Reproducibility of double agar gel immunodiffusion test using stored serum and plasma from paracoccidioidomycosis patients

Background: Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. Methods: The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at ­80o C for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at ­20o C for up to six months. Differences of titers higher than one dilution were considered discordant. Results: First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Conclusions: Storage at ­80o C for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at ­20o C.(AU)

Glaucoma in dogs and cats - management with cyclocryotherapy

Background: The treatment of glaucoma often requires numerous therapeutic modalities to achieve the desired reduction in intraocular pressure (IOP). Cyclodestructive procedures or ciliary body destruction have been performed using techniques with considerable differences in efficacy and complication rates. Among these methods, cyclocryotherapy is non-invasive and simple for the management of uncontrolled glaucoma in dogs and cats. The objective of this case report is to describe the technique of carbon dioxide cyclocryotherapy to reduce intraocular pressure in dogs and cats with glaucoma. Cases: Nine canine patients and one cat with glaucoma were treated with cyclocryotherapy performed under general anaesthesia. Clinical signs patients included blepharospasm, ocular pain, episcleral congestion and ocular hypertension. The patients showed higher levels of IOP, higher than 30 mmHg. Surgical treatment with general anaesthesia was applied. The pre-anaesthesia protocol included acepromazine 0.05 mg/kg with methadone 0.2 mg/kg, followed by intravenous propofol and maintenance with isoflurane and oxygen. An ophthalmological cryocautery unit was used with carbon dioxide as the cryogenic agent and a retinal cryoprobe of 3.2 mm diameter tip for the procedure. The method used was a double cycle of freezing and thawing for 60 s in each site. The cryoprobe was centred approximately 5 mm posterior to the corneoscleral limbus over the ciliary body. The temperature of each cyclocryotherapy spot was between -60°C and -80°C and each site was maintained in place for 60 s; 4 to 6 spots of the double freeze-thaw cycle were made. This technique did not have any serious complications during or after the application of cryotherapy, but chemosis and hyperaemia of the bulbar conjunctiva developed. Subconjunctival anti-inflammatory steroids were injected to minimise swelling and patient discomfort. Satisfactory results were observed; in all cases, the intraocular pressure decreased, with the usual result being a cosmetic and painless eye. Discussion: Even with recent surgical and medical advances, pain and blindness are still common occurrences in glaucoma in human and veterinary practice. The cyclodestructive procedures included cyclodialysis, cyclodiathermy, cyclocryotherapy, and cyclophotocoagulation. The cryosurgery in veterinary ophthalmology has many indications for the treatment of ocular diseases and is effective at decreasing intraocular pressure in patients with persistent uncontrolled glaucoma. Cyclocryotherapy has been shown to reduce intraocular pressure in dogs, cats, rabbits and humans with normotensive and glaucomatous eyes. The application of a cryoprobe over the ciliary processes results in ablating ciliary tissue so that aqueous humour inflow is reduced to acceptable levels. In the clinical cases evaluated, there was a reduction in intraocular pressure reaching acceptable levels, with the usual result being cosmetic and painless eye. Medical therapy remains the predominant method for treating glaucoma in veterinary patients; therefore, cyclocryotherapy is an effective, simple way to lower IOP and is a reasonable treatment option for glaucoma management. Cyclocryotherapy has shown good results, with a low learning curve and it can also be repeated if necessary.

Uso de placa aquecedora visando a incubação do espermatozoide ovino após criopreservação / Use of heating plate aiming at the ovine sperm incubation after cryopreservation

Objetivou-se avaliar a utilização da placa aquecedora, em substituição ao banho-maria, no teste de termorresistência (TTR) de espermatozoides de carneiros após a criopreservação. Foram coletados 10 ejaculados de três carneiros adultos (n=30), por meio de vagina artificial para ovinos. Em seguida, foram diluídos em TrisGema de ovo, a uma concentração final de 200 x106 sptz/mL, e submetidos á curva de resfriamento, para posterior criopreservação em palhetas em nitrogênio líquido. Após descongelação, as amostras foram analisadas quanto à integridade de membrana, de acrossoma e atividade mitocondrial. O sêmen então foi dividido em dois tubos e submetido ao TTR, um em banho-maria e outro em placa aquecedora, e, ainda, avaliado a cada 30 minutos, do tempo zero (pós-descongelação) até 90 minutos de incubação, a 37 °C, quanto à motilidade espermática e à motilidade progressiva. As variáveis foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey a 5% de probabilidade. Após o processo de congelação/descongelação, os espermatozoides apresentaram baixo percentual de integridade de membrana e elevado percentual de atividade mitocondrial e integridade do acrossoma. Não foi observada diferença na motilidade espermática nem na motilidade progressiva ao longo do TTR, quando comparados os espermatozoides que foram incubados em banho-maria aos incubados em placa aquecedora durante o período de 90 minutos. A placa aquecedora pode ser utilizada como meio de incubação dos espermatozoides de carneiros em substituição ao banho-maria.

The objective was to evaluate the use of the hot plate to replace the water bath in the thermo resistance test of ovine sperm after cryopreservation. Ten ejaculates were also collected from three adult sheep (n=30), by means of artificial sheep vagina. The collected samples were diluted in Tris-Egg Yolk, at a final concentration of 200 x106 sptz/mL and subjected to a cooling curve for subsequent cryopreservation in liquid nitrogen straws. Immediately after thawing, the samples were analyzed for membrane and acrosome integrity, and mitochondrial activity. The semen was then divided into two tubes and submitted to TTR, one in a water bath and the other in a hot plate, and evaluated every 30 minutes, from time zero (post-thaw) until 90 minutes of incubation at 37 °C, for sperm motility and progressive motility. The variables were subjected to analysis of variance and the means were compared using the Tukey test at 5% probability. After the freeze/thawing process, sperm showed a low percentage of membrane integrity, high percentage of mitochondrial activity, in addition to maintaining the integrity of acrossome. No difference was observed in sperm motility nor progressive motility, along the TTR, when comparing the sperm that were incubated in a water bath in relation to those incubated in a hot plate during the period of 90 minutes. The heating plate can be used as a means of incubating sheep sperm in replacement of the water bath.

Influence of glycerol addition on the quality of cat epididymal sperm during the freeze-thaw process / Influência da adição do glicerol sobre a qualidade dos espermatozoides epididimários de gato durante as etapas da congelação-descongelação

Cryopreservation of epididymal sperm is a useful tool for preserving the genetic potential of valuable animal specimens. The domestic cat is used as a model to study and develop cryogenics for other felines. However, regulation of the entire cryopreservation process is essential for the success of this biotechnology. Thus, our aim was to evaluate the effects of glycerol equilibration time and freezethaw stages on the quality of epididymal sperm obtained from domestic cats. Epididymal sperm were recovered with TRIS and immediately evaluated for total motility (TM), vigor, viability, membrane functionality (HOST), and morphology. Then, TRIS-20% egg yolk was added to the samples, which were equally divided into two 1.5 mL tubes and refrigerated at 4 ºC for 1 hour. Subsequently, glycerol was added at a final concentration of 5%. The samples were incubated with glycerol (equilibration time) for either 5 or 10 minutes (groups G5 and G10, respectively) and then frozen. Thawing occurred at 37 ºC for 30 seconds. The samples were evaluated at all stages. A reduction in TM was observed only after thawing; however, it was higher in G5 (39.00 ± 4.07%) than in G10 (18.50 ± 4.54%). Vigor declined in both groups after thawing; however, they did not differ from each other. Sperm viability was maintained in G5 after glycerolization (53.60 ± 2.59%); in G10, sperm viability decreased in the glycerolized sample (48.80 ± 2.93%) when compared to that in the fresh sample (59.90 ± 1.74%). Post-thaw viability of G5 (33.80 ± 1.89%) was higher than that of G10 (18.80 ± 3.01%). In the HOST, a decrease in viability was only observed after thawing, with no difference between the groups (41.50 ± 2.84% for G5 and 40.20 ± 3.49% for G10). With regard to sperm morphology, normal sperm decreased while sperm with post-thaw secondary defects increased in both groups. In conclusion, a shorter equilibration time for glycerolization preserves epididymal sperm quality better and the freeze-thaw process is the most critical stage of thawing.(AU)

A criopreservação dos espermatozoides epididimários é uma ferramenta útil para preservar o potencial genético de um animal valioso. Além disso, o gato doméstico é modelo eleito para o estudo e desenvolvimento da criogenia para os demais felinos. Contudo, para o sucesso dessa biotécnica é essencial o controle de todo o processo de criopreservação. Assim, objetivou-se avaliar o efeito do tempo de equilíbrio da glicerolização e das etapas da congelação-descongelação sobre a qualidade dos espermatozoides epididimários de gato doméstico. Para tanto, espermatozoides epididimários foram recuperados com TRIS e imediatamente avaliados quanto à motilidade total (MT), vigor, viabilidade, funcionalidade de membrana (HOST) e morfologia. Em seguida, as amostras foram adicionadas de TRIS-gema a 20%, fracionadas igualmente em dois tubos de 1,5 mL, refrigeradas a 4 ºC por 1 hora e, posteriormente, adicionadas de glicerol na concentração final de 5%. As amostras foram incubadas com glicerol (tempo de equilíbrio) por 5 ou 10 minutos (grupos G5 e G10, respectivamente) e depois congeladas. A descongelação ocorreu a 37 ºC por 30 segundos. As amostras foram avaliadas em todas as etapas. Uma redução na MT foi observada apenas na pós-descongelação, no entanto G5 (39,00 ± 4,07%) foi superior ao G10 (18,50 ± 4,54%). O vigor declinou pós-descongelação em ambos os grupos; contudo, não diferiram entre si. A viabilidade espermática foi mantida no G5 pós-glicerolização (53,60 ± 2,59%), diferentemente do observado em G10, em que a amostra glicerolizada (48,80 ± 2,93%) reduziu em relação à fresca (59,90 ± 1,74%). A viabilidade pós-descongelação de G5 (33,80 ± 1,89%) foi superior à de G10 (18,80 ± 3,01%). No HOST, uma redução da viabilidade só foi observada pósdescongelação, não havendo diferença entre os grupos (41,50 ± 2,84% para G5 e 40,20 ± 3,49% para G10). Em relação à morfologia espermática, os espermatozoides normais diminuíram, enquanto os espermatozoides com defeitos secundários pós-descongelação aumentaram em ambos os grupos. Conclui-se que um menor tempo de equilíbrio para a glicerolização preserva melhor a qualidade dos espermatozoides epididimários e a etapa mais crítica do processo de congelação-descongelação é a descongelação.(AU)

Avaliação estrutural de espermatozoide caprino criopreservado, através da microscopia eletrônica de transmissão / Ultrastructural evaluation of cryopreserved caprine sperm through transmission electronic microscopy

The ultrastructure evaluation allows the analysis of the sperm cell in a subcellular proportion that is not observed in optical microscopy. Transmission electron microscopy (MET) is a tool used to determine the size and shape of inorganic and biological structures based on the interaction of electrons incident on matter. In this sense, with the use of MET, the objective was to evaluate the ultrastructural changes after the semen manipulation, mainly in the freeze-thaw process, which were not observed in tests using optical microscopy, that is, the morphological integrity of the membranes and goat sperm organelles. The evaluation took place at the Institute of Biosciences of the University of Brasília (UnB). The semen straws were thawed and washed in PBS. Then, they were centrifuged and fixed, contrasted in bloc and subjected to dehydration. The samples were placed for polymerization and ultrathin cuts were made and stored until the time of MET evaluation. In the ultrastructural analyzes by MET in the present work, no harmful actions occurred in either the control or experimental groups. The head regions (plasma membrane and acrosome) remained preserved, with no change in DNA. In conclusion, MET is especially useful tool for cell evaluation.

Avaliação estrutural de espermatozoide caprino criopreservado, através da microscopia eletrônica de transmissão / Ultrastructural evaluation of cryopreserved caprine sperm through transmission electronic microscopy

The ultrastructure evaluation allows the analysis of the sperm cell in a subcellular proportion that is not observed in optical microscopy. Transmission electron microscopy (MET) is a tool used to determine the size and shape of inorganic and biological structures based on the interaction of electrons incident on matter. In this sense, with the use of MET, the objective was to evaluate the ultrastructural changes after the semen manipulation, mainly in the freeze-thaw process, which were not observed in tests using optical microscopy, that is, the morphological integrity of the membranes and goat sperm organelles. The evaluation took place at the Institute of Biosciences of the University of Brasília (UnB). The semen straws were thawed and washed in PBS. Then, they were centrifuged and fixed, contrasted in bloc and subjected to dehydration. The samples were placed for polymerization and ultrathin cuts were made and stored until the time of MET evaluation. In the ultrastructural analyzes by MET in the present work, no harmful actions occurred in either the control or experimental groups. The head regions (plasma membrane and acrosome) remained preserved, with no change in DNA. In conclusion, MET is especially useful tool for cell evaluation.(AU)

Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.(AU)

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.(AU)

Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.

Effec t of Freeze-Thaw Cycles on Lipid Oxidation and Myowater in Broiler Chickens

The present study was carried out to investigate the influence of freezing-thawing cycles (0, 2, 4 and 6) on lipid oxidation and myowater contents and distribution. Nine replicates of chicken breast meat samples were used for each cycle. Lipid oxidation was determined by measuring peroxide value, and malondialdehyde (MDA) concentrations, which reflect thiobarbituric acid reactive substance (TBARS). Color was determined with a digital colorimeter. Muscle moisture contents were determined by drip loss and thawing loss, water holding capacity, and nuclear magnetic resonance (NMR). The results showed that, as the number of freeze-thaw cycles increased, meat redness decreased and MDA and peroxide values increased. Drip loss and thawing loss tended to decreasing as the number of freeze-thaw cycles increased. Water holding capacity also decreased as a function of increasing freeze-thaw cycles. NMR relaxometry profile showed freeze-thaw cycles change the water distribution of meat subjected to multiple freeze-thaw cycles. In conclusion, multiple freezing and thawing rate (6 cycles) increased lipid oxidation, decreased myowater, and impaired the color of chicken meat.

Effec t of Freeze-Thaw Cycles on Lipid Oxidation and Myowater in Broiler Chickens

The present study was carried out to investigate the influence of freezing-thawing cycles (0, 2, 4 and 6) on lipid oxidation and myowater contents and distribution. Nine replicates of chicken breast meat samples were used for each cycle. Lipid oxidation was determined by measuring peroxide value, and malondialdehyde (MDA) concentrations, which reflect thiobarbituric acid reactive substance (TBARS). Color was determined with a digital colorimeter. Muscle moisture contents were determined by drip loss and thawing loss, water holding capacity, and nuclear magnetic resonance (NMR). The results showed that, as the number of freeze-thaw cycles increased, meat redness decreased and MDA and peroxide values increased. Drip loss and thawing loss tended to decreasing as the number of freeze-thaw cycles increased. Water holding capacity also decreased as a function of increasing freeze-thaw cycles. NMR relaxometry profile showed freeze-thaw cycles change the water distribution of meat subjected to multiple freeze-thaw cycles. In conclusion, multiple freezing and thawing rate (6 cycles) increased lipid oxidation, decreased myowater, and impaired the color of chicken meat.(AU)

Physicochemical characteristics and lipid oxidation of chicken inner fillets subjected to different thermal processing types

The objective of this study was to evaluate the effects of different types of thermal processing on the physiochemical characteristics and lipid oxidation of chicken inner fillets. The study was divided into three assays. In the first assay, 50 chicken inner fillets were divided into five treatments, totaling 10 samples per treatment. Treatments consisted in cooking in water bath, electric oven, microwave oven, deep frying, or grilling. The analyzed variables were: cooking weight loss (CWL) and lipid oxidation determined by thiobarbituric acid reactive substances (TBARS). In the second assay, 50 chicken inner fillets were divided into five treatments, totaling 10 samples per treatment. Each treatment consisted of the same cooking methods applied in the first assay, and storage for 48 hours under refrigeration and reheating in a microwave oven. The variable analyzed in the second assay was lipid oxidation (TBARS). In the third assay, 30 samples of chicken inner fillets were subjected to one, four and eight freeze-thaw cycles, after which meat pH, myofibrillar fragmentation index (MFI), water retention capacity (WRC), and lipid oxidation (TBARS) were determined. Chicken inner fillets submitted to deep frying and cooked in a microwave oven presented greater lipid oxidation than the other cooking methods, and deep frying resulted in the highest cooking weight loss. Reheating chicken inner fillets in a microwave oven caused the highest meat lipid oxidation. Increasing the number of freeze-thaw cycles increases the pH, MFI, WRC and TBARS values of chicken inner fillets.

Physicochemical characteristics and lipid oxidation of chicken inner fillets subjected to different thermal processing types

The objective of this study was to evaluate the effects of different types of thermal processing on the physiochemical characteristics and lipid oxidation of chicken inner fillets. The study was divided into three assays. In the first assay, 50 chicken inner fillets were divided into five treatments, totaling 10 samples per treatment. Treatments consisted in cooking in water bath, electric oven, microwave oven, deep frying, or grilling. The analyzed variables were: cooking weight loss (CWL) and lipid oxidation determined by thiobarbituric acid reactive substances (TBARS). In the second assay, 50 chicken inner fillets were divided into five treatments, totaling 10 samples per treatment. Each treatment consisted of the same cooking methods applied in the first assay, and storage for 48 hours under refrigeration and reheating in a microwave oven. The variable analyzed in the second assay was lipid oxidation (TBARS). In the third assay, 30 samples of chicken inner fillets were subjected to one, four and eight freeze-thaw cycles, after which meat pH, myofibrillar fragmentation index (MFI), water retention capacity (WRC), and lipid oxidation (TBARS) were determined. Chicken inner fillets submitted to deep frying and cooked in a microwave oven presented greater lipid oxidation than the other cooking methods, and deep frying resulted in the highest cooking weight loss. Reheating chicken inner fillets in a microwave oven caused the highest meat lipid oxidation. Increasing the number of freeze-thaw cycles increases the pH, MFI, WRC and TBARS values of chicken inner fillets.(AU)

Estudo do perfil de miRNAs de ejaculados suínos com diferentes congelabilidades

Resumo da tese em português: A criopreservação espermática é o método mais eficiente para preservar sêmen a longo prazo em mamíferos. No entanto, o congelamento do sêmen de cachaços ainda é o maior desafio para a indústria suína devido à alta sensibilidade ao choque frio das células espermáticas desta espécie e à variação dos resultados pós-descongelamento entre indivíduos e ejaculados do mesmo reprodutor. Para resolver esse problema, investigamos se os microRNAs (miRNAs) presentes em células espermáticas e pequenas vesículas extracelulares (EVs) do plasma seminal de ejaculados de suínos podem predizer se ejaculados permanecerão com alta qualidade após passarem pelo processo de congelação-descongelação. Para tanto, foram coletados 27 ejaculados de alta qualidade de 27 machos (um de cada), obtidas amostras de miRNAs de espermatozoides e das EVs de plasma seminal in natura através de protocolos de centrifugação e, após o processo de criopreservação, determinamos dois grupos com diferentes congelabilidades considerando as análises de estrutura e funcionalidade dos espermatozoides após a descongelação (P <0,05): Alta congelabilidade (AC; n = 04) e baixa congelabilidade (BC; n = 04). Feito isso, finalmente investigamos o perfil de miRNAs de espermatozoides e EVs de plasma seminal em ambos os grupos com diferentes congelabilidades. Nossos principais resultados mostraram que três miRNAs foram diferentemente abundantes em ejaculados BC, sendo o ssc-miR-503 encontrado em níveis mais elevados em células espermáticas. O ssc-miR-130a e o ssc-miR-9 foram mais abundantes em EVs do plasma seminal (P <0,10). Por meio da análise de enriquecimento, foi possível verificar que esses miRNAs estão relacionados as modificações no desenvolvimento das células germinativas masculinas e na produção de energia utilizada pelos espermatozóides para manter sua viabilidade e funcionalidade. Portanto, por meio deste estudo, podemos demonstrar que o ssc-miR-503, ssc-miR-130a e ssc-miR-9 estão relacionados à baixa criotolerância espermática no sêmen de suínos. Deste modo, esses miRNAs podem ser usados como biomarcadores para predizer sua baixa capacidade de tolerar o processo de criopreservação.

Resumo da tese em inglês: Sperm cryopreservation is the most efficient method to preserve sêmen for the long term in mammals. However, freeze boar sêmen is still the biggest challenge for the swine industry due to the high cold shock sensitivity of boar sperm cells and the variance of post-thaw results among individuals and ejaculates from the same boar. To solve this problem, we investigate if microRNAs (miRNAs) present in sperm cells and small extracellular vesicles (E.V.s) from seminal plasma of raw boar ejaculates can predict high-quality ejaculates after underwent the freeze-thaw process. Therefore, we collected 27 high-quality ejaculates of 27 boars (one of each) obtained miRNAs samples of sperm cells and E.V.s from raw seminal plasma through centrifugation protocols. After the cryopreservation process, we determined two groups with different freezability considering the analysis post-thaw of structure and sperm functionality (P <0.05): High freezability (H.F.; n=04) and low freezability (L.F.; n=04). That done, we finally investigate the miRNAs profile of sperm cells and E.V.s from seminal plasma in both groups with different freezability. Our main results showed that three miRNAs were differently abundant in L.F. ejaculates, being the ssc-miR-503 found in higher levels in sperm cells. The ssc-miR-130a and ssc-miR-9 most abundant in E.V.s from seminal plasma (P <0.10). Through enrichment analysis, it was possible to verify that these miRNAs could be performing modifications in the development of male germ cells and in the production of energy to spermatozoa to maintain their viability and functionality. Therefore, through this study, we can demonstrate that ssc-miR-503, ssc-miR-130a, and ssc-miR-9 are related to low sperm cryotolerance in boars semen. So those miRNAs can be used as a biomarker to predict their low ability to tolerate the cryopreservation process.

Influência do tempo de equilíbrio sobre a qualidade do sêmen caprino após a descongelação / Influence of balance time on quality of buck semen after thawing

The animals were divided into three groups G1, G2, G3. Semen collected was diluted in Tris-yolk baseand packaged in 0.25 ml straws, a dose of 100 x 106 sperm. Semen samples were distributed in differentequilibrium time protocols. The group 1 (1h), group 2 (2 h), group 3 (3 h). Mean values for motility were19.37%, 16.87% and 16.25% respectively for G1, G2 and G3. To analyze (TTR) which consisted of placing asemen , and incubated at 37°C for a period of 120 minutes. The force sperm motility and sperm were evaluatedby TTR in different time: 0, 60, 120, 180 minutes. The indices obtained from motility and sperm vigor wereevaluated by PROC GLM (SAS, 2001) and proceeded to the 5% Tukey test. This study demonstrated effect theequilibration period, on the viability of goat semen after the freeze-thaw process, in which the semen exposed toless cooling time has a higher sperm viability.(AU)

Influência do tempo de equilíbrio sobre a qualidade do sêmen caprino após a descongelação / Influence of balance time on quality of buck semen after thawing

The animals were divided into three groups G1, G2, G3. Semen collected was diluted in Tris-yolk baseand packaged in 0.25 ml straws, a dose of 100 x 106 sperm. Semen samples were distributed in differentequilibrium time protocols. The group 1 (1h), group 2 (2 h), group 3 (3 h). Mean values for motility were19.37%, 16.87% and 16.25% respectively for G1, G2 and G3. To analyze (TTR) which consisted of placing asemen , and incubated at 37°C for a period of 120 minutes. The force sperm motility and sperm were evaluatedby TTR in different time: 0, 60, 120, 180 minutes. The indices obtained from motility and sperm vigor wereevaluated by PROC GLM (SAS, 2001) and proceeded to the 5% Tukey test. This study demonstrated effect theequilibration period, on the viability of goat semen after the freeze-thaw process, in which the semen exposed toless cooling time has a higher sperm viability.

Criopreservação do sêmen caprino: efeito do tempo de equilíbrio sobre a qualidade do sêmen após a descongelação / Cryopreservation of goat semen: Balancing time effect on semen quality after thawing

Which was used sixteen samples of adult goats semen collected using an artificial vagina weresubjected to cryopreservation in order to verify the influence of equilibration time at a temperature of 5 °C onthe freezability of goat semen. Were distributed in the different protocols equilibrium time: 1h equilibration time(G1); 2 h equilibration time (G2); 3h equilibration time (G3).After this process the samples were placed inliquid nitrogen vapor , and maintained for 20 min . The end was made the freezing process in cryogenics tank .Thawing was performed in a water bath at 37°C for 30 seconds. The averages for motility were 19.375 %,16.875 % and 16.250 % respectively for G1, G2 and G3. This study demonstrated effect of equilibration period ,on the viability of goat semen after the process of freeze-thaw, where the group of smaller G1 exposure time wasless deleterious effects.(AU)

Criopreservação do sêmen caprino: efeito do tempo de equilíbrio sobre a qualidade do sêmen após a descongelação / Cryopreservation of goat semen: Balancing time effect on semen quality after thawing

Which was used sixteen samples of adult goats semen collected using an artificial vagina weresubjected to cryopreservation in order to verify the influence of equilibration time at a temperature of 5 °C onthe freezability of goat semen. Were distributed in the different protocols equilibrium time: 1h equilibration time(G1); 2 h equilibration time (G2); 3h equilibration time (G3).After this process the samples were placed inliquid nitrogen vapor , and maintained for 20 min . The end was made the freezing process in cryogenics tank .Thawing was performed in a water bath at 37°C for 30 seconds. The averages for motility were 19.375 %,16.875 % and 16.250 % respectively for G1, G2 and G3. This study demonstrated effect of equilibration period ,on the viability of goat semen after the process of freeze-thaw, where the group of smaller G1 exposure time wasless deleterious effects.

COMPARAÇÃO DAS TÉCNICAS DE CRIOCIRURGIA, VASECTOMIA E EPIDIDIMECTOMIA PARA A FORMAÇÃO DE RUFIÕES CAPRINOS (Capra hircus)

O estudo foi realizado durante o período de março de 2019 a fevereiro de 2020, utilizando-se animais provenientes do Setor de Caprinocultura, do Instituto de Zootecnia (IZ) da Universidade Federal Rural do Rio de Janeiro (UFRRJ).Foram utilizados 30 caprinos, mestiços de Saanen x Boer, machos, inteiros, púberes, com idade entre 10 e 24 meses. Todos os animaisforam pesados, identificados com placas numeradas fixadas por meio de coleira na região da orelha e do pescoço, fotografados de frente e perfil e avaliados clinicamente. Os caprinos foram alojados em baias coberta suspensas, com dimensões de 5x3m (15m²), soltos durante o dia em piquete anexo e recolhidos ao entardecer. Na baia fornecidos capim picado e ração formulada pelo próprio setor de Zootecnia. Os animais submetidos ao exame andrológico e ao teste de libido, realizados no dia anterior à primeira intervenção cirúrgica e 90 dias após o procedimento cirúrgico. A coleta do sêmen realizada com o auxílio de uma vagina artificial própria para a espécie, uma semana antes de realizar os procedimentos cirúrgicos, 15 dias após os mesmos, e a seguir, semanalmente, até total azoospermia (máximo de 90 dias), verificados o aspecto e a coloração do ejaculado e uma pequena alíquota do mesmo depositada sobre lâmina de vidro coberta com lamínula, onde foram avaliados: motilidade espermática progressiva, vigor, turbilhonamento e posteriormente a concentração em câmara de Neubauer. Na semana anterior ao procedimento cirúrgico,foi realizado exame ultrassonográfico de testículo, epidídimo e plexo pampiniforme, com posterior acompanhamento mensal, de até 90 dias. Em todos os procedimentos experimentais, os animais foram sedados com midazolam (0,1mg/kg) e cloridrato de xilazina 2% (0,05mg/kg) e realizado um bloqueio local com lidocaína 2% sem vasoconstrictor no local da incisão. No pós-operatório foram administrados oxitetraciclina (0,1ml/kg) e flunexin meglumine (1,1mg/kg), por via intramuscular, por 2 dias. Os animais foram divididos em grupos segundo os procedimentos cirúrgico: criocirurgia (n=10), vasectomia (n=10) e epididimectomia (n=10). Todos os animais foram submetidos ao procedimento de orquiectomia após os 90 dias de avaliação. O Objetivo do estudo foi comparar a eficiência da criocirurgia com as técnicas cirúrgicas de vasectomia e epididimectomia para formação de rufião caprino, avaliando a eficácia da criocirugia na cauda do epidídimo para a obtenção de esterilização irreversível de machos, avaliação da presença de libido nos animais e avaliação histopatológica, após 90 dias em todos os grupos.

The study will be carried out during the period of 2019/2020, using animals from the Department of Animal Production of the Institute of Animal Science (IZ) of the Federal Rural University of Rio de Janeiro (UFRRJ). It will be used 30 goats, crossbred Saanen x Boer, males, whole, pubescent, aged between 6 and 8 months. All animals will be weighed, identified with numbered plaques fixed by collar in the neck region and photographed front and profile and clinically evaluated. The goats will be housed in suspended covered bays, with dimensions of 5x3m (15 m²), loose during the day in attached picket and collected at dusk. In the bay will be provided chopped grass and feed formulated in the Animal Husbandry sector. The animals will undergo andrological examination, performed at two different times: the day before the first surgical intervention and 90 days after the surgical procedure. The semen collection will be performed with the aid of an artificial vagina suitable for the species, one week before performing the surgical procedures and after 15 days of the same, once a week until total azoospermia (maximum 90 days). Initially, the appearance and color of the ejaculate will be checked. A small aliquot of the ejaculate will be deposited on a sheet of glass covered with coverslip, where they will be evaluated: progressive sperm motility, vigor and swirling and later concentration in a hematimetric chamber (Neubauer chamber). The animals will be submitted to the libido test at two different times, T0 and T90 days after the procedures. In all 3 experimental procedures, the animals will be sedated with 0.2 mg / kg of midazolam and 0.4 mg / kg of xylazine hydrochloride 2%. Afterwards, a local blockade with 2% lidocaine without vasoconstrictor will be performed at the incision site. In the postoperative period, 2mg / kg of tramadol hydrochloride and 25mg / kg of sodium dipyrone will be administered intramuscularly. Technical group of cryosurgery - After palpation and stipulation of the point of introduction of the needle, the region of the tail of the epididymis will be transfixed mid-way in the lateral medial direction with the use of a disposable sterile hypodermic needle 40mm x 0.8 The needle, when inserted into the tissue, will be coupled to a circuit to the cryosurgery apparatus. Immediately after the application of two freeze / thaw cycles, the time of 60 seconds / 45 seconds, respectively. Technical group of epididymectomy - the skin will be incised in the region of the tail of the epididymis, proceeding to the dissection of the structure and ligation of the vas deferens with inabsorbable wire, performing flat to flat raffia with inabsorbable wire. Vasectomy technique group - An incision will be made between the testis and the inguinal ring to locate the spermatic cord and posterior incision in the vaginal tunica, and the vas deferens will be isolated. A double ligation will be made in the vas deferens and incision of a part thereof and ending with the raffia of the subcutaneous tissue and the skin. The objective of the study is to compare the efficiency of minimally invasive cryosurgery with the surgical techniques of vasectomy and epididymectomy in goats.

CRIOPRESERVAÇÃO DE SÊMEN DE ZEBRAFISH

O zebrafish apresenta diversas características interessantes, que fazem da espécie um modelo biológico popular para estudos de diversas áreas de pesquisa, resultando em um elevado desenvolvimento de novas linhagens que precisam ser mantidas e preservadas. A criopreservação pode atender essa necessidade de manter os recursos genéticos da espécie, o que torna o desenvolvimento de pesquisas na área de suma importância. Esta tese tem como objetivo principal realizar um levantamento do histórico da pesquisa sobre criopreservação em zebrafish e realizar o desenvolvimento de protocolos de criopreservação de sêmen de zebrafish utilizando diversos tipos de avaliações sobre os danos causados pelo processo criopreservação. O objetivo do primeiro estudo foi realizar uma revisão sistemática sobre a criopreservação de tecidos e células reprodutivas de zebrafish. Na revisão sistemática foi possível identificar e quantificar as principais características dos protocolos de criopreservação já utilizados em diferentes tipos de amostras. Com os resultados obtidos nesse estudo foi possível identificar limitações e tendências para novas pesquisas, além de confirmar que assim como em outras espécies de peixes, a criopreservação de sêmen é o método mais efetivo para preservação do material genético do zebrafish. No segundo estudo objetivou-se avaliar a combinação e interação de diferentes extensores, crioprotetores permeáveis e não permeáveis, analisando os efeitos da criopreservação sobre os espermatozoides através de uma abordagem ampla de variáveis. Nesse estudo foi possível identificar que a utilização do leite em pó na solução crioprotetora promoveu maior proteção aos espermatozoides, especialmente quanto aos danos oxidativos e de DNA. No terceiro estudo o objetivo foi avaliar o estresse oxidativo e os danos no DNA em diferentes etapas do processo de criopreservação. Observou-se que a etapa de congelamento/descongelamento gerou mais danos que a exposição do sêmen aos crioprotetores durante o equilíbrio. Foi possível identificar que a utilização de leite em pó diminuiu a geração de espécies reativas de oxigênio e a fragmentação de DNA, além de promover maior atividade antioxidante. Com todos os resultados obtidos foi possível observar que a criopreservação de sêmen de zebrafish atualmente é a alternativa mais concreta para preservação do material genético da espécie, e que os protocolos de criopreservação de sêmen devem ser otimizados levando em consideração uma abordagem mais ampla das variáveis. Os bons resultados obtidos com a inclusão do leite em pó na solução crioprotetora indicam que a combinação entre crioprotetor permeável e não permeável pode melhorar os resultados obtidos após o processo de criopreservação.

The zebrafish has several interesting characteristics, which make the species a popular biological model for studies in several research areas, resulting in a high development of new strains that need to be maintained and preserved. Cryopreservation can meet the need to maintain the genetic resources of the species, which makes the development of research in the area of extreme importance. The main objective of this thesis is to carry out a survey of the history of research on cryopreservation in zebrafish and to develop protocols for cryopreservation of zebrafish semen using different types of assessments on the damage caused by the cryopreservation process. The aim of the first study was to conduct a systematic review on the cryopreservation of zebrafish reproductive tissues and cells. In the systematic review, it was possible to identify and quantify the main characteristics of the cryopreservation protocols already used in different types of samples. With the results obtained in this study, it was possible to identify limitations and trends for new research, in addition to confirming that as in other fish species, cryopreservation of semen is the most effective method for preserving zebrafish genetic material. In the second study, the objective was to evaluate the combination and interaction of different extenders, permeable and non-permeable cryoprotectants, analyzing the effects of cryopreservation on sperm through a wide approach of variables. In this study, it was possible to identify that the use of powdered milk in the cryoprotective solution promoted greater protection for sperm, especially regarding oxidative and DNA damage. In the third study, the objective was to assess oxidative stress and DNA damage at different stages of the cryopreservation process. It was observed that the freeze/thaw stage generated more damage than the semen exposure to cryoprotectants during equilibrium. It was possible to identify that the use of powdered milk decreased the generation of reactive oxygen species and the fragmentation of DNA, in addition to promoting greater antioxidant activity. With all the results it was observed that the zebrafish sperm cryopreservation is currently the most concrete alternative to preserving the genetic material of the species, and that semen cryopreservation protocols must be optimized considering a broader approach of the variables. The good results obtained with the inclusion of powdered milk in the cryoprotective solution indicate that the combination of permeable and non-permeable cryoprotectants can improve the results obtained after the cryopreservation process.

COLETA E CRIOPRESERVAÇÃO DE SÊMEN DE CACHORRO-DO-MATO (CERDOCYON THOUS)

O desenvolvimento de técnicas reprodutivas que possam ser utilizadas nos animais em vida-livre permite coletar e preservar material genético que convencionalmente não teríamos acesso, permitindo aumentar a variabilidade de amostras em um biobanco. Porém, infelizmente, muitas das técnicas conhecidas e amplamente utilizadas, foram desenvolvidos em ambientes laboratoriais controlados e que quando aplicados a campo, não é possível obter os mesmos resultados, sendo necessário a criação de novos protocolos para essas situações. Entre os meios comerciais, é de maior interesse para uso a campo, um diluente que possa ser armazenado à temperatura ambiente, sem necessidade de armazenamento a 5°C e que obtenha bons resultados com aumento do tempo de equilíbrio. É por isto que neste estudo, objetivou-se avaliar a viabilidade de coletar sêmen de Cachorro-do-mato (Cerdocyon thous), in situ, utilizando a técnica da cateterização uretral após indução farmacológica com medetomidina na dose 0,1 mg/kg e avaliar se o meio de congelamento de sêmen comercial (optiXcell) que permite armazenamento a temperatura ambiente para uso em canídeos, facilitando sua manutenção e utilização a campo. Além disso, testou-se também dois períodos de equilíbrio (duas e quatro horas) no resfriamento com o objetivo de avaliar se é possível prolongar esse tempo para quatro horas, o que tornaria mais favorável seu uso a campo. Com o primeiro experimento, utilizando cães domésticos como modelo experimental, foi avaliado a qualidade seminal pós-descongelamento em três diluentes comerciais (CaniPlus Freeze, Triladyl, OptiXcell) após duas e quatro horas de resfriamento através de análise subjetiva, por sistema computadorizado de análise seminal e por citometria de fluxo. Neste experimento, obtivemos resultados favoráveis ao uso do optixcell com quatro horas de tempo de equilíbrio, este tratamento demonstrou boas taxas de manutenção das membranas espermáticas, além disso obteve resultados próximos aos outros dois meios já utilizados em cães domésticos, podendo ser uma nova opção prática. Para o segundo experimento, a coleta farmacológica em cachorros-do-mato obteve quatro coletas com qualidade para criopreservação, demonstrando que este método pode ser utilizado em momentos que a manipulação digital ou eletroejaculação não possam ser utilizados. Também foi observado que os melhores resultados de coleta foram obtidos nos meses de junho a setembro, podendo indicar sazonalidade reprodutiva. Não foi possível realizar o descongelamento de sêmen de cachorros-do-mato com qualidade, muito provavelmente devido aos intemperes de sua manipulação em ambiente não controlado, demonstrando a necessidade e urgência de estabelecermos protocolos aplicáveis a campo.

The development of reproductive techniques that can be used in free-living animals allows the collection and preservation of genetic material that we would not conventionally have access to, allowing to increase the variability of samples in a biobank. However, unfortunately, many of the known and widely used techniques were developed in controlled laboratory environments and that when applied to the field, it is not possible to obtain the same results, being necessary to create new protocols for these situations. Among commercial freezing médium for semen, a diluent that can be stored at room temperature, without the need for storage at 5°C and that obtains good results with extended equilibrium time, is of most interest for use in the field. That is why this study aimed to evaluate the viability of collecting semen from Crab-eating-fox (Cerdocyon thous), in situ, using the technique of urethral catheterization after pharmacological induction with medetomidine at a dose of 0.1 mg/kg and evaluate whether the comercial semen freezing medium (optiXcell) that allows storage at room temperature can be used in canids, facilitating their maintenance and use in the field. In addition, two periods of equilibrium (two and four hours) in the cooling were also tested in order to assess whether it is possible to extend this time to four hours making it more effective in the field. With the first experiment, using domestic dogs as an experimental model, the post-thaw seminal quality was evaluated in three commercial diluents (CaniPlus Freeze, Triladyl, OptiXcell) after two and four hours of cooling. They were analyzed through computerized seminal analysis system for sperm motility and by flow cytometry. In this experiment, we obtained favorable results for the use of optixcell in dogs sperm with four hours of equilibrium time, this treatment demonstrated good maintenance rates of sperm membranes, and in addition, the OptiXcell medium obtained results close to the other two extenders already used in domestic dogs, which can be a new practical option. For the second experiment, pharmacological collection in crab-eatingfoxes obtained four collections with quality for cryopreservation, demonstrating that this method can be used at times when digital manipulation or electroejaculation cannot be used. It was also observed that the best collection results were obtained between June and September, which may indicate reproductive seasonality. Although the optixcell medium has been validated for use in domestic dogs, it was not possible to thaw semen from wild dogs withquality and viability of use, most likely due to the weathering of its handling in an uncontrolled environment, demonstrating the need and urgency to establish protocols applicable to the field.