed using domestic animals as models. Current concepts and controversies in the restoration of gametes in adult females does not exclude the needs to preserve ovarian tissue. Importantly, ovarian tissue can be collected and preserved even after the death of the donors, being applied when finding dead females. Furthermore, collection of ovarian biopsies is also feasible and will not affect reproductive function. Among the cryopreservation methods, the vitrification has been indicated due to practical logistic, as well as because it will avoid the formation of large intracellular ice crystals, and it is claimed that ovarian stromal damage will be decreased under vitrification. Considering the number of threatened prim

Vitrification of ovarian tissue from non-human primates

Background: One of the strategies to preserve genetic material from nonhuman primates (NHP) consists in the implementation of germplasm banks, for future application in reproductive biotechniques, as well as for biomedical research. Based on the success rates achieved in human, there is a prominent possibility to succeed also with NHP. However, studies with NHP are still scarce, especially regarding the cryopreservation of ovarian tissue.Review: Neotropical non-human primates, especially males, have been used in research related to reproductive biotechniques in Brazil. Regarding research on female reproduction and ovarian tissue preservation, most studies were performed using domestic animals as models. Current concepts and controversies in the restoration of gametes in adult females does not exclude the needs to preserve ovarian tissue. Importantly, ovarian tissue can be collected and preserved even after the death of the donors, being applied when finding dead females. Furthermore, collection of ovarian biopsies is also feasible and will not affect reproductive function. Among the cryopreservation methods, the vitrification has been indicated due to practical logistic, as well as because it will avoid the formation of large intracellular ice crystals, and it is claimed that ovarian stromal damage will be decreased under vitrification. Considering the number of threatened primate species and the needs to preserve their habitat, but also their gametes, development of preservation protocols are needed. Among the procedures, vitrification appears as a practical method to be applied in the near future. Although a low number of studies is reported, most of them were performed in the recent years. In this context, this article reviews recent information on the vitrification of ovarian tissue of non-human primates. Due to the limited number of studies in these species, observed data are compared with the literature in domestic and human mammals.[...](AU)

VITRIFICAÇÃO DE TECIDO OVARIANO DE ZEBRAFISH (Danio rerio) ENCAPSULADO EM HIDROGEL DE ALGINATO DE SÓDIO

O zebrafish (Danio rerio) é um importante modelo animal e tem se desta ado na esquisa iom di a or sua omo ogia fisiológica e genética aos humanos. Com isso, diversas linhagens e animais de grande valor genético têm sido desenvolvidos, possuindo assim, grande necessidade de sua preservação. o entanto a rio reser aç o de oócitos de peixe ainda é limitada e necessita aprimoramento. O hidrogel de alginato de sódio além de fornecer suporte para as células, tem demonstrado ser um potencial crioprotetor. Portanto, o objetivo deste estudo foi avaliar a eficiência da técnica de encapsulamento em hidrogel de alginato de sódio durante o procedimento de vitrificação do tecido ovariano de zebrafish. No Experimento 1, foram avaliados a forma de encapsulamento (imersão ou em 30 L de alginato), o momento de exposição ao crioprotetor (antes ou após o encapsulamento) e a temperatura de aquecimento (28, 37 e 50ºC) e foi avaliada a integridade da membrana pela coloração azul de tripan. Os dados mostraram que o tecido ovariano encapsulado por imersão, exposto ao crioprotetor após o encapsulamento e aquecido a 28ºC, apresentou maior integridade de membrana em todas as fases de desenvolvimento dos oócitos (PG: 37.71 ± 3.86 %; CA: 29.93 ± 4.18 %; Vtg1: 18.61 ± 4.69 %) e foi utilizado no Experimento 2. No Experimento 2 foram avaliados quatro grupos vitrificados (VS: Metanol 1,5 M + Me2SO 5,5 M + sacarose 0,5 M; VS1-A: Metanol 1,5 M + Me2SO 5,5 M + sacarose 0,5 M - encapsulado em alginato; VS2-A: Metanol 0,75 M + Me2SO 2,75 M + sacarose 0,25 M - encapsulado em alginato; VA: encapsulado em alginato) e foram avaliados a integridade da membrana (SYBR-14/PI), morfologia (histologia HE), atividade mitocondrial (MTT) e peroxidação lipídica (TBARS). O tratamento VA demonstrou menor percentagem de integridade de membrana, enquanto VS demonstrou maior integridade de membrana no qual não diferenciou do tratamento VS1-A. A atividade mitocondrial foi maior no tratamento não encapsulado (VS) e os tratamentos encapsulados tiveram menor valor. O tratamento VA obteve o maior nível de peroxidação lipídica, enquanto VS1-A e VS obtiveram os menores valores no qual VS não se diferenciou do tratamento VS2-A. Os resultados obtidos neste trabalho demonstram que a técnica de encapsulamento em hidrogel de alginato de sódio não teve ação crioprotetora e não permitiu a redução da concentração de crioprotetores. No entanto, a partir de observações durante os experimentos, foi encapsulamento dos fragmentos de tecido ovariano em hidrogel de alginato, possibilitou suporte e evitou perda de células durante o processo de vitrificação.

Zebrafish (Danio rerio) is an important animal model and has stood out in biomedical research for its physiological and genetic homology to humans. Thereby, several lines and animals of great genetic value have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the efficiency of the sodium alginate hydrogel encapsulation technique during the zebrafish ovarian tissue vitrification procedure. In Experiment 1, were evaluated the encapsulation form (immersion or 30 L alginate bead), the moment of exposure to vitrification solution (before or after encapsulation) and the warming temperature (28, 37 and 50 ºC) and was evaluated the membrane integrity by trypan blue stain. Data showed that ovarian tissue encapsuled by immersion, exposed to vitrification solution after encapsulation and warmed at 28ºC, had higher membrane integrity of all oocyte developmental stage (PG: 37.71 ± 3.86 %; CA: 29.93 ± 4.18 %; Vtg1: 18.61 ± 4.69 %) and was used in Experiment 2. In Experiment 2 were evaluated four vitrified groups (VS: 1.5M Methanol + 5.5M Me2SO + 0.5M sucrose; VS1-A: 1.5M Methanol + 5.5M Me2SO + 0.5M sucrose encapsulated in alginate; VS2-A: 0.75M Methanol + 2.75M Me2SO + 0.25M sucrose encapsulated in alginate; VA: encapsulated in alginate) and were evaluated the membrane integrity (SYBR-14/PI), morphology (histology HE), mitochondrial activity (MTT), and lipid peroxidation (TBARS). The VA treatment showed a lower percentage of membrane integrity, while VS demonstrated a higher membrane integrity in which it did not differ from the VS1-A treatment. Mitochondrial activity was greater in the non-encapsulated treatment (VS) and the encapsulated treatments had less value. The VA treatment obtained the highest level of lipid peroxidation, while VS1-A and VS obtained the lowest values in which VS was not different from the VS2-A treatment. The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action and did not allow the reduction of the CPA concentration. However, the encapsulation of the fragments of ovarian tissue in alginate hydrogel, enabled support and prevented loss of cells during the vitrification process.

UTILIZAÇÃO DO RESVERATROL ASSOCIADO A NANOPARTÍCULAS DE SÍLICA PARA A VITRIFICAÇÃO DE TECIDO OVARIANO BOVINO

Os folículos pré-antrais constituem a principal reserva reprodutiva em fêmeas e são classificados em primordiais, de transição, primários e secundários. A criopreservação tem sido uma ferramenta promissora para a preservação da fertilidade feminina, principalmente em mulheres submetidas a tratamentos contra o câncer ou com problemas reprodutivos. Nesse tipo de abordagem, é de extrema importância a preservação da reserva folicular assim como a funcionalidade tecidual, pois posteriormente a reserva folicular poderá ser submetida a biotécnicas de reprodução assistida como cultivo in vitro e transplante. Diversos protocolos e substâncias são rotineiramente avaliados para o desenvolvimento do processo de criopreservação para posterior transplante. Dentre eles, o resveratrol possui ação antioxidante minimizando a produção de espécies reativas de oxigênio (EROS) durante a vitrificação e cultivo in vitro. Os recentes avanços em nanotecnologia expandiram suas possíveis aplicações na biomedicina, assim as nanoparticulas vem sendo utilizadas em tratamentos oncológicos como um carreador de fármacos. Outros estudos reportaram o desenvolvimento de diversas formulações de nanopartículas com potencial antioxidante, anti-inflammatório e antibiótico. Dessa forma, as nanopartículas têm demonstrado resultados promissores na criopreservação de sêmen e cultivo de folículos pré-antrais.

The preantral follicles constitute the female reproductive reserve and are classified as primordial, transitional, primary, and secondary. Cryopreservation has been a promising tool for the preservation of female fertility, especially in women undergoing cancer treatment or with reproductive problems. In this type of approach, it is extremely important to preserve the follicular reserve, as well as the functionality of the tissue, since the follicular reserve will be subjected to assisted reproductive techniques such as in vitro culture and transplantation. Several protocols and substances are routinely evaluated for the development of the cryopreservation process. Among them, resveratrol has antioxidant action, minimizing the production of ROS during vitrification and in vitro culture. Recent advances in nanotechnology have expanded its possible applications in biomedicine, therefore nanoparticles have been used in cancer treatments as a drug carrier. Other studies have reported the development of several nanoparticle formulations with antioxidant, anti-inflammatory and antibiotic potential. Thus, nanoparticles have shown promising results in cryopreservation of semen and in vitro culture of preantral follicles.

AVALIAÇÃO DA QUALIDADE FOLICULAR PÓS-CRIOPRESERVAÇÃO TESTANDO: UM MÉTODO ALTERNATIVO DE CONGELAMENTO LENTO E HIPOTAURINA NO MEIO DE TRANSPORTE, DE TECIDO GONADAL DE GATAS

Esta pesquisa teve por justificativa ampliar os estudos acerca da criopreservação de tecido ovariano de gatas domésticas, juntamente com o uso de antioxidante no meio de transporte, podendo servir de modelo experimental para felinos selvagens. Objetivou-se validar um método de congelamento lento sem a utilização de máquina (Capítulo 2). E comparar os efeitos da criopreservação sobre diferentes métodos vitrificação e congelamento lento, com e sem a adição de antioxidante hipotaurina no meio de transporte, em diferentes tempos de exposição, dos ovários (Capítulo 3). Foram utilizados os ovários de 320 gatas domésticas, submetidas à ovariectomia eletiva. O tecido ovariano foi dissecado e cortado em fragmentos de tamanhos pré-determinados 7 mm × 3 mm × 3 mm (63 mm3). Os fragmentos cortados foram divididos aleatoriamente sendo que metade foi direcionado para a avaliação a fresco e outra metade para criopreservação pelos métodos lento e rápido. Para o congelamento lento utilizou-se meio de cultivo MEM (earle) sem glutamina, com bicarbonato de sódio e antibiótico acrescido de dimetil sulfóxido. A vitrificação foi realizada empregando dimetil sulfóxido (DMSO) e solução de DAP 123 (acetamida 1M, DMSO 2M e propilenoglicol 3M). Fez-se análise dos fragmentos teciduais por histomorfologia, imuno-histoquímica utilizando a caspase-3 clivada. Os resultados adquiridos demonstraram a eficiência do protocolo de congelação testado, de maneira que não houve diferença entre o congelamento testado e o automatizado (p<0,05). Para hipotaurina no meio de transporte notou-se sua eficiência quando utilizada na refrigeração por 24 horas e criopreservação pelo método vitrificação (p<0,05). Assim, concluímos que o método alternativo de congelamento lento é eficiente e a hipotaurina ajuda a preservar a viabilidade folicular quando há a necessidade de refrigeração e vitrificação do fragmento ovariano.

This research had as justification to expand the studies about the cryopreservation of ovarian tissue of domestic cats, together with the use of antioxidant in the means of transport, being able to serve as an experimental model for wild cats. The objective was to validate a slow freezing method without using a machine (Chapter 2). And to compare the effects of cryopreservation on different methods of vitrification and slow freezing, with and without the addition of antioxidant hypotaurin in the transport medium, at different exposure times, of the ovaries (Chapter 3). The ovaries of 320 domestic cats, submitted to elective ovariectomy, were used. The ovarian tissue was dissected and cut into fragments of predetermined sizes 7 mm × 3 mm × 3 mm (63 mm3). The cut fragments were divided randomly, half of which was directed to fresh evaluation and the other half to cryopreservation by slow and fast methods. For slow freezing, MEM culture medium (earle) without glutamine, sodium bicarbonate and antibiotic plus dimethyl sulfoxide was used. Vitrification was performed using dimethyl sulfoxide (DMSO) and DAP 123 solution (1M acetamide, 2M DMSO and 3M propylene glycol). Tissue fragments were analyzed by histomorphology, immunohistochemistry using cleaved caspase-3. The ovaries of 320 domestic cats, submitted to elective ovariectomy, were used. The ovarian tissue was dissected and cut into fragments of predetermined sizes 7 mm × 3 mm × 3 mm (63 mm3). The cut fragments were divided randomly, half of which was directed to fresh evaluation and the other half to cryopreservation by slow and fast methods. For slow freezing, MEM culture medium (earle) without glutamine, sodium bicarbonate and antibiotic plus dimethyl sulfoxide was used. Vitrification was performed using dimethyl sulfoxide (DMSO) and DAP 123 solution (1M acetamide, 2M DMSO and 3M propylene glycol). Tissue fragments were analyzed by histomorphology, immunohistochemistry using cleaved caspase-3. The acquired results demonstrated the efficiency of the tested freezing protocol, so that there was no difference between the tested and the automated freezing (p <0.05). For hypotaurin in the transport medium, its efficiency was noted when used in refrigeration for 24 hours and cryopreservation by the vitrification method (p <0.05). Thus, we conclude that the alternative method of slow freezing is efficient and the hypotaurin helps to preserve follicular viability when there is a need for refrigeration and vitrification of the ovarian fragment.

Current status and future perspective of canine and feline ovarian tissue culture / Status atual e perspectiva futura de cultura de tecido ovariano canino e felino

The culture of ovarian follicles is an important tool for understanding of the mechanisms controlling follicle development and differentiation of its oocyte. The benefit from recovering developmentally competent oocytes from small, immature follicles (primordial, primary, secondary and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in female cancer survivors. To-date, live offspring from cultured primordial follicles has occurred only in the mouse. Progress in larger, more complex species has been limited because these animal models have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. In this presentation, we highlight current status of this topic for domestic carnivores (i.e., dogs and cats) as well as future research priorities.(AU)

A cultura de folículos ovarianos é uma ferramenta importante para o entendimento dos mecanismos que controlam o desenvolvimento de folículos e a diferenciação de oócitos. O benefício da recuperação de oócitos competentes em termos de desenvolvimento de folículos pequenos e imaturas (primordiais, primários, secundários e antral precoce) também seria significativo, variando do resgate de genoma de espécies em extinção à preservação da fertilidade em sobreviventes de câncer femininos. Até o momento, descendentes de folículos primordiais cultivados ocorreram somente em ratos. O progresso em espécies maiores e mais complexas tem sido limitado porque estes modelos de animais tem durações de foliculogênese mais longa, requerendo maior tempo de cultura para gerar folículos e oócitos completamente crescidos. Nesta apresentação ressaltamos o status atual deste tópico para carnívoros domésticos (i.e. cães e gatos), além de prioridades futuras de pesquisa.(AU)

Natural infection in ovarian structures by bovine herpesvirus 1: molecular and serological detection / Infecção natural em estruturas ovarianas pelo Herpesvírus bovino 1: detecção molecular e sorológica

In this study, the polymerase chain reaction (PCR) was used to evaluate the presence of viral DNA in ovarian tissue, in the cumulus-oocyte complex (COC), follicular liquid, and blood of animals naturally infected with bovine herpesvirus-1 (BoHV-1). The serum profile of the sampled animals was also evaluated. Samples of serum, blood, ovarian tissue, follicular liquid, and COC were collected from 147 slaughterhouse animals that were not vaccinated against BoHV-1. Contaminated or insufficient samples were disregarded. Serological tests allowed the identification of serum-positive animals with neutralizing antibodies against BoHV-1. Analysis of samples by PCR revealed the presence of viral DNA in 0.9% (1/115) of the COC samples, in 4.3% (5/117) of the ovarian tissue samples, and in 2.8% (3/108) of the blood samples. Viral DNA was not detected in any of the follicular liquid samples. In serological samples, a positivity of 83.6% (117/140) was observed for BoHV-1. All PCR-positive animals, regardless of the samples analyzed, showed positivity in the serum neutralization test for the detection of BoHV-1-specific antibodies. According to these results, a high prevalence of antibodies against BoHV-1 was detected in naturally infected animals from different herds, and the molecular tests revealed the presence of viral DNA in bovine ovarian tissue, providing evidence that this might be...(AU)

Neste trabalho foi avaliada a presença do DNA viral, por meio da Reação em Cadeia da Polimerase (PCR), no tecido ovariano, nos oócitos, líquido folicular e sangue de vacas naturalmente infectadas. Também foi avaliado o perfil sorológico dos animais amostrados. Foram coletadas amostras de soro, sangue, tecido ovariano, líquido folicular e complexo cumulus-oócitos de 147 animais abatidos em frigorífico não vacinados contra o herpesvirus bovino 1 (BoHV-1). Amostras tóxicas ou insuficientes foram descartadas. Os testes sorológicos foram realizados permitindo a identificação dos animais soropositivos para anticorpos neutralizantes contra o BoHV-1. Foram realizadas as PCRs onde foi observada a presença do DNA viral em 0,9% (1/115) dos oócitos, em 4,3% (5/117) do tecido ovariano e em 2,8% (3/108) do sangue. Em nenhuma das amostras de líquido folicular foi detectado o DNA viral. Nas amostras sorológicas observou-se 83,6% (117/140) de positividade para o BoHV-1. Dentre os animais positivos na PCR, independente das amostras, todos apresentavam positividade no teste de soroneutralização para detecção de anticorpos para BoHV-1. Conclui-se que em animais de diferentes rebanhos analisados foi detectada alta prevalência de anticorpos contra o BoHV-1 e que nos testes moleculares houve a presença do DNA viral em amostras de tecidos ovarianos de bovinos, evidenciando que estas estruturas poderia...(AU)

Current status and future perspective of canine and feline ovarian tissue culture / Status atual e perspectiva futura de cultura de tecido ovariano canino e felino

The culture of ovarian follicles is an important tool for understanding of the mechanisms controlling follicle development and differentiation of its oocyte. The benefit from recovering developmentally competent oocytes from small, immature follicles (primordial, primary, secondary and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in female cancer survivors. To-date, live offspring from cultured primordial follicles has occurred only in the mouse. Progress in larger, more complex species has been limited because these animal models have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. In this presentation, we highlight current status of this topic for domestic carnivores (i.e., dogs and cats) as well as future research priorities.

A cultura de folículos ovarianos é uma ferramenta importante para o entendimento dos mecanismos que controlam o desenvolvimento de folículos e a diferenciação de oócitos. O benefício da recuperação de oócitos competentes em termos de desenvolvimento de folículos pequenos e imaturas (primordiais, primários, secundários e antral precoce) também seria significativo, variando do resgate de genoma de espécies em extinção à preservação da fertilidade em sobreviventes de câncer femininos. Até o momento, descendentes de folículos primordiais cultivados ocorreram somente em ratos. O progresso em espécies maiores e mais complexas tem sido limitado porque estes modelos de animais tem durações de foliculogênese mais longa, requerendo maior tempo de cultura para gerar folículos e oócitos completamente crescidos. Nesta apresentação ressaltamos o status atual deste tópico para carnívoros domésticos (i.e. cães e gatos), além de prioridades futuras de pesquisa.

AUTOTRANSPLANTE HETEROTÓPICO DE TECIDO OVARIANO EQUINO

O tecido ovariano quando coletado por procedimentos de biópsia permite a realização de diversos estudos com aplicações clínicas no campo da preservação da fertilidade feminina. O objetivo do presente estudo foi avaliar a influência da idade da doadora e dos sítios de transplante intramuscular (IM) e subvulvar (SVU) sobre a qualidade, ativação e densidade folicular em fragmentos de tecido ovariano equino autotransplantados após 3 e 7 dias. Fragmentos de tecido ovariano foram obtidos (n = 8) por meio de biópsia e, em seguida, autotransplantados aleatoriamente nos sítios IM e SVU e recuperados após 3 e 7 dias. Os principais achados foram: (i) a viabilidade e ativação folicular foram menores nos fragmentos transplantados no sítio SVU; (ii) a depleção folicular pós-transplante variou entre animais e não foi influenciada pela idade e; (iii) a presença de estruturas ovarianas (folículos antrais e corpo lúteo) no momento da biópsia tem associação com a densidade folicular nos fragmentos obtidos.

Ovarian tissue samples collected by biopsy pick-up method can be used to perform several studies related to fertility preservation. The aim of this study was to evaluate the effects of donor age and intramuscular (IM) and subvulvar (SVU) sites on the follicular viability, activation, and density in ovarian fragments autotransplanted. Biopsy pick-up was performed in mares (n = 7) and ovarian tissue samples were autotransplanted at IM and SVU sites by 3 or 7 days. In summary, our findings indicate that: (i) follicular viability and activation were lower in the ovarian fragments grafted at SVU site; (ii) the follicular depletion post-graft differed among animals and was not influenced by donor age; and (iii) the area of ovarian structures (antral follicles and corpus luteum) presents in the ovary at biopsy procedure had a positive effect on follicular density.

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

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

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).