Spectrophotometric method for determination of Colossoma macropomum (tambaqui) sperm concentration / Determinação da concentração espermática de tambaqui (Colossoma macropomum) por espectrofotometria

To assist the reproductive management of tambaqui (Colossoma macropomum) males in laboratory and commercial fish farming, a linear regression model was obtained from concentration curves using the spectrophotometric method. Twenty-two tambaqui males with an average age of three years old were selected and divided into two groups containing 11 animals each. Both groups alternately received a single dose of carp pituitary extract (CPE; 2.0 mg/kg body weight, intracoelomic). Sperm was collected 14 h after hormonal treatment and diluted (1:4000; sperm:formaldehyde saline). The concentration was estimated by counting spermatozoa in a Neubauer chamber and by using a spectrophotometer (λ=540 nm). Individual sperm concentration ranged from 11.40 to 71.13 × 109 sperm/mL. The degree of transmittance ranged from 62.1% to 95.0%. There was a significant correlation (r2 = 0.966; p < 0.0001) between sperm concentration analyzed in a Neubauer chamber and transmittance at 540 nm. Analysis by spectrophotometry and the prediction provided by the equation Y=100.293 - 0.509X proved to be an efficient and fast method for estimating sperm concentration in tambaqui and can be used in routine procedures in artificial fish reproduction laboratories.

Visando auxiliar o manejo reprodutivo de machos de tambaqui (Colossoma macropomum) em piscicultura de laboratório e comercial, obteve-se um modelo de regressão linear a partir de curvas de concentração por método espectrofotométrico. Foram selecionados 22 machos de tambaqui com idade média de três anos. Eles foram divididos em dois grupos contendo 11 animais cada. Ambos os grupos receberam alternadamente uma única dose de extrato de hipófise de carpa (EHC; 2,0 mg/kg de peso corporal, intracelomático). O esperma foi coletado 14 horas após o tratamento hormonal e diluído (1:4000; esperma: solução salina formaldeído). A concentração foi estimada por contagem de espermatozoides em câmara de Neubauer e por espectrofotômetro (λ=540 nm). A concentração espermática individual variou de 11,40 a 71,13 × 109 espermatozoides/mL. O grau de transmitância variou de 62,1 a 95,0%. Houve correlação significativa (r2 = 0,966; p < 0,0001) entre a concentração espermática analisada em câmara de Neubauer e a transmitância em 540 nm. A análise por espectrofotometria e a predição pela equação Y=100,293 - 0,509X mostrou ser um método eficiente e rápido para estimar a concentração espermática de tambaqui, podendo ser utilizado em procedimentos de rotina em laboratórios de reprodução artificial de peixes.

Assessment of coconut water added to Numida meleagris egg yolk as cryoprotectant for goat semen

Effects were assessed of the dilutants TRIS and ACP - 101c® with the addition of different guinea fowl (Numida meleagris) egg yolk concentrations. Fifteen ejaculates were collected from five goats of the Anglo Nubian breed. The ejaculates were pooled and then divided into 12 groups, two control groups (GC1 TRIS, with 2.5% Gallus gallus domesticus hen egg yolk GOGD), (GC2 Control Group ACP - 101c®, with the addition of 2.5% Gallus gallus domesticus hen egg yolk GOGD) and ten experimental groups (EG), containing TRIS and ACP added with different concentrations of egg yolk from guinea hen (Numida meleagris) (TRIS 2,5% GONM; TRIS 5% GONM; TRIS 10% GONM; TRIS 15% GONM; TRIS 20% GONM; ACP® 2,5% GONM; ACP® 5% GONM; ACP® 10% GONM; ACP® 15% GONM; ACP® 20% GONM). Then cryopreservation was carried out and the samples stored in liquid nitrogen (-196 °C). After seven days, the samples were thawed and assessed for spermatic kinetics, immunofluorescence and sperm morphology. Analysis of GOMN by the CASA system showed that the various parameters were similar to those of GOGD (P>0.05). The membrane integrity, mitochondrial potential and the acrosome were not influenced by the treatment (P>0.05) nor by the dilutant used for cryopreservation (P>0.05). The spermatic morphology was also preserved by the different GOGD and GONM concentrations in the ACP® and TRIS dilutants, with no statistically significant differences (P<0.05). It was concluded that Numida meleagris egg yolk, as external membrane cryoproctant added to the dilutants ACP-101c® and TRIS, improved goat semen quality.(AU)

Plasma rico em plaquetas e células tronco no tratamento de desmopatia dos ligamentos colaterais distais em equino / Platelet-rich plasma and stem cells in the treatment of desmopathy of the distal collateral ligaments in equine

Os equinos são animais muito utilizados em eventos esportivos, sendo, desta forma, propensos a sofrer acidentes que podem levar a lesões articulares e ósseas. Na rotina clínica, artifícios como o uso de raio X e ultrassonografia são de grande importância para que o correto diagnóstico seja feito e a conduta clínica possa ser melhor planejada. Além disso, alguns biomateriais, como o plasma rico em plaquetas (PRP) e células-tronco mesenquimais (CTMs), têm se inserido nos protocolos terapêuticos, demonstrando grande efetividade no tratamento desse tipo de lesão, devido à grande quantidade de fatores de crescimento presentes em ambos os biomateriais. O PRP é um derivado sanguíneo, caracterizado pela alta concentração plaquetária. Foi produzido a partir de duas centrifugações, a primeira para separar o plasma das hemácias e a segunda para concentrar as plaquetas. As CTMs foram isoladas de tecido adiposo, cultivadas, transportadas e aplicadas de forma autóloga, assim como o PRP. No presente relato, um equino, fêmea, Brasileiro de Hipismo com 8 anos, foi diagnosticado com desmopatia nos ligamentos colaterais da articulação interfalângica distal e foi tratado com PRP e CTMs, de forma associada e sequencial. Foi encontrada uma melhora do quadro clínico, significativa, em comparação aos dados encontrados na literatura, demonstrando grande potencialidade do uso associado de PRP e CTMs no tratamento de lesões ligamentares.

Horses are animals widely used in sporting events and are therefore prone to accidents that can lead to joint and bone injuries. In the clinical routine, devices such as the use of X-ray and ultrasound are of great importance for the correct diagnosis to be made and the clinical conduct can be better planned. In addition, some biomaterials such as platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) have been included in the therapeutic protocols, demonstrating great effectiveness in the treatment of this type of injury due to the large amount of growth factors present in both biomaterials. PRP is a blood derivative, characterized by high platelet concentration. It is produced from two centrifugations, the first to separate plasma from red blood cells and the second to concentrate platelets. MSCs were isolated from adipose tissue, cultured, transported and applied autologously, as well as PRP. In the present report, an 8-year-old female Brazilian equestrian horse was diagnosed with desmopathy in the collateral ligaments of the distal interphalangeal joint and was treated with PRP and MSCs in an associated and sequential manner. A significant improvement in the clinical picture was found in comparison to the data found in the literature, demonstrating great potential of the associated use of PRP and MSCs in the treatment of ligament injuries.

Effect of adding anethole to the cryopreservation medium (powdered coconut water) on morphology, kinetics, and oxidative stress of buck sperm / Efeito da adição de anethole ao meio de criopreserção (água de coco em pó) na morfologia, cinética e estresse oxidativo do sêmen de bode

The quality of post-thawing goat sperm is critical to the success of artificial insemination protocols and may be influenced by extenders, cryoprotectants, and antioxidant substances. Therefore, the objective of this study was to evaluate the effects of the antioxidant anethole on goat sperm diluted in preservation medium based on powdered coconut water (ACP-101c) and frozen. For that, each ejaculate was submitted to the following treatments: ACP-101c (control); control plus supplementation with 30, 300, or 2000 µg/ mL anethole. The samples were thawed and evaluated for morphology, kinetics, membrane integrity, and reactive oxygen species (ROS). The addition of anethole increased morphological abnormalities (P < 0.05), however, it did not affect sperm kinetics. Flow cytometry analysis showed that sperm cells cryopreserved with 300 µg/mL anethole had lower acrosome integrity than those cryopreserved in other treatments. Evaluation of oxidative stress revealed that cells stored in the presence of 2000 µg/mL anethole had small amounts of ROS when compared to those preserved in the control medium alone or supplemented with 300 µg/mL anethole (P < 0.05). After cryopreservation of sperm with 2000 µg/mL anethole, the highest percentage of viable sperm without ROS was observed (P < 0.05). In conclusion, despite reducing ROS levels, the supplementation of anethole in ACP-101c did not affect sperm kinetics or membrane integrity post-thawing, however, it did cause morphological damage to sperm.(AU)

A qualidade do espermatozoide caprino pós-descongelação é crítica para o sucesso dos protocolos de inseminação artificial e pode ser influenciada por extensores, crioprotetores e substâncias antioxidantes. Portanto, o objetivo deste estudo foi avaliar os efeitos do antioxidante anetole sobre espermatozoides caprinos diluídos em meio de conservação à base de água de coco em pó (ACP-101c) e congelados. Para tanto, cada ejaculado foi submetido aos seguintes tratamentos: ACP-101c (controle); controle mais suplementação com 30, 300 ou 2000 µg / mL de anetole. As amostras foram descongeladas e avaliadas quanto à morfologia, cinética, integridade de membranas e espécies reativas de oxigênio. A adição de anetole aumentou as anormalidades morfológicas (P < 0,05), no entanto, não afetou a cinética dos espermatozoides. A análise da citometria de fluxo mostrou que as células de esperma criopreservadas com 300 µg / mL anethole tinham integridade acrosma menor do que aquelas criopreservadas em outros tratamentos. A avaliação do estresse oxidativo revelou que as células armazenadas na presença de 2000 µg / mL anethole apresentaram pequenas quantidades de ROS quando comparadas às preservadas em meio de controle isoladamente ou suplementadas com 300 µg / mL anethole (P < 0,05). Após a criopreservação de espermatozoides com 2000 µg / mL anethole, observou-se a maior porcentagem de espermatozoides viáveis sem ROS (P < 0,05). A população com espermatozoides viáveis sem ROS foi maior quando utilizado 2.000 µg / mL (P < 0,05). Em conclusão, apesar de reduzir os níveis de ROS, a suplementação de anetole em ACP-101c não afetou a cinética espermática e a integridade da membrana pós-descongelação, entretanto, causou danos morfológicos nos espermatozoides.(AU)

Sêmen ovino e caprino imunossexado e diluído em meio de conservação à base de água de coco em pó (ACP101/102c) / Ram and goat semen immunosexed and diluted in powdered coconut water-based preservation medium (ACP101/102c)

Sperm sexing aims to separate sperm populations in carriers of the "X" or "Y" chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c. Ejaculates from five rams and five goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with "Y" chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species. The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.(AU)

Biotecnologias como ferramentas para o desenvolvimento do Nordeste do Brasil / Biotechnologies as tools for the development of northeast Brazil

A biotecnologia tem sido um ramo de estudo diferencial para diversos setores da sociedade, apresentando, através de bioprodutos e bioprocessos, melhorias para o avanço e desenvolvimento da região Nordeste do Brasil. Vale pontuar que um importante meio que traz anualmente acréscimos inovadores à área biotecnológica é o setor acadêmico, que através de cursos stricto sensu a nível de mestrado e doutorado, promovem pesquisas relevantes para vários setores como economia, agroindústria, saúde, dentre outros. Exemplos disso são: o curso de Doutorado em Biotecnologia da RENORBIO) e o Programa Profissional de Pós-Graduação em Biotecnologia em Saúde Humana e Animal (PPGBiotec). O presente artigo se sobre o percurso trilhado pelos cursos stricto sensu mencionados, bem como ressalta a relevância da Biotecnologia para a região Nordeste do Brasil, em seus diferentes campos de investigação, com ênfase nos Recursos Naturais, Agropecuária e Saúde. Exemplificamos as biotecnologias utilizando a água de coco que vêm sendo trabalhadas desde os anos 1980’s e sua evolução até os dias atuais. Com base em toda a potencialidade da Região Nordeste para a geração de bioprodutos e bioprocessos, ressaltamos que os mesmos só serão úteis se realmente forem tratados como inovação tecnológica, gerarem nota fiscal e impactarem positivamente para o bem estar da sociedade.

Biotechnology has been a branch of differential study for various sectors of society, presenting, through bioproducts and bioprocesses, improvements for the advancement and development of the Northeast region of Brazil. It is worth noting that an important means that annually brings innovative additions to the biotechnological area is the academic sector, which through stricto sensu courses at the master's and doctoral level, promote relevant research for various sectors such as economics, agribusiness, health, among others. Examples of this are: the Doctorate course in Biotechnology (RENORBIO) and the Professional Graduate Program in Biotechnology in Human and Animal Health (PPGBiotec). This article is about the path taken by the stricto sensu courses mentioned, as well as emphasizing the relevance of Biotechnology for the Northeast region of Brazil, in its different research fields, with emphasis on Natural Resources, Agriculture and Health. We exemplify biotechnologies using coconut water that has been worked since the 1980's and its evolution to the present day. Based on all the potential of the Northeast Region for the generation of bioproducts and bioprocesses, we emphasize that they will only be useful if they are really treated as technological innovation, generate invoices and have a positive impact on society's well-being.

Biotecnologias como ferramentas para o desenvolvimento do Nordeste do Brasil / Biotechnologies as tools for the development of northeast Brazil

A biotecnologia tem sido um ramo de estudo diferencial para diversos setores da sociedade, apresentando, através de bioprodutos e bioprocessos, melhorias para o avanço e desenvolvimento da região Nordeste do Brasil. Vale pontuar que um importante meio que traz anualmente acréscimos inovadores à área biotecnológica é o setor acadêmico, que através de cursos stricto sensu a nível de mestrado e doutorado, promovem pesquisas relevantes para vários setores como economia, agroindústria, saúde, dentre outros. Exemplos disso são: o curso de Doutorado em Biotecnologia da RENORBIO) e o Programa Profissional de Pós-Graduação em Biotecnologia em Saúde Humana e Animal (PPGBiotec). O presente artigo se sobre o percurso trilhado pelos cursos stricto sensu mencionados, bem como ressalta a relevância da Biotecnologia para a região Nordeste do Brasil, em seus diferentes campos de investigação, com ênfase nos Recursos Naturais, Agropecuária e Saúde. Exemplificamos as biotecnologias utilizando a água de coco que vêm sendo trabalhadas desde os anos 1980s e sua evolução até os dias atuais. Com base em toda a potencialidade da Região Nordeste para a geração de bioprodutos e bioprocessos, ressaltamos que os mesmos só serão úteis se realmente forem tratados como inovação tecnológica, gerarem nota fiscal e impactarem positivamente para o bem estar da sociedade.(AU)

Biotechnology has been a branch of differential study for various sectors of society, presenting, through bioproducts and bioprocesses, improvements for the advancement and development of the Northeast region of Brazil. It is worth noting that an important means that annually brings innovative additions to the biotechnological area is the academic sector, which through stricto sensu courses at the master's and doctoral level, promote relevant research for various sectors such as economics, agribusiness, health, among others. Examples of this are: the Doctorate course in Biotechnology (RENORBIO) and the Professional Graduate Program in Biotechnology in Human and Animal Health (PPGBiotec). This article is about the path taken by the stricto sensu courses mentioned, as well as emphasizing the relevance of Biotechnology for the Northeast region of Brazil, in its different research fields, with emphasis on Natural Resources, Agriculture and Health. We exemplify biotechnologies using coconut water that has been worked since the 1980's and its evolution to the present day. Based on all the potential of the Northeast Region for the generation of bioproducts and bioprocesses, we emphasize that they will only be useful if they are really treated as technological innovation, generate invoices and have a positive impact on society's well-being.(AU)

Utilização de proteínas anticongelantes na criopreservação seminal de peixes / Use of anti-freezing proteins in seminal fish cryopreservation

A criopreservação seminal é uma das biotécnica reprodutivas mais promissoras para a aquicultura, pois permite o transporte de gametas, garante melhorias genéticas em espécies desejadas e minimiza a assincronia entre machos e fêmeas reprodutores. Apesar dos benefícios, danos celulares podem ser ocasionados durante o processo de congelação, causando a diminuição da qualidade dos espermatozoides. Sendo assim, estudos que busquem alternativas que minimizem tais danos são de grande importância. As proteínas anticongelantes (AFPs) são importantes compostos originados a partir de processos evolutivos em organismos que vivem em ambientes polares. As AFPs podem ser utilizadas como possíveis agentes crioprotetores, pois são capazes de diminuir os cristais de gelo e proteger a membrana plasmática das células durante o processo de congelação. Dessa forma, esta revisão tem como objetivo reunir informações acerca da utilização de proteínas anticongelantes na conservação do sêmen de peixes e apresentar os principais resultados encontrados na literatura. Observou-se que a utilização dessas substâncias na criopreservação seminal em peixes promoveu melhorias nas taxas de motilidade espermática, velocidades e integridade de membrana plasmática. Tais melhorias foram observadas de maneira interespecífica. Portanto, ainda são necessários estudos mais aprofundados com intuito de verificar o efeito das AFPs na capacidade de fertilização, buscando a melhor opção para suplementação do meio.

Seminal cryopreservation is one of the most promising reproductive biotechniques for aquaculture, as it allows the transport of gametes, ensures genetic improvements in desired species and minimizes asynchrony between breeding males and females. Despite the benefits, cell damage can be caused during the freezing process, causing decreased quality of sperm. Therefore, studies that seek alternatives that minimize such damage are of great importance. Antifreeze proteins (AFPs) are important compounds originated from evolutionary processes in organisms that live in polar environments. AFPs can be used as possible cryoprotective agents, as they are able to decrease ice crystals and protect the plasma membrane of cells during the freezing process. Thus, this review aims to gather information about the use of antifreeze proteins in the conservation of fish semen and present the main results found in the literature. It was observed that the use of these substances in seminal cryopreservation in fish promoted improvements in the rates of sperm motility, velocities and integrity of the plasma membrane. Such improvements were observed in an interspecific manner. Therefore, further studies are needed in order to verify the effect of AFPs on fertilization capacity, seeking the best option for supplementing the medium.

Utilização de proteínas anticongelantes na criopreservação seminal de peixes / Use of anti-freezing proteins in seminal fish cryopreservation

A criopreservação seminal é uma das biotécnica reprodutivas mais promissoras para a aquicultura, pois permite o transporte de gametas, garante melhorias genéticas em espécies desejadas e minimiza a assincronia entre machos e fêmeas reprodutores. Apesar dos benefícios, danos celulares podem ser ocasionados durante o processo de congelação, causando a diminuição da qualidade dos espermatozoides. Sendo assim, estudos que busquem alternativas que minimizem tais danos são de grande importância. As proteínas anticongelantes (AFPs) são importantes compostos originados a partir de processos evolutivos em organismos que vivem em ambientes polares. As AFPs podem ser utilizadas como possíveis agentes crioprotetores, pois são capazes de diminuir os cristais de gelo e proteger a membrana plasmática das células durante o processo de congelação. Dessa forma, esta revisão tem como objetivo reunir informações acerca da utilização de proteínas anticongelantes na conservação do sêmen de peixes e apresentar os principais resultados encontrados na literatura. Observou-se que a utilização dessas substâncias na criopreservação seminal em peixes promoveu melhorias nas taxas de motilidade espermática, velocidades e integridade de membrana plasmática. Tais melhorias foram observadas de maneira interespecífica. Portanto, ainda são necessários estudos mais aprofundados com intuito de verificar o efeito das AFPs na capacidade de fertilização, buscando a melhor opção para suplementação do meio.(AU)

Seminal cryopreservation is one of the most promising reproductive biotechniques for aquaculture, as it allows the transport of gametes, ensures genetic improvements in desired species and minimizes asynchrony between breeding males and females. Despite the benefits, cell damage can be caused during the freezing process, causing decreased quality of sperm. Therefore, studies that seek alternatives that minimize such damage are of great importance. Antifreeze proteins (AFPs) are important compounds originated from evolutionary processes in organisms that live in polar environments. AFPs can be used as possible cryoprotective agents, as they are able to decrease ice crystals and protect the plasma membrane of cells during the freezing process. Thus, this review aims to gather information about the use of antifreeze proteins in the conservation of fish semen and present the main results found in the literature. It was observed that the use of these substances in seminal cryopreservation in fish promoted improvements in the rates of sperm motility, velocities and integrity of the plasma membrane. Such improvements were observed in an interspecific manner. Therefore, further studies are needed in order to verify the effect of AFPs on fertilization capacity, seeking the best option for supplementing the medium.(AU)

Morfologia do espermatozoide ovino após resfriamento em diluentes à base de água de coco durante o período chuvoso e seco no Nordeste do Brasil / Morphology of ovine spermatozoa after cooling in extenders based in coconut water during the rainy and dry season in the Northeast of Brazil

The aim of this work went to evaluate the spermatic morphology of ram semen cooled to 4 °C in nature (INCW) and in powdered coconut water (ACP-102®) during the rainy and dry season in the Northeast of Brazil. The semen of four rams was collected, divided into two fractions and diluted in INCW and ACP-102®. The samples were conditioned in refrigerator to 4 °C and after 2, 24 and 48 hours of cooling were submitted at thermo resistant test (TT). Semen slides were executed in the beginning and in the end of TT to evaluation of the spermatic morphology (SM). The SM parameters, within different preservation times (2, 24 and 48h) and extenders (INCW and ACP-102®), were expressed in media and standard deviation (SD) and submitted to Tukey test (p<0.05). According to the diluted samples in ACP-102®, was observed a percentage increase of morphology normal spermatozoon in the rainy season as was verified in the dry season. In conclusion, the ACP-102® extender present good preserve capacity. Agreed with this study, the raining season did not have influence on the characteristics of spermatic morphology.

Morfologia do espermatozoide ovino após resfriamento em diluentes à base de água de coco durante o período chuvoso e seco no Nordeste do Brasil / Morphology of ovine spermatozoa after cooling in extenders based in coconut water during the rainy and dry season in the Northeast of Brazil

The aim of this work went to evaluate the spermatic morphology of ram semen cooled to 4 °C in nature (INCW) and in powdered coconut water (ACP-102®) during the rainy and dry season in the Northeast of Brazil. The semen of four rams was collected, divided into two fractions and diluted in INCW and ACP-102®. The samples were conditioned in refrigerator to 4 °C and after 2, 24 and 48 hours of cooling were submitted at thermo resistant test (TT). Semen slides were executed in the beginning and in the end of TT to evaluation of the spermatic morphology (SM). The SM parameters, within different preservation times (2, 24 and 48h) and extenders (INCW and ACP-102®), were expressed in media and standard deviation (SD) and submitted to Tukey test (p<0.05). According to the diluted samples in ACP-102®, was observed a percentage increase of morphology normal spermatozoon in the rainy season as was verified in the dry season. In conclusion, the ACP-102® extender present good preserve capacity. Agreed with this study, the raining season did not have influence on the characteristics of spermatic morphology.(AU)

Aspectos econômicos e avaliação do período de viabilidade de espermatozoides ovino imunossexados e diluídos em meio de conservação à base de água de coco em pó (ACP-102c) / Economic aspects and evaluation of the viability period of immunosexed and diluted ram sperm in powdered coconut water conservation extender (ACP-102c)

Background: Sperm sexing is increasing in use because pre-determining the sex of the calf allows greater profitability and promotes significant gains in the productive systems that utilize the technique. Deployment of a low-cost and practical preservation methodol-ogy may further favor the cost-benefit ratio. Flow cytometry, the most commonly used sexing technique, has high costs and is very restricted. As an alternative, immunosexing has been studied, which uses sex-specific monoclonal antibodies. Thus, the objective of this study was to evaluate the immunosexing technique in conjunction with cryopreservation in ACP-102c and examine its economic aspects with regard to ram semen.Materials, Methods & Results: Ejaculates from two ram individuals were collected with the aid of an artificial vagina, evaluated, and submitted to the immunosexing protocol, according to the manufacturer’s recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro-RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP (ACP-102c + 20% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated to 4°C, stabilized for 30 min, frozen in liquid nitrogen vapors (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were thawed and evaluated for sperm kinetics both by using computerized semen analysis with SCA® software (Sperm Class Analyzer version 5.0) and subjectively comparing specimens from the two animals using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique...

Uso da polpa desidratada do fruto de Mauritia flexuosa como suplemento ao diluente de congelação do sêmen caprino / Use of dehydrated pulp of Mauritia flexuosa fruit as supplement to freezing extender of goat semen

Objetivou-se avaliar a qualidade in vitro do sêmen caprino descongelado utilizando diluentesuplementado com a polpa desidratada do fruto de Mauritia flexuosa. O experimento foi dividido emduas etapas. Na primeira, foram utilizados 15 pools,fracionados em 13 tratamentos com diferentesconcentrações do extrato bruto. Os melhores resultados de viabilidade espermática obtidos na primeiraetapa foram utilizadas na segunda etapa (criopreservação). Para isto, foram formados dois gruposutilizando 15 pools, sendo um diluente constituído (TRIS + 7% glicerol + melhores concentrações doextrato bruto) e outro pelo diluente (TRIS + 2,5% gema de ovo + 7% glicerol + melhores concentraçõesdo extrato bruto). Na primeira etapa os grupos contendo baixa quantidade do extrato não diferiram dogrupo controle (P≤0,05). Todavia na segunda etapa, após descongelação, os grupos TRIS contendo 2,5%ou 0% de gema de ovo apresentaram diferença significativa, onde o grupo TB06GLGE foi superior aogrupo controle. Portanto, a polpa desidratada do fruto de Mauritia flexuosa,nas concentrações de 0,25% a1%, não atuou de forma benéfica sobre parâmetros espermáticos do sêmen caprino após acriopreservação/descongelação.(AU)

The objective was to evaluate the in vitro quality of the thawed goat semen using diluentsupplemented with the dehydrated pulp of the Mauritia flexuosa fruit. The experiment was divided intotwo stages. In the first, 15 fractionated pools were used in 13 treatments with different concentrations ofthe crude extract. The best results of sperm viability obtained in the first experimental stage were used inthe second experimental stage (cryopreservation). Afterwards, two groups were formed using 15 pools,one constituent (TRIS + 7% glycerol + best concentrations of the crude extract) and another by thediluent (TRIS + 2,5% egg yolk + 7% glycerol + best concentrations of the crude extract). In the firststage, the groups containing low amount of extract did not differ from the control group (P≤0.05).However, in the second stage, after thawing, TRIS groups containing 2.5% or 0% egg yolk presented asignificant difference, where the TB06GLGE group was superior to the control group. Therefore, thedehydrated fruit pulp of Mauritia flexuosa at concentrations of 0.25% to 1% did not benefit goat semenparameters after cryopreservation/thawing.(AU)

Uso da polpa desidratada do fruto de Mauritia flexuosa como suplemento ao diluente de congelação do sêmen caprino / Use of dehydrated pulp of Mauritia flexuosa fruit as supplement to freezing extender of goat semen

Objetivou-se avaliar a qualidade in vitro do sêmen caprino descongelado utilizando diluentesuplementado com a polpa desidratada do fruto de Mauritia flexuosa. O experimento foi dividido emduas etapas. Na primeira, foram utilizados 15 pools,fracionados em 13 tratamentos com diferentesconcentrações do extrato bruto. Os melhores resultados de viabilidade espermática obtidos na primeiraetapa foram utilizadas na segunda etapa (criopreservação). Para isto, foram formados dois gruposutilizando 15 pools, sendo um diluente constituído (TRIS + 7% glicerol + melhores concentrações doextrato bruto) e outro pelo diluente (TRIS + 2,5% gema de ovo + 7% glicerol + melhores concentraçõesdo extrato bruto). Na primeira etapa os grupos contendo baixa quantidade do extrato não diferiram dogrupo controle (P≤0,05). Todavia na segunda etapa, após descongelação, os grupos TRIS contendo 2,5%ou 0% de gema de ovo apresentaram diferença significativa, onde o grupo TB06GLGE foi superior aogrupo controle. Portanto, a polpa desidratada do fruto de Mauritia flexuosa,nas concentrações de 0,25% a1%, não atuou de forma benéfica sobre parâmetros espermáticos do sêmen caprino após acriopreservação/descongelação.

The objective was to evaluate the in vitro quality of the thawed goat semen using diluentsupplemented with the dehydrated pulp of the Mauritia flexuosa fruit. The experiment was divided intotwo stages. In the first, 15 fractionated pools were used in 13 treatments with different concentrations ofthe crude extract. The best results of sperm viability obtained in the first experimental stage were used inthe second experimental stage (cryopreservation). Afterwards, two groups were formed using 15 pools,one constituent (TRIS + 7% glycerol + best concentrations of the crude extract) and another by thediluent (TRIS + 2,5% egg yolk + 7% glycerol + best concentrations of the crude extract). In the firststage, the groups containing low amount of extract did not differ from the control group (P≤0.05).However, in the second stage, after thawing, TRIS groups containing 2.5% or 0% egg yolk presented asignificant difference, where the TB06GLGE group was superior to the control group. Therefore, thedehydrated fruit pulp of Mauritia flexuosa at concentrations of 0.25% to 1% did not benefit goat semenparameters after cryopreservation/thawing.

Concentração de proteínas do plasma seminal e sua relação com a criopreservação do sêmen ovino em diferentes diluentes / Protein concentration of seminal plasma and their relation with cryopreservation of ram semen using differents extenders

O objetivo deste trabalho foi avaliar a relação entre a concentração de proteínas no plasma seminal ovino com parâmetros de qualidade do sêmen criopreservado em diluentes TRIS e em Água de Coco em Pó (ACP102c). Um total de 12 coletas/animal em ovinos Santa Inês (n=2) e Dorper (n=2) foram realizadas para obtenção do plasma seminal e para criopreservação nos diluentes de trabalho. Foi utilizado o método de Bradford para determinação da concentração de proteína. Para avaliação do sêmen fresco e criopreservado foram utilizados os testes de viabilidade por eosina-nigrosina e teste hiposmótico (HOST), além dos parâmetros de motilidade em sistema de análise de sêmen auxiliada por computador (CASA). Baseado no valor médio da concentração de proteína, os ejaculados foram agrupados em alta (AP) e baixa (BP) concentração proteica. Correlações significativamente positivas foram encontradas entre a concentração de proteína com os parâmetros motilidade progressiva (PROG) e congelabilidade (CONGEL), e negativa para deslocamento lateral de cabeça (ALH) nas amostras criopreservadas em ambos os diluentes, além de correlação positiva para motilidade total (MOT) em TRIS. Sêmen de ejaculados do grupo AP criopreservados em TRIS apresentaram MOT, PROG e CONGEL significativamente superior ao grupo BP e inferiores para ALH. Quando criopreservado em ACP102c, sêmen do grupo AP foi superior ao BP para PROG e CONGEL. Variações na qualidade do sêmen criopreservado entre ejaculados podem estar associados à concentração de proteínas do plasma seminal, e estas podem atuar na manutenção da progressividade de espermatozoides ovinos criopreservados em diluentes TRIS e ACP102c.

The objective of this study was to evaluate the relation between the protein concentration in ram seminal plasma and quality parameters of the semen cryopreserved in TRIS and Coconut Water Powder (ACP102c) extenders. A total of 12 semen collections/animal of Santa Ines (n=2) and Dorper (n=2) rams were performed to obtain seminal plasma and semen cryopreservation in work extenders. The protein content was determined using the Bradford ́s method. The eosin-nigrosin and hyposmotic (HOST) viable tests and motility parameters obtained by computer assisted semen analysis were evaluated for fresh and cryopreserved semen. The ejaculates were grouped in high (AP) and low (BP) protein content, based on the mean value of protein concentration. Significantly positive correlations were found between the protein content and progressive motility (PROG), freezability (CONGEL) and negative for lateral head displacement (ALH) in the semen cryopreserved in both extenders. Therefore, positive correlation was found for total motility (MOT) in TRIS. Semen samples from AP group cryopreserved in TRIS extender were significantly better than BP group for MOT, PROG and CONGEL, and inferior for ALH. When criopreserved in ACP102c extender, the AP group were better than BP group for PROG and CONGEL. Variations in the quality of cryopreserved semen from different ejaculates may be associated with the concentration of seminal plasma proteins and may act to maintain the progressiveness of ram sperm cryopreserved in TRIS and ACP102c extenders.

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3’-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...(AU)

Concentração de proteínas do plasma seminal e sua relação com a criopreservação do sêmen ovino em diferentes diluentes / Protein concentration of seminal plasma and their relation with cryopreservation of ram semen using differents extenders

O objetivo deste trabalho foi avaliar a relação entre a concentração de proteínas no plasma seminal ovino com parâmetros de qualidade do sêmen criopreservado em diluentes TRIS e em Água de Coco em Pó (ACP102c). Um total de 12 coletas/animal em ovinos Santa Inês (n=2) e Dorper (n=2) foram realizadas para obtenção do plasma seminal e para criopreservação nos diluentes de trabalho. Foi utilizado o método de Bradford para determinação da concentração de proteína. Para avaliação do sêmen fresco e criopreservado foram utilizados os testes de viabilidade por eosina-nigrosina e teste hiposmótico (HOST), além dos parâmetros de motilidade em sistema de análise de sêmen auxiliada por computador (CASA). Baseado no valor médio da concentração de proteína, os ejaculados foram agrupados em alta (AP) e baixa (BP) concentração proteica. Correlações significativamente positivas foram encontradas entre a concentração de proteína com os parâmetros motilidade progressiva (PROG) e congelabilidade (CONGEL), e negativa para deslocamento lateral de cabeça (ALH) nas amostras criopreservadas em ambos os diluentes, além de correlação positiva para motilidade total (MOT) em TRIS. Sêmen de ejaculados do grupo AP criopreservados em TRIS apresentaram MOT, PROG e CONGEL significativamente superior ao grupo BP e inferiores para ALH. Quando criopreservado em ACP102c, sêmen do grupo AP foi superior ao BP para PROG e CONGEL. Variações na qualidade do sêmen criopreservado entre ejaculados podem estar associados à concentração de proteínas do plasma seminal, e estas podem atuar na manutenção da progressividade de espermatozoides ovinos criopreservados em diluentes TRIS e ACP102c.(AU)

The objective of this study was to evaluate the relation between the protein concentration in ram seminal plasma and quality parameters of the semen cryopreserved in TRIS and Coconut Water Powder (ACP102c) extenders. A total of 12 semen collections/animal of Santa Ines (n=2) and Dorper (n=2) rams were performed to obtain seminal plasma and semen cryopreservation in work extenders. The protein content was determined using the Bradford ́s method. The eosin-nigrosin and hyposmotic (HOST) viable tests and motility parameters obtained by computer assisted semen analysis were evaluated for fresh and cryopreserved semen. The ejaculates were grouped in high (AP) and low (BP) protein content, based on the mean value of protein concentration. Significantly positive correlations were found between the protein content and progressive motility (PROG), freezability (CONGEL) and negative for lateral head displacement (ALH) in the semen cryopreserved in both extenders. Therefore, positive correlation was found for total motility (MOT) in TRIS. Semen samples from AP group cryopreserved in TRIS extender were significantly better than BP group for MOT, PROG and CONGEL, and inferior for ALH. When criopreserved in ACP102c extender, the AP group were better than BP group for PROG and CONGEL. Variations in the quality of cryopreserved semen from different ejaculates may be associated with the concentration of seminal plasma proteins and may act to maintain the progressiveness of ram sperm cryopreserved in TRIS and ACP102c extenders.(AU)

Seleção de receptoras em um programa de transferência de embriões (pive) em bovinos no nordeste do Brasil / Selection procedure in embryo transfer program in cattle at northeastern Brazil

A seleção de receptoras constitui-se em uma das principais etapas de um programa de transferência de embriões (TE), influenciando substancialmente os resultados econômicos da aplicação dessa biotécnica. Considerando os desafios enfrentados para obtenção deum número de receptoras suficiente para atender à demanda de um programa de TE em bovinos, o presente estudo foi realizado com o objetivo de ampliar os elementos utilizados como critérios de pré-seleção dessas matrizes, visando incrementar o número de fêmeas disponíveis para a etapa de sincronização de ciclo estral. Para tanto, 1017 fêmeas das raças Girolando, Gir Leiteiro e Nelore,oriundas de propriedades localizadas no Nordeste brasileiro, foram avaliadas por meio de exames ginecológicos, desde as etapas preliminares de seleção, até ocorrência de parto a termo; identificando-se dois grupos experimentais,representados pelos animais que apresentavam folículos maduros ou corpos lúteos por ocasião da pré-seleção. Os resultados obtidos revelaram que as matrizes que apresentam folículos maduros por ocasião do exame ginecológico preliminar, de admissão no programa de transferência de embriões na condição dereceptoras, apresentaram rendimentos finais equivalentes aos daquelas admitidas pela presença de corpo lúteo no mesmo momento, independentemente do ovário (direito ou esquerdo). O estudo demonstrou, ainda, que e mbriões em estádios de blastocisto (inicial, tipicamente caracterizado ou expandido) devem ser prioritariamente selecionados para transferência.

Recipent selection comprises an important step for embryo transfer (ET) programs, affecting significantly the economic results of that biotechnology. Considering the challenges for obtaining recipient cows in a number big enough to attend the demand of an ET program, this research was carried out with the aim of to expand criteria of recipient pre-selection, in order to increase the number of females disposable for estrum cycle synchronization. Then, 1017 cows of different breeds (Girolando, GirLeiteiro and Nelore) from farms located in Brazilian Northeast, where evaluated by gynecological exams since preliminary phases of selection up to calving birth, divided into experimental groups represented by females presenting mature follicles or corpus luteal at pre-selection. Results obtained have shown similar final yields for the two experimental groups, revealing that both ovarian structures should be used as criteria for including matrices in ET programs. In addition, embryos in stage of blastocyst (Initial, completely characterized or expanded) must be preferred for ET.

Seleção de receptoras em um programa de transferência de embriões (pive) em bovinos no nordeste do Brasil / Selection procedure in embryo transfer program in cattle at northeastern Brazil

A seleção de receptoras constitui-se em uma das principais etapas de um programa de transferência de embriões (TE), influenciando substancialmente os resultados econômicos da aplicação dessa biotécnica. Considerando os desafios enfrentados para obtenção deum número de receptoras suficiente para atender à demanda de um programa de TE em bovinos, o presente estudo foi realizado com o objetivo de ampliar os elementos utilizados como critérios de pré-seleção dessas matrizes, visando incrementar o número de fêmeas disponíveis para a etapa de sincronização de ciclo estral. Para tanto, 1017 fêmeas das raças Girolando, Gir Leiteiro e Nelore,oriundas de propriedades localizadas no Nordeste brasileiro, foram avaliadas por meio de exames ginecológicos, desde as etapas preliminares de seleção, até ocorrência de parto a termo; identificando-se dois grupos experimentais,representados pelos animais que apresentavam folículos maduros ou corpos lúteos por ocasião da pré-seleção. Os resultados obtidos revelaram que as matrizes que apresentam folículos maduros por ocasião do exame ginecológico preliminar, de admissão no programa de transferência de embriões na condição dereceptoras, apresentaram rendimentos finais equivalentes aos daquelas admitidas pela presença de corpo lúteo no mesmo momento, independentemente do ovário (direito ou esquerdo). O estudo demonstrou, ainda, que e mbriões em estádios de blastocisto (inicial, tipicamente caracterizado ou expandido) devem ser prioritariamente selecionados para transferência.(AU)

Recipent selection comprises an important step for embryo transfer (ET) programs, affecting significantly the economic results of that biotechnology. Considering the challenges for obtaining recipient cows in a number big enough to attend the demand of an ET program, this research was carried out with the aim of to expand criteria of recipient pre-selection, in order to increase the number of females disposable for estrum cycle synchronization. Then, 1017 cows of different breeds (Girolando, GirLeiteiro and Nelore) from farms located in Brazilian Northeast, where evaluated by gynecological exams since preliminary phases of selection up to calving birth, divided into experimental groups represented by females presenting mature follicles or corpus luteal at pre-selection. Results obtained have shown similar final yields for the two experimental groups, revealing that both ovarian structures should be used as criteria for including matrices in ET programs. In addition, embryos in stage of blastocyst (Initial, completely characterized or expanded) must be preferred for ET.(AU)