Cryopreservation efficiency of red-rumped agouti (Dasyprocta leporina) sperm obtained from different origins through epididymal retrograde flushing or electroejaculation.

This study investigated whether the origin of sperm (epididymal vs. ejaculate) affects the cryopreservation efficiency in agouti (Dasyprocta leporina). Five sexually mature agoutis underwent electroejaculation, resulting in obtaining four semen samples. After 15 days, the same animals were euthanized, and through retrograde flushing, sperm samples were obtained from the epididymis tails. In both collection methods, samples were evaluated for sperm parameters (sperm concentration, motility, vigor, membrane integrity, osmotic response, and morphology). Then, samples were diluted in ACP 109c, added with 20% egg yolk, and a final concentration of 6% glycerol. Finally, the samples were packaged in 0.25 mL straws and frozen in liquid nitrogen. After one week, samples were thawed and evaluated in the same way as fresh samples, with the addition of membrane integrity analysis using fluorescent probes (C-FDA/PI) and computerized analysis (CASA). Immediately after obtaining the sperm, samples obtained directly from the epididymis presented higher values (P ≤ 0.05) than those obtained by electroejaculation concerning the parameters of volume, sperm concentration, and total number of sperm (1,398.25 ± 206.0 x106 and 184.5 ± 78.0 x106 sperm). On the other hand, in the classical evaluation of the other sperm parameters and the computerized analysis (CASA) after thawing, such as total motility, no statistical differences were observed between sperm from both origins (ejaculate: 16.7 ± 8.2% and epididymal: 24.8 ± 12.0%, P > 0.05). This demonstrates the possibility of direct application of the cryopreservation protocol for agouti (D. leporina) sperm obtained via the epididymis or ejaculate.

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models.

Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.

Influência da temperatura testicular sobre os parâmetros espermáticos de suínos mantido em confinamento no Cariri Cearense / Influence of testicular temperature on sperm parameters of boars keep in confination at cariri cearense

The objective of this work is to evaluate the correlation between testicular temperature and seminal quality of boars kept in confinement in the region of Cariri Ceará. In the period from October 19, 2019 to July 14, 2020 with two monthly collections at 7 am, ejaculates were collected from 4 healthy boars of initial age of 20 months, crossbred from the Landrace and Large White breeds, and marked with the following numbers 20, 22, 23 and 24 were evaluated and their testicular temperature was also measured. The collected semen showed whitish color and pasty consistency ith an average volume of 441.25mL, average concentration of 383.3 x106 sperm, average motilityof 95%, average vigor of 4.6, average viability of 70% and functionality of membrane 65.5% indexes those ideals. With Pearson's correlation test it was possible to demonstrate a positive correlation between temperature 31.37 °C with motility and vigor and two with a negative correlation between temperature 31.85 and 30.23 °C and sperm concentration respectively -0.913 and -0.974. It was also possible to show that the parameters did not differ from the average. Concluding that there is a correlation, but we should try to study a little more on the subject.(AU)

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models

Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.(AU)

Influência da temperatura testicular sobre os parâmetros espermáticos de suínos mantido em confinamento no Cariri Cearense / Influence of testicular temperature on sperm parameters of boars keep in confination at cariri cearense

The objective of this work is to evaluate the correlation between testicular temperature and seminal quality of boars kept in confinement in the region of Cariri Ceará. In the period from October 19, 2019 to July 14, 2020 with two monthly collections at 7 am, ejaculates were collected from 4 healthy boars of initial age of 20 months, crossbred from the Landrace and Large White breeds, and marked with the following numbers 20, 22, 23 and 24 were evaluated and their testicular temperature was also measured. The collected semen showed whitish color and pasty consistency ith an average volume of 441.25mL, average concentration of 383.3 x106 sperm, average motilityof 95%, average vigor of 4.6, average viability of 70% and functionality of membrane 65.5% indexes those ideals. With Pearson's correlation test it was possible to demonstrate a positive correlation between temperature 31.37 °C with motility and vigor and two with a negative correlation between temperature 31.85 and 30.23 °C and sperm concentration respectively -0.913 and -0.974. It was also possible to show that the parameters did not differ from the average. Concluding that there is a correlation, but we should try to study a little more on the subject.

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models

Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants.

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions - NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ±â€¯8.6%); however, the SSV was only efficient with DMSO alone (63.9 ±â€¯7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ±â€¯2.9% viable cells with 2.0 ±â€¯0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.

Adaptação da tecnologia do ovário artificial para os animais silvestres / Adaptation of the artificial ovary technology for wild animals

A biodiversidade mundial tem sido consideravelmente reduzida nos últimos anos, principalmente devido as modificações antrópicas nos habitats. Como consequência, tem-se observado um rápido e contínuo declínio das espécies mamíferas silvestres. Nesse contexto, a biotécnica “Ovário Artificial”, surge como uma ferramenta para a conservação da biodiversidade, pois tem como objetivo a manipulação in vitro de oócitos inclusos em folículos préantrais visando o seu crescimento e maturação, além da preservação de oócitos por meio do resfriamento e/ou criopreservação, permitindo o fornecimento de milhares de oócitos, que poderão ser utilizados posteriormente. Este artigo de revisão descreve os aspectos gerais, importância e avanços do ovário artificial em animais silvestres.(AU)

The world biodiversity has declined in recent years, mainly due to anthropogenic changes in habitats. As consequence, a rapid and steady decline in wild mammalian species is observed. In this context, the "Artificial Ovary" biotechnology emerges as a tool for the biodiversity conservation, since its objective is the in vitro manipulation of oocytes enclosed in preantral follicles, aiming their growth and maturation, as well as their preservation by cooling and / or cryopreservation, allowing the supply of thousands of oocytes, which can be further used. This review describes the general aspects, importance and advances of the artificial ovary in wild animals.(AU)

Adaptação da tecnologia do ovário artificial para os animais silvestres / Adaptation of the artificial ovary technology for wild animals

A biodiversidade mundial tem sido consideravelmente reduzida nos últimos anos, principalmente devido as modificações antrópicas nos habitats. Como consequência, tem-se observado um rápido e contínuo declínio das espécies mamíferas silvestres. Nesse contexto, a biotécnica “Ovário Artificial”, surge como uma ferramenta para a conservação da biodiversidade, pois tem como objetivo a manipulação in vitro de oócitos inclusos em folículos préantrais visando o seu crescimento e maturação, além da preservação de oócitos por meio do resfriamento e/ou criopreservação, permitindo o fornecimento de milhares de oócitos, que poderão ser utilizados posteriormente. Este artigo de revisão descreve os aspectos gerais, importância e avanços do ovário artificial em animais silvestres.

The world biodiversity has declined in recent years, mainly due to anthropogenic changes in habitats. As consequence, a rapid and steady decline in wild mammalian species is observed. In this context, the "Artificial Ovary" biotechnology emerges as a tool for the biodiversity conservation, since its objective is the in vitro manipulation of oocytes enclosed in preantral follicles, aiming their growth and maturation, as well as their preservation by cooling and / or cryopreservation, allowing the supply of thousands of oocytes, which can be further used. This review describes the general aspects, importance and advances of the artificial ovary in wild animals.

Conservation of somatic tissue derived from collared peccaries (Pecari tajacu Linnaeus, 1758) using direct or solid-surface vitrification techniques.

Cryopreservation of somatic tissue can be applied in biodiversity conservation, especially for wild species as collared peccary. We aimed to evaluate the effect of vitrification techniques of ear tissue of collared peccary [direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the layers of epidermis and dermis by conventional histology and cell ability during the in vitro culture. Thus, both the vitrification methods were able to maintain normal patterns of the epidermis as the cornea and granular layers, furthermore the intercellular space and dermal-epidermal junction of the spinous layer when compared to fresh control. Nevertheless, DVC and SSV percentage of normality decreased in the morphological integrity of cytoplasm (37.5 and 25.0%) of spinous layer, respectively, as compared to the fresh fragments (100%, p < 0.05). Moreover, other differences between the fresh control (100%) and DVC tissues were verified in the intra-epidermal cleavage of the spinous (37.5%) and basal (37.5%) layers. In general, DVC and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters for the in vitro culture (p > 0.05). In addition, only at time of 72 h (D3), in the growth curve, DVC fragments showed a reduced cell concentration than fresh control. In conclusion, SSV was found to be a more efficient method for vitrifying collared peccary skin tissue when compared to DVC. These results are relevant for the tissue cryopreservation from collared peccary and could also be useful for mammals with phylogenetic relationships.

Sperm characteristics following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins in the collared peccary (Pecari tajacu).

The aim of the current study was to compare sperm quality characteristics of the collared peccary (Pecari tajacu) following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins (LDL). Semen from 11 adult males was obtained by electroejaculation and evaluated for sperm motility, vigor, morphology as well as membrane integrity analyzed by the hypo-osmotic swelling (HOS) test and a fluorescent staining. Moreover, the semen was diluted in a Tris-based extender containing 20% egg yolk (control group) or 5, 10 or 20% LDL (treatment groups). The semen samples were frozen in liquid nitrogen and thawed in a water bath for 60s at 37°C. The treatments did not affect (p>0.05) sperm vigor, morphology or membrane integrity analyzed by the HOS test. However, post-thaw sperm motility was significantly higher (p<0.05) in the extender supplemented with 20% LDL (36.4 ± 5.3%) compared with the egg yolk extender and extender supplemented with 10% LDL. Furthermore, the percentage of membrane-intact frozen-thawed spermatozoa analyzed by the fluorescent staining was significantly higher (p<0.05) in the extender supplemented with 20% LDL (27.4 ± 6.5%) than in the other groups. In conclusion, 20% LDL can be used to substitute the whole egg yolk as a cryoprotective additive for freezing semen of the collared peccary.

Formação de bancos de germoplasma e sua contribuição para a conservação de espécies silvestres no Brasil / Formation of germplasm banks and its contribution to the wildlife conservation in Brazil

A biodiversidade vem sofrendo grande diminuição nas últimas décadas, apesar do incremento de algumas iniciativas, que visam o uso sustentável da diversidade biológica. A criação de bancos de germoplasma, os quais abrigam coleções-base para a conservação de ampla variabilidade genética vegetal e animal, surge como alternativa para manutenção da biodiversidade. O presente trabalho apresenta dados acerca da legislação vigente que regulariza a conservação e uso de recursos genéticos, bem como os aspectos técnicos relativos a formação de coleções de gametas e embriões, particularmente, visando a preservação de espécies silvestres no Brasil.(AU)

Biodiversity has suffered great loss in recent decades, despite the increased number of initiatives that aims the sustainable use of biological diversity. The creation of germplasm banks, which are collections that conserves the broad genetic variability of plants and animals, is an alternative to maintaining biodiversity. This paper presents data about the legislation that regulates the use and conservation of genetic resources, as well as the technical aspects of the formation of collections of gametes and embryos, particularly in the preservation of wild species in Brazil.(AU)

Formação de bancos de germoplasma e sua contribuição para a conservação de espécies silvestres no Brasil / Formation of germplasm banks and its contribution to the wildlife conservation in Brazil

A biodiversidade vem sofrendo grande diminuição nas últimas décadas, apesar do incremento de algumas iniciativas, que visam o uso sustentável da diversidade biológica. A criação de bancos de germoplasma, os quais abrigam coleções-base para a conservação de ampla variabilidade genética vegetal e animal, surge como alternativa para manutenção da biodiversidade. O presente trabalho apresenta dados acerca da legislação vigente que regulariza a conservação e uso de recursos genéticos, bem como os aspectos técnicos relativos a formação de coleções de gametas e embriões, particularmente, visando a preservação de espécies silvestres no Brasil.

Biodiversity has suffered great loss in recent decades, despite the increased number of initiatives that aims the sustainable use of biological diversity. The creation of germplasm banks, which are collections that conserves the broad genetic variability of plants and animals, is an alternative to maintaining biodiversity. This paper presents data about the legislation that regulates the use and conservation of genetic resources, as well as the technical aspects of the formation of collections of gametes and embryos, particularly in the preservation of wild species in Brazil.

Aplicação das biotécnicas de MOIFOPA, transgênese eclonagem na reprodução de caprinos / Accomplishment of the biotechniques MOIFOPA, transgenesis and clonage in caprine reproduction

A pecuária corresponde hoje a mais da metade da produção agrícola em países desenvolvidos e mais de um quarto em países em desenvolvimento. Os caprinos têm grande importância econômica pela sua contribuição na produção de carne, leite e pele em diversos países. Este crescimento tem requerido novos conhecimentos técnicos e científicos, especialmente relacionados ao aumento da eficiência produtiva e reprodutiva dos rebanhos, o que levou ao desenvolvimento de várias biotécnicas ligadas a reprodução. Estas biotecnologias têm um papel direto na obtenção de maior número de animais de elevada qualidade. Entretanto algumas delas ainda não possuem aplicabilidade direta, permanecendo restritas aos centros de pesquisa, tais como a transgênese, clonagem e MOIFOPA. No futuro poderão ter aplicações na produção de produtos farmacológicos, na produção de modelos para estudos de doenças animais ou humanas, na melhoria de características de produção animais, no estudo da regulação e a expressão gênica, entre muitas outras, estando direta ou indiretamente relacionadas ao bem-estar do homem. Assim, o objetivo do presente trabalho foi realizar uma breve revisão sobre algumas biotécnicas aplicadas à reprodução da espécie caprina.

The animal business corresponds to more than a half of the agricultural production in developed countries and to more than a quart in developing ones. Goats have a huge economic importance because of its contribution on meat, milk and skin production in many countries. This growth requires new technical and scientific acknowledgments, especially those related to improvement of productive and reproductive efficiency of cattles, that lead the development of some biotechniques related to reproduction. These biotechnologies have a directly role on the gain of a larger number of animals with high quality. However some of them still don't have directly application, they are still restricted at research centers such as transgenesis, clone and MOIFOPA. On the future it could contributes for medicine production, for models to animals and human disease studies, on the study of genetics regulation and expression, being directly or indirectly related to human well being. On this way, the objective of the present work was to perform a brief review of some biotechniques applied to goat reproduction.

Aplicação das biotécnicas de transferência de embriões e fertilização in vitro na reprodução de caprinos / Accomplishment of the biotechniques embryo transfer and in vitro fertilization on caprine reproduction

A caprinocultura vem apresentando um ciclo de crescimento mundial, o qual vem se intensificando principalmente nos países em desenvolvimento. Nesse contexto, há a necessidade da aplicação de técnicas de reprodução assistida com o objetivo de aumentar a eficiência reprodutiva e produtiva dos rebanhos. Apesar de algumas biotécnicas existirem há mais de 50 anos, nos últimos dez anos houve um grande aprimoramento das já existentes e o desenvolvimento de novas. Atualmente, algumas técnicas já apresentam grande aplicabilidade a campo como a inseminação artificial, a sexagem de sêmen, a transferência e criopreservação de embriões e a produção in vitro de embriões. Estas biotécnicas contribuem para acelerar o ganho genético de animais superiores bem como permitem superar alguns obstáculos de eficiência reprodutiva. Dessa forma, o objetivo do presente trabalho foi realizar uma breve revisão sobre algumas biotécnicas aplicadas à reprodução da espécie caprina.

The goat business is presenting a world improve cycle, which has been intensified especially in developing countries. On this context, it is needed to apply assisted reproduction techniques with the aim to increase the reproductive and productive efficiency of cattles. In spite of some techniques has existed far from 50 years ago, in the last ten years it was a huge improvement of them and the development of other ones. Currently, some techniques already present large use at farm such as artificial insemination, semen sorting, embryo transfer and cryopreservation and embryo in vitro production. These techniques contribute to accelerate the genetic gain in superior animals such as allow to overcome some obstacles of reproductive efficiency. On this way, the objective of the present work was to perform a brief review of some biotechniques applied to goat reproduction.

Conservação do sêmen canino sob refrigeração em diferentes caixas isotérmicas / Canine semen conservation under cooling in different isothermal boxes

O presente estudo objetivou determinar o período máximo de conservação em diferentes caixas isotérmicas sobre a viabilidade do sêmen canino refrigerado. O sêmen de oito cães foi coletado por manipulação digital. A fração espermática foi avaliada quanto ao volume, coloração, motilidade, vigor, morfologia espermática, integridade acrossomal, resposta osmótica e concentração espermática. Em seguida, foi diluída em Leite UHT desnatado acrescido de 20% de gema de ovo e dividida em alíquotas, que foram armazenadas em caixas isotérmicas de poliestireno expandido de 3L ou 5L, contendo gelo biológico. As referidas caixas foram abertas após 12h, 18h, 24h e 30h de armazenagem. Nessa ocasião, o sêmen foi reavaliado quanto às características já citadas. A temperatura interna das caixas e a temperatura ambiente foram monitoradas. Como principais resultados, a motilidade espermática progressiva foi conservada de maneira aceitável até as 30 horas em ambas as caixas, as quais não apresentaram diferenças quanto à conservação de nenhuma das características seminais (P > 0,05). Porém, a temperatura interna da caixa de 3L apresentou uma elevação significativa a partir das 18h (P < 0,05), sendo que na caixa de 5L, a temperatura interna manteve-se constante por todo o experimento. Conclui-se que o sêmen canino diluído em leite UHT desnatado pode ser eficientemente conservado por até 30h em caixas isotérmicas de 3L ou 5L, contendo gelo reciclável. Mas salienta-se que a caixa de 5L apresenta maior segurança para a viabilidade espermática, pois é mais eficiente na manutenção de uma temperatura constante.

The aim of this study was to determine the maximum time of conservation in different isothermal boxes of the diluted canine semen under cooling. Semen from eight dogs was collected by digital manipulation. The sperm fraction was evaluated for volume, color, motility, vigor, sperm morphology, acrossomal integrity, osmotic answer and sperm concentration. Then, it was diluted in skimmed UHT milk plus egg yolk (20%) and divided in samples, which were stored in 3L or 5L expanded polystyrene isothermal boxes, containing recyclable ice. These boxes were open after 12h, 18h, 24h and 30h of storage and semen was reevaluated for the characteristics already mentioned. The temperature into the boxes and the environmental temperature were monitored. As main results, the progressive sperm motility was acceptably conserved up to 30h in both boxes, which did not present differences in the conservation of none of the seminal characteristics (P>0.05). However, the temperature into the 3L-box showed a significant elevation starting from the 18h (P <0.05), and the temperature into the 5L-box remained constant for whole the experiment. It is concluded that the canine semen diluted in skimmed UHT milk can be efficiently conserved for up to 30h in 3L or 5L isothermal boxes, containing recyclable ice. But it is pointed out that the 5L-box presents larger safety for the sperm viability, because it is more efficient in the maintenance of a constant temperature.