Recuperação, caracterização e criopreservação de espermatozoides epididimários de animais silvestres do bioma Caatinga / Recovery, characterization and cryopreservation of epididymal sperm from wild animals of the Caatinga biome

A Caatinga, bioma exclusivamente brasileiro, abriga grande diversidade biológica, porém sofre com graves ameaças ambientais. Por isso, é iminente a necessidade de desenvolvimento de técnicas voltadas para a conservação dos animais que nela habitam, bem como se seu germoplasma. Quando do súbito óbito de um animal biologicamente valioso, a recuperação de espermatozoides epididimários pode se apresentar como a única possibilidade para salvaguardar gametas. Ainda, esta biotécnicas configura-se em uma ferramenta possível de ser utilizada quando não há outra forma de se coletar o sêmen em determinada espécie. Os espermatozoides coletados podem ser armazenados por meio da criopreservação, e posteriormente utilizados em outras biotecnologias. Neste sentido, esta revisão tem como objetivo apresentar os aspectos da recuperação, caracterização e criopreservação de espermatozoides epididimários em animais silvestres com principal foco em espécies do bioma Caatinga, como os preás, cutias, catetos e emas.(AU)

The Caatinga, an exclusively Brazilian biome, is home to great biological diversity, but suffers from serious environmental threats. Therefore, there is an imminent need to develop techniques aimed at the conservation of the animals that inhabit it, as well as their germplasm. When the sudden death of a biologically valuable animal, the recovery of epididymal spermatozoa may present itself as the only possibility to safeguard gametes. Still, this biotechnique is a tool that can be used when there is no other way to collect semen in each species. Collected spermatozoa can be stored through cryopreservation, and later used in other biotechnologies. In this sense, this review aims to present aspects of recovery, characterization, and cryopreservation of epididymal spermatozoa in wild animals with a focus on species from the Caatinga biome, such as cavies, agoutis, collared peccary, and rheas.(AU)

Biologia reprodutiva das abelhas africanizadas (Apis melífera) e biotecnologias aplicadas à sua conservação / Reproductive biology of Africanized bees (Apis mellifera) and biotechnologies applied to their conservation

A biodiversidade das abelhas africanizadas (Apis melífera) encontra-se sob grande ameaça e, neste cenário as investigações acerca da biologia reprodutiva, alguns aspectos ainda incipientes, bem como métodos aprimorados para a inseminação instrumental e criopreservação de gametas podem ser ferramentas preciosas para a conservação in situ e ex situ de subespécies e ecótipos. O entendimento e adoção de ferramentas como estas mencionadas, podem auxiliar na seleção de características de interesse para os criadores, assim como, nos esforços para a conservação de populações de abelhas ameaçadas.(AU)

The biodiversity of Africanized bees (Apis mellifera) is under great threat and, in this scenario, investigations about reproductive biology, some aspects are still incipient, as well as improved methods for instrumental insemination and cryopreservation of gametes can be precious tools for ex situ and in conservation. situ of subspecies and ecotypes can be contemplated. The understanding and adoption of tools such as those mentioned can help in the selection of characteristics of interest to breeders, as well as those committed to the conservation of endangered bee populations.(AU)

In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

Impacto de antimicrobianos nos parâmetros cinéticos de espermatozoides criopreservados de catetos / Impact of antimicrobials on the kinetic parameters of collared peccary frozen sperm

The objective was to verify the impact of the addition of antimicrobials on the kinetic parameters of sperm in the cryopreserved semen of collared peccaries. Ejaculates from 10 adult male, obtained by electroejaculation, were used. The samples had their kinetic parameters evaluated by computer analysis (CASA). Subsequently, they were cryopreserved in Tris plus egg yolk (20%) and glycerol (3%), whether or not (control) added gentamicin (70µg/mL) or the combination penicillin (1000 IU/mL) and streptomycin (1mgE/mL) (P+E). After one week, the samples were thawed and evaluated similarly to fresh semen. In fresh semen, total motility of 95.3±0.8% and 72.1±3.5% progressive motility were observed. After thawing, there were no differences between treatments, except for the cross-beat frequency (BCF) parameter, which was negatively influenced by P+E, in relation to fresh semen (p <0.05). In conclusion, it is suggested the use of gentamicin as an antimicrobial for the cryopreservation of semen from peccaries.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.(AU)

Criopreservação e cultivo de tecido testicular como ferramenta na conservação de mamíferos silvestres / Testicular tissue cryopreservation and culture as a tool for the conservation of wild mammals

A criopreservação do tecido testicular se apresenta como ferramenta promissora para a reprodução assistida, possibilitando o armazenamento de fragmentos contendo grande número de células germinativas em várias fases de desenvolvimento, incluindo espermatogônias indiferenciadas que podem ser cultivadas, garantindo a produção ilimitada de espermatozoides. Nesta revisão, são abordados aspectos técnicos relativos ao processamento e aplicabilidade da criopreservação e do cultivo de tecido testicular em mamíferos silvestres, ressaltando seus desafios e perspectivas.(AU)

Cryopreservation of the testicular tissue is a promising tool for assisted reproduction, allowing the storage of fragments containing a large number of germ cells in various stages of development, including undifferentiated spermatogonia that can be cultured, guaranteeing the unlimited production of spermatozoa. In this review, technical aspects related to the processing and applicability of testicular tissue cryopreservation and culture in wild mammals are discussed, highlighting their challenges and perspectives.(AU)

Criopreservação e cultivo de tecido testicular como ferramenta na conservação de mamíferos silvestres / Testicular tissue cryopreservation and culture as a tool for the conservation of wild mammals

A criopreservação do tecido testicular se apresenta como ferramenta promissora para a reprodução assistida, possibilitando o armazenamento de fragmentos contendo grande número de células germinativas em várias fases de desenvolvimento, incluindo espermatogônias indiferenciadas que podem ser cultivadas, garantindo a produção ilimitada de espermatozoides. Nesta revisão, são abordados aspectos técnicos relativos ao processamento e aplicabilidade da criopreservação e do cultivo de tecido testicular em mamíferos silvestres, ressaltando seus desafios e perspectivas.

Cryopreservation of the testicular tissue is a promising tool for assisted reproduction, allowing the storage of fragments containing a large number of germ cells in various stages of development, including undifferentiated spermatogonia that can be cultured, guaranteeing the unlimited production of spermatozoa. In this review, technical aspects related to the processing and applicability of testicular tissue cryopreservation and culture in wild mammals are discussed, highlighting their challenges and perspectives.

Reproduction in agouti (Dasyprocta spp): A review of reproductive physiology for developing assisted reproductive techniques

Dasyprocta spp. (agouti) include wild rodents with highlighted ecological and economic importance, and are considered experimental models for endangered hystricognath rodents. Of late, development of techniques to conserve their genetic material as well as the formation of biobanks is increasing. In this context, this review describes the main advances in the knowledge of the reproductive morphophysiological specificities of agouti as well as the development and improvement of assisted reproductive techniques aimed at conservation, multiplication, and exploitation of their reproductive potential under captivity.

Reproduction in agouti (Dasyprocta spp): A review of reproductive physiology for developing assisted reproductive techniques

Dasyprocta spp. (agouti) include wild rodents with highlighted ecological and economic importance, and are considered experimental models for endangered hystricognath rodents. Of late, development of techniques to conserve their genetic material as well as the formation of biobanks is increasing. In this context, this review describes the main advances in the knowledge of the reproductive morphophysiological specificities of agouti as well as the development and improvement of assisted reproductive techniques aimed at conservation, multiplication, and exploitation of their reproductive potential under captivity.(AU)

Superoxide dismutase and catalase activity in collared peccary (Pecari tajacu) seminal plasma and their relation to sperm quality / Atividade da superóxido dismutase e catalase no plasma seminal de catetos (Pecari tajacu) e sua relação com a qualidade espermática

The study of the seminal plasma help us to understand the mechanisms by which reactive oxygen species (ROS) affect the sperm. The antioxidant enzymes, as the superoxide dismutase - SOD and catalase - CAT, are capable of removing the oxidative agents before they produce injuries. The aim of the current study was to investigate the activity of antioxidant enzymes SOD and CAT in seminal plasma, and their association with sperm quality in collared peccaries. Study was conducted during the dry period (August and September) on a region characterized by a semiarid climate, with an average annual temperature of 27°C and irregular rainfall (Mossoro, RN, Brazil; 5°10´S and 37°10´W). Nine ejaculates were obtained from sexually mature males (1 sample per animal) by electroejaculation. Semen was evaluated for microscopic parameters and the activity of SOD and CAT was measured by spectrophotometry. All ejaculates were white in color. Mean values for concentration were of 207 ± 160 x106 sperm/mL, motility of 83.0 ± 20.9% and viability of 72.5 ± 10.4%. In regards to the enzymatic activity, none was observed for the CAT enzyme. Trace levels of SOD (0.033 ± 0.049 AU/mgP) were detected in the ejaculates of all individuals; however, no correlation was observed between SOD levels and the sperm motility (R = 0.35; P = 0.931), vigor (R = 0.29; P = 0.133), viability (R = 0.16; P = 0.29), functional membrane (R = 0.04; P = 0.617) or morphology (R = 0.03; P = 0.637). In conclusion, we demonstrated the first description of antioxidant enzyme activity in seminal plasma of fresh ejaculates obtained from collared peccaries. SOD antioxidant activity was evident during the dry period of a semiarid region, but no relationship between SOD and semen parameters was observed.

O estudo do plasma seminal nos auxilia a compreender os mecanismos pelos quais as espécies reativas de oxigênio (EROs) afetam o espermatozoide. As enzimas antioxidantes, como a superóxido dismutase - SOD e a catalase - CAT, são capazes de remover os agentes oxidativos antes que causem injúrias. O objetivo deste estudo foi investigar a atividade das enzimas SOD e CAT no plasma seminal, e sua associação com a qualidade espermática em catetos. O estudo foi conduzido durante o período seco (agosto a setembro) em uma região caracterizada pelo clima semiárido, com temperatura média anual de 27°C e pluviosidade irregular (Mossoró, RN, Brasil; 5°10´S e 37°10´W). Nove ejaculados foram obtidos de machos sexualmente maduros (1 amostra por animal) por eletroejaculação. O sêmen foi avaliado quanto aos parâmetros microscópicos e a atividade da SOD e da CAT foi mensurada por espectrofotometria. Todos os ejaculados apresentavam coloração branca. Os valores médios para a concentração foram de 207 ± 160 x106 espermatozoides/mL, motilidade de 83,0 ± 20,9% e viabilidade de 72,5 ± 10,4%. No que diz respeito à atividade enzimática, nenhuma foi observada para a enzima CAT. Traços de SOD (0,033 ± 0,049 AU/mgP) foram detectados nos ejaculados de todos os indivíduos; entretanto, nenhuma correlação foi observada entre os níveis de SOD e a motilidade espermática (R = 0,35; P = 0,931), o vigor (R = 0,29; P = 0,133), a viabilidade (R = 0,16; P = 0,29), a função de membrana (R = 0,04; P = 0,617) ou a morfologia espermática (R = 003; P = 0,637). Em conclusão, nós demonstramos a primeira descrição da atividade enzimática antioxidante no plasma seminal de ejaculados frescos obtidos de catetos. A atividade da SOD foi evidente durante o período seco de uma região semiárida, mas nenhuma relação entre a SOD e os parâmetros seminais foi observada.

Superoxide dismutase and catalase activity in collared peccary (Pecari tajacu) seminal plasma and their relation to sperm quality / Atividade da superóxido dismutase e catalase no plasma seminal de catetos (Pecari tajacu) e sua relação com a qualidade espermática

The study of the seminal plasma help us to understand the mechanisms by which reactive oxygen species (ROS) affect the sperm. The antioxidant enzymes, as the superoxide dismutase - SOD and catalase - CAT, are capable of removing the oxidative agents before they produce injuries. The aim of the current study was to investigate the activity of antioxidant enzymes SOD and CAT in seminal plasma, and their association with sperm quality in collared peccaries. Study was conducted during the dry period (August and September) on a region characterized by a semiarid climate, with an average annual temperature of 27°C and irregular rainfall (Mossoro, RN, Brazil; 5°10´S and 37°10´W). Nine ejaculates were obtained from sexually mature males (1 sample per animal) by electroejaculation. Semen was evaluated for microscopic parameters and the activity of SOD and CAT was measured by spectrophotometry. All ejaculates were white in color. Mean values for concentration were of 207 ± 160 x106 sperm/mL, motility of 83.0 ± 20.9% and viability of 72.5 ± 10.4%. In regards to the enzymatic activity, none was observed for the CAT enzyme. Trace levels of SOD (0.033 ± 0.049 AU/mgP) were detected in the ejaculates of all individuals; however, no correlation was observed between SOD levels and the sperm motility (R = 0.35; P = 0.931), vigor (R = 0.29; P = 0.133), viability (R = 0.16; P = 0.29), functional membrane (R = 0.04; P = 0.617) or morphology (R = 0.03; P = 0.637). In conclusion, we demonstrated the first description of antioxidant enzyme activity in seminal plasma of fresh ejaculates obtained from collared peccaries. SOD antioxidant activity was evident during the dry period of a semiarid region, but no relationship between SOD and semen parameters was observed.(AU)

O estudo do plasma seminal nos auxilia a compreender os mecanismos pelos quais as espécies reativas de oxigênio (EROs) afetam o espermatozoide. As enzimas antioxidantes, como a superóxido dismutase - SOD e a catalase - CAT, são capazes de remover os agentes oxidativos antes que causem injúrias. O objetivo deste estudo foi investigar a atividade das enzimas SOD e CAT no plasma seminal, e sua associação com a qualidade espermática em catetos. O estudo foi conduzido durante o período seco (agosto a setembro) em uma região caracterizada pelo clima semiárido, com temperatura média anual de 27°C e pluviosidade irregular (Mossoró, RN, Brasil; 5°10´S e 37°10´W). Nove ejaculados foram obtidos de machos sexualmente maduros (1 amostra por animal) por eletroejaculação. O sêmen foi avaliado quanto aos parâmetros microscópicos e a atividade da SOD e da CAT foi mensurada por espectrofotometria. Todos os ejaculados apresentavam coloração branca. Os valores médios para a concentração foram de 207 ± 160 x106 espermatozoides/mL, motilidade de 83,0 ± 20,9% e viabilidade de 72,5 ± 10,4%. No que diz respeito à atividade enzimática, nenhuma foi observada para a enzima CAT. Traços de SOD (0,033 ± 0,049 AU/mgP) foram detectados nos ejaculados de todos os indivíduos; entretanto, nenhuma correlação foi observada entre os níveis de SOD e a motilidade espermática (R = 0,35; P = 0,931), o vigor (R = 0,29; P = 0,133), a viabilidade (R = 0,16; P = 0,29), a função de membrana (R = 0,04; P = 0,617) ou a morfologia espermática (R = 003; P = 0,637). Em conclusão, nós demonstramos a primeira descrição da atividade enzimática antioxidante no plasma seminal de ejaculados frescos obtidos de catetos. A atividade da SOD foi evidente durante o período seco de uma região semiárida, mas nenhuma relação entre a SOD e os parâmetros seminais foi observada.(AU)

Conservação de roedores histricognatos silvestres da América do Sul utilizando estratégias reprodutivas / Conservation of South American wild hystricognath rodents using reproductive strategies

A fauna silvestre, especialmente os roedores histricognatos, representa uma importante fonte econômicade carne, couro e pelos para a população humana. Nesse sentido, um aumento produtivo dessa fonte estádiretamente associado ao emprego de estratégias reprodutivas, visando tanto a conservação da biodiversidade,como a multiplicação adequada de indivíduos em cativeiro. Para ambas as situações, a primeira etapa consiste naeficiente conservação de germoplasma com a formação de criobancos. Assim, o objetivo desta revisão éapresentar os principais avanços alcançados na conservação de roedores histricognatos silvestres, especialmenteaqueles da América do Sul, evidenciando os resultados na criopreservação de células e tecidos obtidos em cutiase preás.(AU)

The wild fauna, especially hystricognath rodents, represents an important economic source of meat,leather and hairs for the human population. In this sense, a productive increase of this source is directlyassociated with the use of reproductive strategies, aiming both the conservation of biodiversity and the adequatemultiplication in captivity. For both situations, the first step consists in the efficient conservation of germplasmwith the formation of cryobanks. Thus, the aim of this review is to present the main advances in the conservationof wild hystricognath rodents, especially those from South America, evidencing the results in thecryopreservation of cells and tissues obtained from agoutis and spix's yellow-toothed cavies.(AU)

Conservação de roedores histricognatos silvestres da América do Sul utilizando estratégias reprodutivas / Conservation of South American wild hystricognath rodents using reproductive strategies

A fauna silvestre, especialmente os roedores histricognatos, representa uma importante fonte econômicade carne, couro e pelos para a população humana. Nesse sentido, um aumento produtivo dessa fonte estádiretamente associado ao emprego de estratégias reprodutivas, visando tanto a conservação da biodiversidade,como a multiplicação adequada de indivíduos em cativeiro. Para ambas as situações, a primeira etapa consiste naeficiente conservação de germoplasma com a formação de criobancos. Assim, o objetivo desta revisão éapresentar os principais avanços alcançados na conservação de roedores histricognatos silvestres, especialmenteaqueles da América do Sul, evidenciando os resultados na criopreservação de células e tecidos obtidos em cutiase preás.

The wild fauna, especially hystricognath rodents, represents an important economic source of meat,leather and hairs for the human population. In this sense, a productive increase of this source is directlyassociated with the use of reproductive strategies, aiming both the conservation of biodiversity and the adequatemultiplication in captivity. For both situations, the first step consists in the efficient conservation of germplasmwith the formation of cryobanks. Thus, the aim of this review is to present the main advances in the conservationof wild hystricognath rodents, especially those from South America, evidencing the results in thecryopreservation of cells and tissues obtained from agoutis and spix's yellow-toothed cavies.

Relações entre a câmara de Neubauer a espectrofotometria utilizadas para a determinação da concentração espermática de catetos (Pecari tajacu) / Relationship between Neubauer counting chamber and spectrophotometer used for the sperm concentration determination in collared peccaries (Pecari tajacu)

Em ejaculados provenientes de 28 catetos, verificou-se a existência de relações entre a concentração espermática determinada por meio da câmara de Neubauer e a tramitância observada por espectrofotometria, utilizando comprimentos de onda variando de 530 a 590nm. Os ejaculados apresentaram uma concentração média de 283,9±30,8x106 espermatozoides mL-1, com variação de 30 a 640x106 espermatozoides mL-1. Os valores para tramitância variaram entre 36,9 a 96,3, nos diferentes comprimentos de onda. Não foram detectadas relações significativas entre os dois métodos (P>0,05). Dessa forma, não se recomenda a espectrofotometria para os procedimentos de rotina na determinação da concentração espermática em catetos.(AU)

In ejaculates derived from 28 collared peccaries, we verified the existence of relationships between sperm concentration determined by the Neubauer counting chamber and the tramitance verified by spectrophotometer, under wavelengths varying from 530 to 590nm. Ejaculates presented a concentration of 283.9±30.8x106sperm m-1, varying from 30 to 640x106sperm mL-1. Values for tramitance varied from 36.9 to 96.3, under different wavelengths. No significant relationship was verified between two methods (P>0.05). Thus, the spectrophotometer is not recommended for routine procedures of sperm concentration measurement in collared peccaries.(AU)