Ética e condicionamento de animais selvagens para a aplicação de técnicas de reprodução assistida / Ethics and conditioning of wild animals for the application of assisted reproduction techniques

A relação humano-animal é conhecida desde o período Mesolítico e vem evoluindo ao longo dos anos pela busca de interações cada vez mais positivas. A forma como os animais e os ambientes são tratados abre muitas discussões éticas e morais em várias correntes filosóficas. O fato é que a biodiversidade vem apresentando uma redução expressiva dentro de um contexto de sexta extinção em massa do planeta e, para muitas espécies, trabalhar manejo populacional envolvendo coleções ex situ, seria a única alternativa de sobrevivência. As informações sobre a biologia das espécies selvagens ainda são muito insipientes e, portanto, faz-se necessário desenvolver pesquisas básicas que colaborem com as Técnicas de Reprodução Assistida (ARTs). A possibilidade de se trabalhar dentro do conceito de Conservação Única possibilita uma interconexão entre os planos de ação in situ e ex situ, as ações antrópicas no meio ambiente (sustentabilidade) e as pesquisas que englobam a conservação. Para tanto, utilizar as técnicas de condicionamento, que são prática voltada ao manejo de bem-estar, associadas às biotecnologias reprodutivas tem sido um manejo cada vez mais utilizado, com sucesso para muitas espécies ameaçadas. Animais manejados dentro dos protocolos de boas práticas são grandes candidatos a fazer parte dos programas de conservação integrada.(AU)

The human-animal relationship has been known since the Mesolithic period and has evolved over the years in search of increasingly positive interactions. How animals and environments are treated opens up many ethical and moral discussions in various philosophical currents. The fact is that biodiversity has been showing a significant reduction within the context of the sixth mass extinction on the planet, and, for many species, working on population management involving ex situ collections would be the only alternative for survival. Information on the biology of wild species is still very incipient; therefore, it is necessary to develop basic research that collaborates with Assisted Reproduction Techniques (ARTs). Working within the One Conservation concept enables an interconnection between in situ and ex situ action plans, anthropic environmental actions (sustainability), and research that encompasses conservation. Therefore, using conditioning techniques­a practice aimed at welfare management­associated with reproductive biotechnologies has been increasingly used, with success for many endangered species. Animals handled within suitable practice protocols are great candidates to be part of integrated conservation programs.(AU)

Cryopreservation of jaguar (Panthera onca) sperm cells using different cryoprotectants and different thawing temperatures

The cryopreservation of jaguar semen must be improved to produce high-quality biobanking doses. Until now, the rare studies of semen freezing in the species have only evaluated glycerol, always with a significant reduction in sperm quality in thawed semen. The purpose of this study was to assess the efficacy of three cryoprotectants, dimethylsulfoxide (DMSO), glycerol (GLY), and methanol (MET), in the cryopreservation of jaguar semen in an LDL-based extender, as well as the effect of thawing temperature on dosage quality. Five mature males with a history of reproduction were used. On the males, an infrared thermal image (IRT) was captured, the spicules and testes were analyzed, and the CASA system was used to evaluate the quality of fresh and thawed sperm. The superficial IRT was 4.6 ± 1.2 °C cooler than the anal sphincter, and the semen measured between 27.3 and 28.7 °C shortly after exiting the urethra. The total motility of fresh sperm was 55.3 ± 22.6%, and progressive motility was 36.3 ± 18%. The total motility of thawed sperm was 5.28 ± 2.51%, 4.49 ± %2.49, and 0.51 ± 0.62% for DMSO, GLY, and MET, respectively. DMSO and GLY performed better than MET, and there was no difference in thawing temperature (37°C 30 s vs. 50°C 12 s). All animals exhibit a considerable level of morphological changes in sperm. Low amounts of total and progressive motility were found in the thawed sperm. Males with a high level of sperm morphological changes were found to be fertile, but the lone male with normospermia was infertile. Thus, we contest the applicability of the commonly used morphological classification for bovines to felid species.(AU)

Surgical sterilization of free-ranging capybaras (Hydrochoerus hydrochaeris): "Passos Nunes" uterine horn ligature

Capybaras are the primary hosts of Amblyomma sculptum tick, vectors of Rickettsia rickettsia bacteria, and the zoonotic agent of Brazilian Spotted Fever (BSF). In this context, contraceptive methods have been suggested for population control in order to reduce the number of free-ranging capybaras cohabiting with humans in urban and rural areas and acting as disease amplifiers. To maintain the group's expected behavior and social hierarchy, sterilization techniques that preserve the gonads are recommended. On 126 female capybaras in the Brazilian state of São Paulo, a new surgical technique named "Passos Nunes" uterine horn ligature was performed after adequate general anesthesia. It achieved effective surgical sterilization, with an incision length of about 3 cm in the periumbilical linea alba, cranial to the pubis. After entering the abdominal cavity, the urinary bladder is pulled laterally to access the uterine horns and the cervix. The uterine horn is folded up, forming a strap; the distal portion of the strap is ligated and its distal end sectioned. The exact process is performed on the opposite horn. After the surgical procedure, the musculature is sutured in a sultan pattern and the subcutaneous tissue with a horizontal mattress pattern. The skin is sutured in a separate simple format, using nylon 2.0 for all steps. The wide exposure of the uterine horns facilitates the confirmation of pregnancy, allowing the surgeon to choose between salpingo hysterectomy or ligature of the uterine horns. The present study presents a new technique of surgical sterilization that can be used in female free-ranging mammals in which maintenance of the gonads is recommended, and births of offspring should not occur.(AU)

One Conservation: the integrated view of biodiversity conservation

Abstract The current global situation requires urgent decision-making to reverse processes of mass extinction of thousands of species. As a way of showing the importance of joint actions in this process, we aim to present the concept of One Conservation as a new proposal for the integration of sustainability, in situ and ex situ conservation for the restoration of ecosystems. According to the United Nations, we are beginning the decade of ecosystem restoration and in association with the International Union for Conservation of Nature guidelines, we can join efforts in the conservation of the planet. The survival of many species of wild animals depends on the management of populations currently maintained in ex situ conditions (under human care). To facilitate the exchange of genetic material between in situ and ex situ populations, reproductive biotechniques have become a great tool, making it possible to restore species in their natural environments. For effective conservation to occur, there must be an integrated view of the problem as a whole, and action for solutions must take place jointly by different spheres of society. Even more, conservation must be carried out by the public sector, the private sector, the third sector, and not less importantly, the agricultural sector. Therefore, One Conservation is defined as an interconnection between ex situ and in situ conservation plans, anthropic actions in the environment, and research in different areas that encompass conservation.

One Conservation: the integrated view of biodiversity conservation

The current global situation requires urgent decision-making to reverse processes of mass extinction of thousands of species. As a way of showing the importance of joint actions in this process, we aim to present the concept of One Conservation as a new proposal for the integration of sustainability, in situ and ex situ conservation for the restoration of ecosystems. According to the United Nations, we are beginning the decade of ecosystem restoration and in association with the International Union for Conservation of Nature guidelines, we can join efforts in the conservation of the planet. The survival of many species of wild animals depends on the management of populations currently maintained in ex situ conditions (under human care). To facilitate the exchange of genetic material between in situ and ex situ populations, reproductive biotechniques have become a great tool, making it possible to restore species in their natural environments. For effective conservation to occur, there must be an integrated view of the problem as a whole, and action for solutions must take place jointly by different spheres of society. Even more, conservation must be carried out by the public sector, the private sector, the third sector, and not less importantly, the agricultural sector. Therefore, One Conservation is defined as an interconnection between ex situ and in situ conservation plans, anthropic actions in the environment, and research in different areas that encompass conservation.(AU)

Avanços na andrologia de grandes felinos neotropicais / Advances in neotropical big cats andrology

O desenvolvimento de tecnologias reprodutivas e conhecimentos sobre a andrologia de grandes felinos caminham a pequenos passos, com avanços na última década. Estudos sobre o comportamento de onças-pintadas (Panthera onca) revelam as sequências de comportamentos sócio-sexuais e levantam a possibilidade de a ovulação poder ocorrer por estímulos sensoriais, e não somente pela estimulação mecânica durante a cópula. Um grande avanço na andrologia dos felinos foi o desenvolvimento da colheita farmacológica com α2-agonistas, que já se mostrou eficiente também nos grandes felinos neotropicais. Este foi um verdadeiro divisor de águas para a criopreservação de sêmen, especialmente em animais de vida livre. Pouco se avançou, no entanto, no meio de criopreservação, ainda hoje os meios indicados são à base de TRIS-gema-glicerol, porém a gema não é estável suficiente para uso em animais de vida livre, sendo necessária avaliação de substitutivos como as lipoproteínas de baixa densidade e lecitina de soja. O aprimoramento de reprodução assistida nos felinos neotropicais é pungente, em especial a onça-pintada visto que em alguns biomas a espécie está criticamente em perigo.

The development of reproductive technologies and knowledge about the andrology of big cats are taking small steps, with advances in the last decade. Studies on the sexual behavior of jaguars (Panthera onca) reveal the sequences of sexual behaviors and raise the possibility that ovulation may occur through sensory stimuli and not only through mechanical stimulation during copulation. A significant advance in feline andrology was the development of pharmacological semen collection with α2-agonists, which has proved efficient in neotropical big cats. It was disruptive for semen cryopreservation, especially in free-living animals. Little progress has been made; however, in the cryopreservation environment, even today, the indicated means are based on TRIS-yolk-glycerol. However, the yolk is not stable enough for use in free-living animals, requiring the evaluation of substitutes such as low-density lipoproteins and soy lecithin. The improvement of assisted reproduction in neotropical felines is poignant, especially the jaguar, since in some biomes, the species is critically endangered.

Uma visão integrada das biotecnologias reprodutivas com o conceito de One Conservation / An integrated view of reproductive biotechnologies with the One Conservation's concept

O preocupante status de conservação de milhares de espécies desencadeia uma força tarefa de conservação integrada para evitar o colapso da biodiversidade em muitos nichos ecológicos. A possibilidade de se trabalhar dentro do conceito de one conservation possibilita uma interconexão entre os planos de ação in situ e ex situ, as ações antrópicas no meio ambiente (sustentabilidade) e as pesquisas que englobam a conservação. Associar agricultura e a pecuária às formas sustentáveis de produção é uma maneira efetiva de contribuir com conservação da biodiversidade e dos recursos naturais, além de ofertar melhores meios de vida e bem-estar humano. As populações de fauna mantidas ex situ também são alvo dos programas de conservação integrada. É preciso trabalhar protocolos de manejo de bem-estar para que estes animais sejam viáveis dentro dos programas de reprodução que visam aumento da variabilidade genética para a espécie. Além disto, desafios constantes permeiam a reprodução destes animais, como o desconhecimento de informações comportamentais, fisiológicos, bem como seus mecanismos adaptativos frente a situações de mudanças. A possibilidade de armazenar informações genéticas de espécies silvestres, através dos bancos de germoplasma é uma forma de salvaguardar espécies até que as biotécnicas reprodutivas estejam com delineamentos bem eficazes para serem aplicadas.

The worrying conservation status of thousands of species triggers an integrated conservation task force to prevent biodiversity from collapsing in many ecological niches. The possibility of working within the One Conservation concept enables interconnection between in situ and ex situ action plans, anthropogenic actions in the environment (sustainability), and research that encompasses conservation. Associating agriculture and livestock with sustainable forms of production is an effective way to contribute to the conservation of biodiversity and natural resources and offer better livelihoods and human well-being. Integrated conservation programs also target ex situ fauna populations. It is necessary to work on welfare management protocols to make these animals viable within breeding programs to increase genetic variability for the species. In addition, constant challenges permeate the reproduction of these animals, such as the lack of behavioral and physiological information and their adaptive mechanisms in changing situations. The possibility of storing genetic information of wild species through germplasm banks is a way of safeguarding species until reproductive biotechniques have very effective designs to be applied.

Desafios para a reprodução assistida em animais de vida livre / Challenges for assisted reproduction in free-living animals

A reprodução assistida se faz necessária em programas de conservação de espécies ameaçadas de extinção, sendo um facilitador de transporte e troca de material genético. Neste contexto, o acesso ao material de animais de vida livre é essencial para incrementar o banco genético da espécie em questão, no entanto adaptar os métodos possíveis à realidade do campo torna essa área de pesquisa desafiadora. Ainda hoje os espermatozoides são os gametas mais acessados em animais de vida livre, porém com pouco uso efetivo para criopreservação e produção de filhotes. É pungente a necessidade de mais pesquisas nesta área, uma vez que há centenas de espécies brasileiras ameaçadas, com especificidades fisiológicas e que habitam habitats variados, o que demanda adaptações espécie-específicas e hábitat específicas.

Assisted reproduction is necessary for conservation programs for endangered species, facilitating transport and exchange of genetic material. In this context, access to material from free-living animals is essential to increase the genetic bank of the species in question. However, adapting the possible methods to the reality of the fieldwork makes this area of research a challenge. Even today, sperm are the most accessed gametes in free-living animals, but with little effective use for cryopreservation and production of offspring. The need for more research in this area is acute, as there are hundreds of Brazilian species under threat, with physiological specificities, and that inhabit varied habitats, which demand species-specific adaptations and specific habitats.

O que já sabemos sobre a reprodução de fêmeas de grandes felídeos? / What do we already know about the reproduction of big cats' females?

Os grandes felídeos são predadores de topo de cadeia com um papel essencial nos ecossistemas globais. O conceito de Conservação Única propõe a reprodução artificial como uma das ferramentas para reduzir a vulnerabilidade dessas espécies. Este manuscrito teve como objetivo avaliar o que há de novo na reprodução de grandes felídeos na última década. O conhecimento da fisiologia e do comportamento reprodutivo é o primeiro passo para o desenvolvimento de tecnologias reprodutivas em animais selvagens. Nos grandes felídeos, o comportamento copulatório é de fundamental importância, pois necessitam de mecanismos de indução da ovulação, que podem ser mecânicos, sensoriais ou via administração de hormônio luteinizante. O sucesso no cuidado neonatal representa o sucesso da tecnologia reprodutiva em fêmeas. Na última década, o sucesso da inseminação artificial foi relatado apenas em tigres-siberianos e leopardos da Anatólia, e a inseminação de onças-pintadas é foco de pesquisa do Instituto Reprocon, trocando material genético entre ambientes in situ e ex situ por meio de inseminação artificial. Para obter oócitos viáveis de alta qualidade, a técnica de escolha é a colheita de oócitos por laparoscopia. A produção de embriões in vitro enfrenta desafios para a maturação eficiente de oócitos e sua vitrificação eficiente. As técnicas reprodutivas precisam de estudos aprofundados em grandes felídeos para atingir a repetibilidade necessária para uma aplicação eficiente na conservação.

Big cats are apex predators with an essential role in global ecosystems. The One Conservation concept proposes artificial reproduction as one of the tools to reduce the vulnerability of these species. This manuscript aimed to assess what is new in big cat reproduction in the last decade. Knowledge of reproductive physiology and behavior is the first step towards developing reproductive technologies in wild animals. In big cats, copulatory behavior is of fundamental importance because they need ovulation induction mechanisms, which can be mechanical, sensory, or via the administration of the luteinizing hormone. The success in neonatal care represents the success of reproductive technology in females. In the last decade, successful artificial insemination was only reported in Siberian tigers and Anatolian leopards. Jaguar artificial insemination focuses on research at the Reprocon Institute, exchanging genetic material between in situ and ex situ environments thru artificial insemination. The technique of choice is laparoscopic ovum pick-up to obtain high-quality viable oocytes. The production of in vitro embryos faces challenges for the efficient maturation of oocytes and their efficient vitrification. Reproductive technologies need in-depth studies in big cats to achieve the repeatability necessary for efficient application in conservation.

Can detomidine replace medetomidine for pharmacological semen collection in domestic cats?

Abstract Among the different methods used for semen collection from domestic cats, the pharmacological collection by urethral catheterization becomes disruptive. Medetomidine is the elected α2-adrenoceptor agonist for that, but in several countries, it is not commercially available. This study aimed to evaluate the efficacy of detomidine compared to medetomidine in collecting semen by urethral catheterization in domestic cats. Urethral catheterization was performed on 13 mongrel cats using a disposable semi-rigid tomcat urinary catheter. Of the 19 semen collections performed with medetomidine induction, 94.7% were successful, while with detomidine induction, only 56.3% of 16 were successful. The values semen samples variables were as follows for volume - 10.56 ± 0.4 vs 8.88 ± 0.5 mL, motility - 171.67 ± 0.79 vs 49.77 ± 3.45%, vigor - 4.1 ± 0.03 vs 3.10 ± 0.1 and concentration - 3.24 ± 0.19 vs 2.15 ± 0.13 ×109 sperm/mL respectively for medetomidine and detomidine group. The failure in semen collections with detomidine was mainly due to azoospermic samples, poor urethral relaxation, insufficient volume, or contamination of urine. The sperm concentration was also lower in the detomidine group (P <0.05) when compared to medetomidine. However, when the volume of semen collected was compared, we found no statistical differences. Despite its low performance in collecting semen from cats, detomidine may be an alternative when medetomidine is not accessible.

Can detomidine replace medetomidine for pharmacological semen collection in domestic cats?

Among the different methods used for semen collection from domestic cats, the pharmacological collection by urethral catheterization becomes disruptive. Medetomidine is the elected α2-adrenoceptor agonist for that, but in several countries, it is not commercially available. This study aimed to evaluate the efficacy of detomidine compared to medetomidine in collecting semen by urethral catheterization in domestic cats. Urethral catheterization was performed on 13 mongrel cats using a disposable semi-rigid tomcat urinary catheter. Of the 19 semen collections performed with medetomidine induction, 94.7% were successful, while with detomidine induction, only 56.3% of 16 were successful. The values semen samples variables were as follows for volume - 10.56 ± 0.4 vs 8.88 ± 0.5 mL, motility - 171.67 ± 0.79 vs 49.77 ± 3.45%, vigor 4.1 ± 0.03 vs 3.10 ± 0.1 and concentration - 3.24 ± 0.19 vs 2.15 ± 0.13 ×109 sperm/mL respectively for medetomidine and detomidine group. The failure in semen collections with detomidine was mainly due to azoospermic samples, poor urethral relaxation, insufficient volume, or contamination of urine. The sperm concentration was also lower in the detomidine group (P<0.05) when compared to medetomidine. However, when the volume of semen collected was compared, we found no statistical differences. Despite its low performance in collecting semen from cats, detomidine may be an alternative when medetomidine is not accessible.(AU)

Cryptorchidism in free-living jaguar (Panthera onca): first case report

Cryptorchidism is a genital alteration wherein one or both testicles fail to descend into the scrotum and has multifactorial causes. A free-range adult male was captured twice in the Pantanal of Nhecolândia to put a GPS collar and semen collection. Pharmacological semen collection, andrological examination and semen analysis were performed. At the first capture and during the andrological examination only the left testis was found, and the male qualified as cryptorchid. The penis had no penile spines at either procedure. The semen volume obtained at first and second capture was 435 and 160 L, respectively, with a concentration of 618 and 100 x 106 sperm/mL, progressive motility of ~ 5% and ~ 1% and total morphological sperm abnormalities of 74% and 86%. The male was monitored by a GPS collar, but the signal was lost, making it difficult to re-captures and perform new seminal and ultrasound evaluations to discard monorchidism exceedingly rare in felids. Genetic studies to assess the individual's homozygosity are necessary to verify whether cryptorchidism in this individual has a genetic factor.

Cryptorchidism in free-living jaguar (Panthera onca): first case report

Cryptorchidism is a genital alteration wherein one or both testicles fail to descend into the scrotum and has multifactorial causes. A free-range adult male was captured twice in the Pantanal of Nhecolândia to put a GPS collar and semen collection. Pharmacological semen collection, andrological examination and semen analysis were performed. At the first capture and during the andrological examination only the left testis was found, and the male qualified as cryptorchid. The penis had no penile spines at either procedure. The semen volume obtained at first and second capture was 435 and 160 L, respectively, with a concentration of 618 and 100 x 106 sperm/mL, progressive motility of ~ 5% and ~ 1% and total morphological sperm abnormalities of 74% and 86%. The male was monitored by a GPS collar, but the signal was lost, making it difficult to re-captures and perform new seminal and ultrasound evaluations to discard monorchidism exceedingly rare in felids. Genetic studies to assess the individual's homozygosity are necessary to verify whether cryptorchidism in this individual has a genetic factor.(AU)