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)

Viability of Toxoplasma gondii in cattle semen cryopreserved with different concentrations of cryoprotectant / Viabilidade do Toxoplasma gondii em sêmen de bovino criopreservado em diferentes concentrações de crioprotetores

ABSTRACT: Toxoplasma gondii can be eliminated in bovine semen. Cryopreserved semen is often used due to the fact that artificial insemination in dairy and beef cattle provides benefits in terms of production. However, little is known regarding the viability and infectivity of T. gondii tachyzoites in cryopreserved bovine semen. In the present study, cattle semen negative for T. gondii were contaminated with 1 x 106 tachyzoites (RH strain) and cryopreserved with and without different cryoprotectants, such as DMSO (concentrations of 2.5%, 5.0%, 7.5%, 8.0% and 10.0%) and glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%), followed by freezing in liquid nitrogen (-196°C). After 24 hours, the samples were thawed and inoculated in 10 mice per cryoprotectant concentration. The mice were evaluated for clinical signs of toxoplasmosis (rough coat, diarrhea, hypoactivity and sudden death) as well as serum titers of IgM and IgG and the presence of tachyzoites in the peritoneal lavage. The results revealed that T. gondii remained infective in all samples. Clinical signs of toxoplasmosis were observed in the mice beginning with the 6th day post-inoculation (DPI) and 100% lethality was found between the 7th and 9th DPI. Viable tachyzoites were recovered from peritoneal exudate of dead mice (except for the control group), with higher mean of tachyzoite counts in the intraperitoneal lavage for 5% DMSO (±3.32 x 106), 8% DMSO (±3.53 x 106), 3% glycerol (±4.75 x 106), 7.5% glycerol (±6.26 x 106) and the absence of cryoprotectant (±3.11 x 106). Seroconversion occurred in the treated groups, with titers of IgG from 1:16 to 1:128 and IgM from 1:16 to 1:512. T. gondii viability and infectivity were maintained in cattle semen during 24 hours of cryopreservation at -196°C with and without cryoprotectant. However, further studies are necessary to determine whether cryopreserved semen contributes to the spread of toxoplasmosis through artificial insemination.

RESUMO: Sabe-se que Toxoplasma gondii pode ser eliminado no sêmen bovino. A inseminação artificial em bovinos leiteiros e de corte proporcionou avanços e benefícios nas produções e para isso o sêmen criopreservado é frequentemente utilizado. No entanto, pouco se sabe sobre a viabilidade e infectividade dos taquizoítos de T. gondii em sêmen bovino criopreservado. Para isso o sêmen bovino, negativo para T. gondii, foi contaminado com 1x106 taquizoítos (cepa RH), criopreservados com ou sem diferentes crioprotetores como DMSO (2.5%, 5.0%, 7.5%, 8.0% e 10.0%) e Glicerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% e 10.0%) e congelados em nitrogênio líquido (-196°C). Após 24 horas, essas amostras foram descongeladas e inoculadas em 10 camundongos por diluente de concentração de crioprotetor. Os camundongos foram avaliados quanto a sinais clínicos de toxoplasmose (pele áspera, diarreia, hipoatividade e morte súbita), títulos séricos de IgM e IgG e presença de taquizoítos no lavado peritoneal. Os resultados mostraram que T. gondii se manteve infectante em todas as amostras, inclusive naquelas sem crioprotetor. Sinais clínicos de toxoplasmose foram observados nos camundongos a partir do 6º dia pós-inoculação (DPI) e 100% de letalidade foi verificada entre o 7º ao 9º DPI. Nos camundongos mortos, exceto no grupo controle, taquizoítos viáveis foram recuperados do exsudato peritoneal, com maior média de taquizoítos quantificados na lavagem intraperitoneal para DMSO a 5% (±3.32x106), 8% (±3.53x106) e glicerol 3% (±4.75x106), 7,5% (±6.26x106) e livre de crioprotetor (±3.11x106). A soroconversão ocorreu nos grupos tratados com títulos de IgG (1:16 a 1:128) e IgM (1:16 a 1:512). A viabilidade e infectividade do T. gondii no sêmen bovino durante as 24 horas de criopreservação a -196°C foram mantidas com ou sem crioprotetor. No entanto, mais estudos são necessários para verificar se o sêmen criopreservado contribui para a disseminação da toxoplasmose na inseminação artificial.

Comparação entre diferentes técnicas empregadas na conservação e manutenção de peças anatômicas / Comparison between different techniques used in the conservation and maintenance of anatomical parts

Dentre as diversas formas de obter a conservação de peças anatômicas para estudo, a formolização é a mais utilizada. Esta técnica altera a coloração e textura, dificultando o processo de ensino-aprendizagem. Além disso, proporciona um ambiente insalubre que pode comprometer a saúde de indivíduos expostos constantemente ao formol. Existem técnicas alternativas como a glicerinação, criodesidratação e corrosão, as quais reduzem os riscos à saúde ocasionados pela exposição aos materiais, proporcionam um ambiente livre de odores desagradáveis e facilitam a manipulação e o armazenamento das peças, quando comparadas com a formolização. O presente trabalho visa à apresentação de técnicas alternativas para a conservação e manutenção de peças anatômicas e seus devidos procedimentos, buscando a substituição da utilização do formol como forma principal de conservação. A técnica de glicerinação apresenta grande eficiência quanto à preservação das características de coloração e formato das vísceras, além de ser uma técnica de baixo custo e não apresentar riscos à saúde, assim como a técnica de criodesidratação, a criodesidratação difere em relação à tonalidade das vísceras, entretanto demonstra facilidade quanto à conservação, armazenamento e manutenção das mesmas. Já a injeção e corrosão de órgãos facilitam a visualização da arquitetura interna de órgãos ocos, além de permitir fazer o modelo de vascularização de órgãos. No entanto, esta pode ser considerada uma técnica cara a depender do polímero utilizado.

Among the several ways to obtain the conservation of anatomical parts for study, formalization is the most used. This technique changes the color and texture, making the teaching-learning process difficult. In addition, it provides an unhealthy environment that may compromise the health of individuals constantly exposed to formaldehyde. There are alternative techniques such as glycerination, cryodehydration, and corrosion, which reduce health risks caused by exposure to materials, provide an environment free of unpleasant odors and facilitate handling and storage of parts, when compared to formalization. This work aims to present alternative techniques for the conservation and maintenance of anatomical parts and their proper procedures, seeking to replace the use of formaldehyde the main form of conservation. The glycerination technique presents great efficiency in preserving the viscera's color and shape characteristics, in addition to being a low-cost technique and not presenting health risks, as well as the cryodehydration technique. The cryodehydration differs in relation to the tonality of the viscera; however, it demonstrates convenience in terms of their conservation, storage, and maintenance. On the other hand, the injection and corrosion of organs facilitate the visualization of the internal architecture of hollow organs, in addition to allowing the model of organ vascularization. However, it can be considered an expensive technique depending on the polymer used.

Glycerin supplementation strategies for three or seven days affects oxidative stress, follicle dynamics and ovulatory response in Morada Nova sheep

The Ziwuling black goat is an indigenously in China, their offspring are frequently affected by congenital cryptorchidism. The extracellular matrix (ECM) contains cytokines and growth factors that regulate the development of the testis, and component changes often result in pathological changes. Cryptorchidism is closely related to structural changes in ECM. In this study, the histochemical staining, immunohistochemical, immunofluorescence and Western blot combined with semi-quantitative analysis was used to describe the distribution of the important ECM components Collagen type IV (Col IV), laminin (LN)and heparan sulfate proteoglycans (HSPG) in the normal and cryptorchid testes of Ziwuling black goats. Results showed that: The histochemical staining showed that the dysplasia of seminiferous tubules and decreased number of Sertoli cells in cryptorchidism, as well as sparse collagen fiber. Meanwhile, the distribution of reticular fibers is relatively rich. Furthermore, the PAS and AB staining in the interstitial vessels and lamina propria of seminiferous tubules is weak. The immunohistochemical and immunofluorescence revealed that Col IV, LN was strongly expressed in Leydig, Sertoli cells of normal testes and moderately positive in the spermatogonia and spermatids, but HSPG was not expressed in the spermatogonia. However, cryptorchidism, the expression of Col IV, LN and HPSG in Leydig, Sertoli cells significantly decreased, as well as the expression of Col IV and LN in capillary endothelial cells, but HSPG was moderately expressed in spermatogonia. Based on these data, the underdevelopment of spermatogenic epithelium, decreased synthesis function of collagen fibers and Leydig cells develop usually in the cryptorchidism were shown to be closely related to the abnormal metabolism of Col IV and LN. The positive expressed of HSPG in the spermatogonia of cryptorchid testes is related to the compensatory development of spermatogonia.(AU)

Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality

Although cryopreservation is an efficient method for maintaining the biological and genetic resources of sperm, the sperm damage during the cryopreservation process cannot be ignored. It should be possible to obtain the most effective cryopreservation performance by accurately grasping the effects of various factors on the cryopreservation of sperm. The previous study demonstrated that a suitable standard protocol for cryopreservation of Korean native brindled cattle (Chikso) does not exist, based on the methods for semen cryopreservation of Chikso differ in each research center. The most obvious difference between most of protocols is the addition of glycerol before and after cooling during the Chikso cryopreserved semen process. Therefore we focused on the effects of glycerol addition time on the quality of cryopreserved Chikso sperm. In the present study, 27 individual Chikso samples were collected by transrectal massage and divided into two parts: the "cryopreservation method A" group (adding glycerol before cooling) and the "cryopreservation method B" group (adding glycerol after cooling). Meanwhile, the values of various sperm parameters were derived from each group, including sperm motility, kinematics, capacitation status, cell viability, and intracellular ATP levels, which we used to compare and evaluate sperm function. The results of this study indicated that during the semen cryopreservation process of the Chikso, the addition of glycerol after cooling yielded superior results in a variety of sperm parameters, such as sperm motility, progressive motility, rapid motility, VCL, VSL, VAP, ALH, capacitation status, viability, and intracellular ATP level after freezing and thawing. Our study is suggested that the glycerol addition time during the cryopreservation process for Chikso should be considered. In addition, our results may be provided reference to develop suitable the cryopreservation procedure of the Chikso sperm.(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)

Seed and seedling morphology of Bauhinia scandens L / Morfologia de semente e plântula de Bauhinia scandens L

Bauhinia scandens has potential importance as an ornamental and medicinal plant. Researchers have isolated and identified 1-O-alkylglycerol in the leaves of the B. scandens plant, and established antitumor properties using the Brine Shrimp toxicity test, an internationally accepted bioassay. Although this species has high potential, little is known about the viability of seedling production and the morphology of these plants, particularly in terms of seed characteristics and initial stages of germination. The objective of this study was to characterize the seed morphology, germination, and seedlings of B. scandens. Seed water content, weight, and coloration were evaluated. This study also included a description of seed biometrics, external and internal structures, germination, and seedling morphology. Internal seed morphology was evaluated by the anatomical sectioning and X-ray methods. The morphology data obtained were subjected to descriptive statistical analysis and germination data were determined using Cramér's V. B. scandens seeds have a coloration ranging from very dark grayish-red to dark reddish-brown, flat oblong shapes, and rounded bases and apexes with full or slightly undulating margins. Healthy seedlings are produced mainly by seeds with well-formed internal structures. The reddish petiole of the seedling leaves is a taxonomic character for B. scandens identification. The non-domestication and genetic variability of this species reflect on the seed and seedling color and size variation.(AU)

Bauhinia scandens tem potencial como planta ornamental e importância medicinal. Nas folhas da planta de B. scandens, foi relatado pelos pesquisadores, o isolamento e identificação de glicerol 1-O-alquilo, os mesmos estabeleceram a propriedade antitumoral por um bioensaio, aceito internacionalmente denominado teste de toxicidade de Brine Shrimp. Por ser ainda pouco conhecida e com alto potencial exploratório, a viabilidade da produção de mudas e maior conhecimento sobre sua morfologia se faz necessária. Faltam informações sobre as características das sementes e estágios iniciais de germinação. O objetivo deste estudo foi caracterizar o tipo morfológico de sementes, germinação e plântulas de B. scandens. Foram avaliados o teor de água, o peso de mil sementes e a coloração das sementes. Este estudo também incluiu a descrição e biometria da semente, estrutura (externa e interna), germinação e morfologia das plântulas. A morfologia interna das sementes foi avaliada por corte anatômico e método de raios-X. Os dados morfológicos obtidos foram submetidos à análise estatística descritiva e os dados de germinação foram testados utilizando Cramér's V. B. scandens apresentam coloração que varia de vermelho acinzentado muito escuro a marrom avermelhado escuro e o tipo de semente é de forma oblonga plana, base e ápice são arredondados e cheios ou margens ligeiramente onduladas. Plãntulas normais são produzidas majoritariamente por sementes com uma estrutura interna bem formada. O pecíolo avermelhado das folhas da plântula é um caráter taxonômico para identificação de B. scandens. A não domesticação e a variabilidade genética desta espécie refletem na variação da cor e tamanho das sementes e plântulas.(AU)

Uso da galactose na vitrificação de sêmen ovino em palhetas / Use of galactose to vitrify ram semen in straws

A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

Effects of glycerol and sodium pentaborate containing new formulation on sleeve gastrectomy model in rats

ABSTRACT Purpose: The development of cutting surface leakage and postoperative peritoneal adhesions (PPA) after sleeve gastrectomy (SG) are the most serious operative complications. We investigated the effectiveness of the newly developed glycerol and sodium pentaborate containing formulation on the prevention of these complications. Methods: Sixteen Sprague Dawley rats (mean weight 310 ± 50 g, mean age 3 months old) were divided into two groups, consisting of eight rats in each. SG and a double-layer suture technique were performed for each group. In study group, there was the mixture of 2 mL 3% glycerol plus 3% sodium pentaborate formulation, and in the control group 2 mL 0.9% NaCl was injected into the peritoneal cavity. Rats were sacrificed after 30 days, then macroscopic adhesion grade scoring and histopathological evaluations were assessed. Results: Macroscopic PPA scores in the control and study groups were 2.75 ± 0.16 and 1.50 ± 0.327, respectively (p = 0.004). Histopatologic fibrosis scores in the control and study groups were 0.87 ± 0.125 and 2.00 ± 0.26, respectively (p = 0.002). Conclusions: In SG operation model, glycerol plus sodium pentaborate compound decreased PPA formation and also increased stomach cut surface line fibrosis. This new formulation is hopeful for more safe SG operations.

Uso da túnica vaginal canina conservada em glicerina a 98% como curativo biológico no tratamento de feridas cutâneas produzidas em ratos Wistar / Use of canine vaginal tunic preserved in 98% glycerin as biological curative in the treatment of skin produced in wound Wistar rats

Na procura por material alternativo no tratamento de feridas cutâneas, buscou-se com este estudo avaliar o comportamento da túnica vaginal canina conservada em glicerina a 98% como curativo biológico no tratamento de feridas cutâneas provocadas experimentalmente em ratos. Foram utilizados 16 ratos Wistar, nos quais foram induzidas experimentalmente duas lesões cutâneas na região dorsal, sendo uma cranial e a outra caudal. Uma ferida serviu de controle, a qual não recebeu tratamento, a outra recebeu a túnica vaginal recobrindo toda extensão da lesão. A seleção de qual das feridas, cranial ou caudal, iria receber a membrana foi feita de forma aleatória. Os animais foram distribuídos em quatro grupos de igual número para análises clínicas, macroscópicas e histológicas decorridos três, sete, 14 e 21 dias de pós-operatório. Na avaliação clínica e macroscópica pode-se observar que o processo de reparação teve evolução semelhante tanto no grupo controle quanto no grupo membrana. Na análise histopatológica foi constatada grande quantidade de infiltrado inflamatório nos períodos iniciais, no entanto, decorridos 21 dias, a epiderme já se encontrava reparada, e em todos os momentos foi observada maior presença de tecido conjuntivo no grupo membrana, sugerindo maior precocidade na cicatrização. Deste modo, concluiu-se que a túnica vaginal canina pode ser utilizada como nova alternativa no tratamento de feridas cutâneas.(AU)

In the search for alternative material in the treatment of cutaneous wounds, this study aimed to evaluate the behavior of canine vaginal tunic conserved in glycerin 98% as a biological dressing in the treatment of cutaneous wounds provoked experimentally in rats. Sixteen animals were used in which two cutaneous lesions were caused in the dorsal region, one cranial and the other caudal. One wound served as a control which did not receive treatment, the other received the vaginal tunic covering all extension of the lesion. The selection of which of the wounds, cranial or caudal, would receive the membrane was made at random. The animals were divided into four groups of equal numbers for clinical, macroscopic, and histological analyzes after three, seven, 14 and 21 postoperative days. In the clinical and macroscopic evaluation, it can be observed that the healing process had similar evolution in both the control group and the membrane group. In the histopathological analysis, a large amount of inflammatory infiltrate was observed in the initial periods; however, after 21 days the epidermis was already healed, and at all times a greater presence of connective tissue was observed in the membrane group, suggesting a greater precocity in healing. Thus, it was concluded that the canine vaginal tunic can be used as a new alternative in the treatment of cutaneous wounds.(AU)

Uso da túnica vaginal canina conservada em glicerina a 98% como curativo biológico no tratamento de feridas cutâneas produzidas em ratos Wistar / Use of canine vaginal tunic preserved in 98% glycerin as biological curative in the treatment of skin produced in wound Wistar rats

Na procura por material alternativo no tratamento de feridas cutâneas, buscou-se com este estudo avaliar o comportamento da túnica vaginal canina conservada em glicerina a 98% como curativo biológico no tratamento de feridas cutâneas provocadas experimentalmente em ratos. Foram utilizados 16 ratos Wistar, nos quais foram induzidas experimentalmente duas lesões cutâneas na região dorsal, sendo uma cranial e a outra caudal. Uma ferida serviu de controle, a qual não recebeu tratamento, a outra recebeu a túnica vaginal recobrindo toda extensão da lesão. A seleção de qual das feridas, cranial ou caudal, iria receber a membrana foi feita de forma aleatória. Os animais foram distribuídos em quatro grupos de igual número para análises clínicas, macroscópicas e histológicas decorridos três, sete, 14 e 21 dias de pós-operatório. Na avaliação clínica e macroscópica pode-se observar que o processo de reparação teve evolução semelhante tanto no grupo controle quanto no grupo membrana. Na análise histopatológica foi constatada grande quantidade de infiltrado inflamatório nos períodos iniciais, no entanto, decorridos 21 dias, a epiderme já se encontrava reparada, e em todos os momentos foi observada maior presença de tecido conjuntivo no grupo membrana, sugerindo maior precocidade na cicatrização. Deste modo, concluiu-se que a túnica vaginal canina pode ser utilizada como nova alternativa no tratamento de feridas cutâneas.

In the search for alternative material in the treatment of cutaneous wounds, this study aimed to evaluate the behavior of canine vaginal tunic conserved in glycerin 98% as a biological dressing in the treatment of cutaneous wounds provoked experimentally in rats. Sixteen animals were used in which two cutaneous lesions were caused in the dorsal region, one cranial and the other caudal. One wound served as a control which did not receive treatment, the other received the vaginal tunic covering all extension of the lesion. The selection of which of the wounds, cranial or caudal, would receive the membrane was made at random. The animals were divided into four groups of equal numbers for clinical, macroscopic, and histological analyzes after three, seven, 14 and 21 postoperative days. In the clinical and macroscopic evaluation, it can be observed that the healing process had similar evolution in both the control group and the membrane group. In the histopathological analysis, a large amount of inflammatory infiltrate was observed in the initial periods; however, after 21 days the epidermis was already healed, and at all times a greater presence of connective tissue was observed in the membrane group, suggesting a greater precocity in healing. Thus, it was concluded that the canine vaginal tunic can be used as a new alternative in the treatment of cutaneous wounds.

Uso da galactose na vitrificação de sêmen ovino em palhetas / Use of galactose to vitrify ram semen in straws

A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

Butter whey and corn steep liquor as sole raw materials to obtain a bioemulsifier from Yarrowia lipolytica for food oil-in-water emulsions / Soro de manteiga e milhocina como únicas matérias primas para obter bioemulsificante de Yarrowia lipolytica para emulsões óleo-em-água alimentícias

A synthetic medium containing glucose, glycerol, yeast extract (YE), and ammonium sulfate (AS) was compared to several low-cost media in their ability to produce high emulsification index (EI). The goal was to reduce the production costs of an emulsifier with application in food oil-in-water emulsions. To this end, agro-industrial by-products were screened for bioemulsifier production from Yarrowia lipolytica. The statistical analysis showed that the EIs of media containing residual frying oil from palm oil (RFO_palm) or soybean oil (RFO_soy), residual liquid from butter production (butter whey, BWhey) or cheese production (cheese whey, CWhey), supplemented with YE and AS were similar to the EI of the synthetic medium. The replacement of YE by corn steep liquor (CSL) also resulted in similar EI, except for RFO_soy. BWhey was tested with CSL without AS and similar EI (66.8%) was detected in comparison to that of the same medium with AS (66.3%). The cell-free broth obtained after Y. lipolytica growth in BWhey+CSL was successfully used to obtain vegetable oil-in-water emulsions indicating its potential application in food products.(AU)

O meio sintético contendo glicose, glicerol, extrato de levedura (YE) e sulfato de amônio (AS) foi comparado a meios de baixo custo para produzir alto índice de emulsificação (EI). Para este fim, os subprodutos agroindustriais foram rastreados quanto à produção de bioemulsificante por Yarrowia lipolytica. A análise estatística mostrou que EI de meios contendo óleo de fritura residual de óleo de palma (RFO_palm) ou óleo de soja (RFO_soy), líquido residual da produção de manteiga (soro de manteiga, BWhey) ou da produção de queijo (soro de queijo, CWhey), suplementado com YE e AS foram semelhantes ao EI do meio sintético. A substituição do YE por milhocina (CSL) também resultou em EI semelhante, exceto no RFO_soy. O BWhey foi testado com CSL sem AS e EI semelhante (66,8%) foi detectado em comparação com o mesmo meio com AS (66,3%). O meio isento de células obtido após o crescimento de Y. lipolytica no meio BWhey + CSL foi utilizado com sucesso para obter emulsões de óleo vegetal em água, indicando sua potencial aplicação em produtos alimentícios.(AU)

Seed and seedling morphology of Bauhinia scandens L / Morfologia de semente e plântula de Bauhinia scandens L

Bauhinia scandens has potential importance as an ornamental and medicinal plant. Researchers have isolated and identified 1-O-alkylglycerol in the leaves of the B. scandens plant, and established antitumor properties using the Brine Shrimp toxicity test, an internationally accepted bioassay. Although this species has high potential, little is known about the viability of seedling production and the morphology of these plants, particularly in terms of seed characteristics and initial stages of germination. The objective of this study was to characterize the seed morphology, germination, and seedlings of B. scandens. Seed water content, weight, and coloration were evaluated. This study also included a description of seed biometrics, external and internal structures, germination, and seedling morphology. Internal seed morphology was evaluated by the anatomical sectioning and X-ray methods. The morphology data obtained were subjected to descriptive statistical analysis and germination data were determined using Cramér's V. B. scandens seeds have a coloration ranging from very dark grayish-red to dark reddish-brown, flat oblong shapes, and rounded bases and apexes with full or slightly undulating margins. Healthy seedlings are produced mainly by seeds with well-formed internal structures. The reddish petiole of the seedling leaves is a taxonomic character for B. scandens identification. The non-domestication and genetic variability of this species reflect on the seed and seedling color and size variation.

Bauhinia scandens tem potencial como planta ornamental e importância medicinal. Nas folhas da planta de B. scandens, foi relatado pelos pesquisadores, o isolamento e identificação de glicerol 1-O-alquilo,os mesmos estabeleceram a propriedade antitumoral por um bioensaio, aceito internacionalmente denominado teste de toxicidade de Brine Shrimp. Por ser ainda pouco conhecida e com alto potencial exploratório, a viabilidade da produção de mudas e maior conhecimento sobre sua morfologia se faz necessária. Faltam informações sobre as características das sementes e estágios iniciais de germinação. O objetivo deste estudo foi caracterizar o tipo morfológico de sementes, germinação e plântulas de B. scandens. Foram avaliados o teor de água, o peso de mil sementes e a coloração das sementes. Este estudo também incluiu a descrição e biometria da semente, estrutura (externa e interna), germinação e morfologia das plântulas. A morfologia interna das sementes foi avaliada por corte anatômico e método de raios-X. Os dados morfológicos obtidos foram submetidos à análise estatística descritiva e os dados de germinação foram testados utilizando Cramér's V. B. scandens apresentam coloração que varia de vermelho acinzentado muito escuro a marrom avermelhado escuro e o tipo de semente é de forma oblonga plana, base e ápice são arredondados e cheios ou margens ligeiramente onduladas. Plântulas normais são produzidas majoritariamente por sementes com uma estrutura interna bem formada. O pecíolo avermelhado das folhas da plântula é um caráter taxonômico para identificação de B. scandens. A não domesticação e a variabilidade genética desta espécie refletem na variação da cor e tamanho das sementes e plântulas.

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Seed and seedling morphology of Bauhinia scandens L / Morfologia de semente e plântula de Bauhinia scandens L

Bauhinia scandens has potential importance as an ornamental and medicinal plant. Researchers have isolated and identified 1-O-alkylglycerol in the leaves of the B. scandens plant, and established antitumor properties using the Brine Shrimp toxicity test, an internationally accepted bioassay. Although this species has high potential, little is known about the viability of seedling production and the morphology of these plants, particularly in terms of seed characteristics and initial stages of germination. The objective of this study was to characterize the seed morphology, germination, and seedlings of B. scandens. Seed water content, weight, and coloration were evaluated. This study also included a description of seed biometrics, external and internal structures, germination, and seedling morphology. Internal seed morphology was evaluated by the anatomical sectioning and X-ray methods. The morphology data obtained were subjected to descriptive statistical analysis and germination data were determined using Cramér's V. B. scandens seeds have a coloration ranging from very dark grayish-red to dark reddish-brown, flat oblong shapes, and rounded bases and apexes with full or slightly undulating margins. Healthy seedlings are produced mainly by seeds with well-formed internal structures. The reddish petiole of the seedling leaves is a taxonomic character for B. scandens identification. The non-domestication and genetic variability of this species reflect on the seed and seedling color and size variation.(AU)

Bauhinia scandens tem potencial como planta ornamental e importância medicinal. Nas folhas da planta de B. scandens, foi relatado pelos pesquisadores, o isolamento e identificação de glicerol 1-O-alquilo,os mesmos estabeleceram a propriedade antitumoral por um bioensaio, aceito internacionalmente denominado teste de toxicidade de Brine Shrimp. Por ser ainda pouco conhecida e com alto potencial exploratório, a viabilidade da produção de mudas e maior conhecimento sobre sua morfologia se faz necessária. Faltam informações sobre as características das sementes e estágios iniciais de germinação. O objetivo deste estudo foi caracterizar o tipo morfológico de sementes, germinação e plântulas de B. scandens. Foram avaliados o teor de água, o peso de mil sementes e a coloração das sementes. Este estudo também incluiu a descrição e biometria da semente, estrutura (externa e interna), germinação e morfologia das plântulas. A morfologia interna das sementes foi avaliada por corte anatômico e método de raios-X. Os dados morfológicos obtidos foram submetidos à análise estatística descritiva e os dados de germinação foram testados utilizando Cramér's V. B. scandens apresentam coloração que varia de vermelho acinzentado muito escuro a marrom avermelhado escuro e o tipo de semente é de forma oblonga plana, base e ápice são arredondados e cheios ou margens ligeiramente onduladas. Plântulas normais são produzidas majoritariamente por sementes com uma estrutura interna bem formada. O pecíolo avermelhado das folhas da plântula é um caráter taxonômico para identificação de B. scandens. A não domesticação e a variabilidade genética desta espécie refletem na variação da cor e tamanho das sementes e plântulas.(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.

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.(AU)