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)

Avanços na andrologia de animais selvagens / Advances in wild animal andrology

As tecnologias de reprodução assistida (TRA) são de fundamental importância para a conexão de indivíduos em diferentes localidades, facilitando assim o intercâmbio genético e favorecendo a variabilidade genética de uma espécie. Por esta razão, as TRAS podem ser ferramentas importantes para a conservação de espécies ameaçadas de extinção. Apesar dos esforços nas últimas décadas, o avanço no desenvolvimento de tais tecnologias está aquém à urgência de reverter processos de baixa variabilidade genética em algumas espécies. A necessidade de refinamento das técnicas para as particularidades fisiológicas e comportamentais de cada espécie, somada à raridade de acesso aos animais são os principais fatores relacionados as dificuldades em se avançar com as TRAS. As técnicas mais recentes desenvolvidas para a recuperação de espermatozoides em animais selvagens são a colheita farmacológica, com uso de alfa-2-agonistas e a criopreservação / vitrificação testicular com posterior cultivo. Pouco de avançou, no entanto, em relação aos métodos de criopreservação, prevalecendo associação clássica de TRIS-gema-glicerol. Discutimos, então os métodos usados para acesso ao gameta masculino em espécies selvagens e suas aplicações na conservação animal.(AU)

Assisted reproduction technologies (ART) are of fundamental importance for connecting individuals in different locations, thus facilitating genetic exchange and favoring the genetic variability of a species. For this reason, TRAS can be important tools for the conservation of endangered species. Despite efforts in recent decades, the advance in the development of such technologies is short of the urgency of reversing processes of low genetic variability in some species. The need to refine the techniques for the physiological and behavioral particularities of each species, added to the rarity of access to animals, are the main factors related to the difficulties in advancing with TRAS. The most recent techniques developed for sperm collection in wild animals are pharmacological collection, with the use of alpha-2-agonists and testicular cryopreservation / vitrification with subsequent cultivation. Little progress has been made, however, in relation to cryopreservation methods, prevailing the classic association of TRIS-yolk-glycerol. We therefore discuss the methods used to access the male gamete in wild species and their applications in animal 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)

Effectiveness of protected areas for jaguars: the case of the Taiamã Ecological Station in Brazil

The largest feline in the Americas and the third largest in the world, the jaguar is an apex predator in the food chain and a key species in the ecosystems where it occurs, developing important ecological functions in maintaining ecosystem balance. In Brazil, the Pantanal is considered an important refuge for the species, and protected areas such as the Taiamã Ecological Station (TES) are relevant for conservation of pristine ecosystem where the species persist. Thus, considering that this area is located in one of the regions with the highest concentration of this large cat in the Pantanal, the objective of this study was to evaluate the effectiveness of the protection given by the TES for the jaguar population. The station is a flooded island in the middle of the Paraguay river and surrounded by extensive wetlands. We monitored ten jaguars using GPS collar at the TES and its surroundings. The samples were separated into high-water season (January to June) and low-water season (July to December), and the estimated home ranges were grouped as: 1) residents only and 2) all monitored individuals. The stabilization of the home ranges of eight jaguars, considered residents, was determined through variograms. When all jaguars were analysed together there was 55% overlap between the clustered areas of the two analysed seasons. In the analysis excluding non-resident individuals there was 72% overlap between the clustered areas. The type of land cover inside these areas was very similar between these periods. The range of this protected area is not sufficient to effectively protect these jaguar populations, since the grouped home ranges of the resident animals studied are 3.5(wet)/2.5(dry) times larger than the area of the TES. However...

Effectiveness of protected areas for jaguars: the case of the Taiamã Ecological Station in Brazil

Abstract The largest feline in the Americas and the third largest in the world, the jaguar is an apex predator in the food chain and a key species in the ecosystems where it occurs, developing important ecological functions in maintaining ecosystem balance. In Brazil, the Pantanal is considered an important refuge for the species, and protected areas such as the Taiamã Ecological Station (TES) are relevant for conservation of pristine ecosystem where the species persist. Thus, considering that this area is located in one of the regions with the highest concentration of this large cat in the Pantanal, the objective of this study was to evaluate the effectiveness of the protection given by the TES for the jaguar population. The station is a flooded island in the middle of the Paraguay river and surrounded by extensive wetlands. We monitored ten jaguars using GPS collar at the TES and its surroundings. The samples were separated into high-water season (January to June) and low-water season (July to December), and the estimated home ranges were grouped as: 1) residents only and 2) all monitored individuals. The stabilization of the home ranges of eight jaguars, considered residents, was determined through variograms. When all jaguars were analysed together there was 55% overlap between the clustered areas of the two analysed seasons. In the analysis excluding non-resident individuals there was 72% overlap between the clustered areas. The type of land cover inside these areas was very similar between these periods. The range of this protected area is not sufficient to effectively protect these jaguar populations, since the grouped home ranges of the resident animals studied are 3.5(wet)/2.5(dry) times larger than the area of the TES. However, the surroundings of the TES have low human impact and no deforestation and, in this way, the landscape context of the station may have a significant effect on the persistence of a large carnivore like the jaguar. However, it is necessary to create more protected areas in order to prevent possible future environmental changes in the areas surrounding TES.

Effectiveness of protected areas for jaguars: the case of the Taiamã Ecological Station in Brazil

The largest feline in the Americas and the third largest in the world, the jaguar is an apex predator in the food chain and a key species in the ecosystems where it occurs, developing important ecological functions in maintaining ecosystem balance. In Brazil, the Pantanal is considered an important refuge for the species, and protected areas such as the Taiamã Ecological Station (TES) are relevant for conservation of pristine ecosystem where the species persist. Thus, considering that this area is located in one of the regions with the highest concentration of this large cat in the Pantanal, the objective of this study was to evaluate the effectiveness of the protection given by the TES for the jaguar population. The station is a flooded island in the middle of the Paraguay river and surrounded by extensive wetlands. We monitored ten jaguars using GPS collar at the TES and its surroundings. The samples were separated into high-water season (January to June) and low-water season (July to December), and the estimated home ranges were grouped as: 1) residents only and 2) all monitored individuals. The stabilization of the home ranges of eight jaguars, considered residents, was determined through variograms. When all jaguars were analysed together there was 55% overlap between the clustered areas of the two analysed seasons. In the analysis excluding non-resident individuals there was 72% overlap between the clustered areas. The type of land cover inside these areas was very similar between these periods. The range of this protected area is not sufficient to effectively protect these jaguar populations, since the grouped home ranges of the resident animals studied are 3.5(wet)/2.5(dry) times larger than the area of the TES. However...(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)

Comparação entre duas concentrações de glicerol para a criopreservação de sêmen de suçuarana (Puma concolor) / Comparison between two glycerol concentrations to cryopreservation of semen of mountain lions (Puma concolor)

O desenvolvimento de biotécnicas de reprodução é uma importante ferramenta para a conservação de animais silvestres ameaçados de extinção. Procedimentos de reprodução assistida em suçuarana, no entanto, são escassos na literatura, em especial aqueles relacionados à criopreservação de sêmen. Neste sentido, o presente trabalho objetivou avaliar a congelabilidade do sêmen de suçuaranas adultas mantidas em cativeiro, por meio da comparação entre duas concentrações de glicerol no meio de congelamento. Foram usados cinco machos adultos de suçuarana, mantidos no Centro de Reabilitação de Animais Silvestres do Mato Grosso do Sul (CRAS/MS). As amostras foram coletadas por eletroejaculação e avaliadas quanto ao seu aspecto físico, volume, vigor, motilidade, concentração e índice espermático. De cada ejaculado duas alíquotas foram diluídas em meio Tris-citrato-gema de ovo, em concentrações finais de 5 e 7,5% de glicerol, resfriadas a uma taxa de -0,55ºC/min e congeladas a uma taxa de -5,8ºC/min. Depois de descongeladas, as amostras foram reavaliadas e submetidas aos testes de termorresistência e hiposmótico. O protocolo de criopreservação e descongelamento de sêmen proposto se mostrou eficiente em ambas as concentrações de glicerol testadas, não havendo diferença (p>0,05) entre estas.(AU)

The development of biotechnologies of reproduction is an important tool for the conservation of wild animals threatened with extinction. Assisted reproduction procedures in mountain lions, however, are scarce, especially those related to sperm cryopreservation. In this context, this study aimed to evaluate the freezing capacity of semen from adult mountain lions in captivity through the comparison of two concentrations of glycerol in the freezing media. Five adult male mountain lions were used, held at the Rehabilitation Center for Wild Animals of Mato Grosso do Sul (CRAS/MS). Samples were collected by electroejaculation and evaluated for physical appearance, volume, sperm progressive status, sperm motility, sperm concentration and sperm motility index. Each ejaculate was spliced into two aliquots and diluted in Tris-citrate-half egg yolk, at final concentrations of 5 and 7.5% glycerol, cooled at a rate of -0.55ºC/min and frozen at a rate of -5.8ºC/min. Once thawed, the samples were re-evaluated and tested for thermoresistance and hypoosmotic swelling. The suggested protocol for cryopreservation and thawing of semen is efficient in both glycerol concentrations tested, with no difference (p>0.05) between them.(AU)

USO DE SONDAS FLUORESCENTES E ENSAIO DE LIGAÇÃO A OVÓCITOS HETERÓLOGOS E A MEMBRANA PERIVITELÍNICA DE OVO DE GALINHA (Gallus gallus) PARA A AVALIAÇÃO DE ESPERMATOZOIDES FRESCOS E DESCONGELADOS DE JAGUATIRICA (Leopardus pardalis)

ARAUJO, Gediendson Ribeiro de, M. Sc., Universidade Federal de Viçosa, fevereiro de 2012. Uso de sondas fluorescentes e ensaio de ligação a ovócitos heterólogos e a membrana perivitelínica de ovo de galinha (Gallus gallus) para a avaliação de espermatozoides frescos e descongelados de jaguatirica (Leopardus pardalis). Orientador: Tarcízio Antônio Rego de Paula. Coorientador: José Domingos Guimarães. Estratégias de conservação ex situ objetivam auxiliar na manutenção de populações geneticamente viáveis, por meio de estratégias de reprodução assistida e criopreservação de materiais genéticos. Objetivou-se a validação do uso de combinação de sondas fluorescentes e do teste de ligação à zona pelúcida de ovócitos heterólogos criopreservados na qualificação de sêmen fresco e descongelados de jaguatiricas. Foram usadas três jaguatiricas mantidas em cativeiro, sendo obtidos ejaculados por meio de eletroejaculação e avaliados por testes clássicos de rotina. Através destes testes (vigor e motilidade espermáticos, hiposmótico e morfologia) observou-se queda (p<0,05) dos padrões qualitativos do sêmen descongelado em relação ao sêmen fresco. A associação das sondas Iodeto de Propídeo, Hoechst 33342 e Pisum Sativum Agglutinin conjugada com Lectina Fluorescente (FITC-PSA), permitiu a identificação de subpopulações espermáticas de acordo com a localização específica de lesões celulares e detectou de forma mais acurada o efeito deletério da criopreservação no sêmen. O uso de ovócitos criopreservados de gatas domésticas permitiu a observação da queda da capacidade ligante (p<0,05) dos espermatozoides descongelados em relação aos espermatozoides a fresco de jaguatiricas, havendo ainda correlação entre o total de ligantes nas amostras de espermatozoides a fresco com padrões qualitativos no sêmen após o descongelamento. A combinação de sondas fluorescentes utilizadas e o uso de ensaio de ligação a ovócitos heterólogos criopreservados de gatas domésticas foram mais específicos e confiáveis na detecção da queda na qualidade espermática de jaguatiricas após o descongelamento. Os espermatozóides de jaguatiricas também foram capazes de se ligar à membrana perivitelínica em ambos os tratamentos. Apesar de não observada diferença na quantidade de espermatozóides ligados nos tratamentos (p>0,05), houve correlação positiva (r= 0,91; p<0,05) entre eles. Foi observada ainda correlação negativa entre a taxa de espermatozoides ligados na membrana perivitelínica no sêmen a fresco e no descongelado com a população de espermatozóides com cauda dobrada no sêmen descongelado (r= -0,81; p<0,05 e r= -0,86; p<0,05, respectivamente) e correlação negativa (r= -0,71; p< 0,05) entre a população de espermatozóides com cauda fortemente dobrada (defeito maior) com a quantidade de espermatozóides ligados na membrana perivitelínica, ambos no sêmen descongelado. Apesar dos espermatozoides de jaguatirica terem se ligado de forma satisfatória à membrana perivitelínica, o reduzido número de amostras avaliadas não permitiu demonstrar correlação com a fertilidade do espermatozoide. No entanto, o mecanismo suporte desenvolvido para a membrana permitiu a manutenção do seu estiramento durante o processamento e a definição de uma área de avaliação. Sendo assim, estudos futuros, com uma maior amostragem, são necessários para a efetiva validação desta técnica. ABSTRACT ARAUJO, Gediendson Ribeiro de, M. Sc., Universidade Federal de Viçosa, February, 2012. Using fluorescent probes and sperm bin