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A Caatinga, bioma exclusivamente brasileiro, abriga grande diversidade biológica, porém sofre com graves ameaças ambientais. Por isso, é iminente a necessidade de desenvolvimento de técnicas voltadas para a conservação dos animais que nela habitam, bem como se seu germoplasma. Quando do súbito óbito de um animal biologicamente valioso, a recuperação de espermatozoides epididimários pode se apresentar como a única possibilidade para salvaguardar gametas. Ainda, esta biotécnicas configura-se em uma ferramenta possível de ser utilizada quando não há outra forma de se coletar o sêmen em determinada espécie. Os espermatozoides coletados podem ser armazenados por meio da criopreservação, e posteriormente utilizados em outras biotecnologias. Neste sentido, esta revisão tem como objetivo apresentar os aspectos da recuperação, caracterização e criopreservação de espermatozoides epididimários em animais silvestres com principal foco em espécies do bioma Caatinga, como os preás, cutias, catetos e emas.(AU)

The Caatinga, an exclusively Brazilian biome, is home to great biological diversity, but suffers from serious environmental threats. Therefore, there is an imminent need to develop techniques aimed at the conservation of the animals that inhabit it, as well as their germplasm. When the sudden death of a biologically valuable animal, the recovery of epididymal spermatozoa may present itself as the only possibility to safeguard gametes. Still, this biotechnique is a tool that can be used when there is no other way to collect semen in each species. Collected spermatozoa can be stored through cryopreservation, and later used in other biotechnologies. In this sense, this review aims to present aspects of recovery, characterization, and cryopreservation of epididymal spermatozoa in wild animals with a focus on species from the Caatinga biome, such as cavies, agoutis, collared peccary, and rheas.(AU)

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The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.(AU)

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The mule is a sterile hybrid domestic animal that results from the breeding of a male donkey with a female horse, understanding the reproductive biology of these species is very critical. The goal of this paper was to perform a comparative and more accurate histomorphometric of the testicles in Barb horse, donkeys and mules. Microscopic examinations and histological description were carried on genital tract of horses, donkeys and mules healthy and mature; this study was conducted during April-May 2018. The histological and the morphological results shows a similarity between the two equine species and the infertile hybrid for the testicles, the epididymis and the vas deferens. However, the difference was presented on the morphometric data; vas deferens was more voluminous in the horse and donkey than a mule. Moreover, the differences were significantly higher for the surface of the seminiferous tubules and for the epididymis. The lumen of the seminiferous tubules in mule was significantly higher than in the horse and donkey. Absence of gametes in the epididymal cavity and lower number of gametes in the mule. Furthermore, we have noted the presence of spermatozoa in one mule 16.67%. Therefore, the mule could complete development of spermatogenesis.(AU)

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The present study investigated the toxic effect of a mixture of three pesticides (cypermethrin, mancozeb, and metalaxyl) on reproduction and oxidative stress parameters in male Wistar rats. Animals were treated at doses 1/60, 1/30, and 1/10 LD50 of each pesticide daily in the diet for 08 weeks. At the end of the treatment period, animals were sacrificed by decapitation. The results indicate a decrease in the absolute weight of testes and epididymis, the serum of testosterone hormone, and cholesterol levels. These parameters were significant reduced in males exposed to the mixed pesticides. A reduction in sperm concentration, motility, and viability also was observed. Besides, the ingestion of mixed pesticides at all three concentrations caused a significant decrease in GSH, GPx levels and an increase in MDA levels compared to the control group. This was accompanied by histopathological changes in testis and epididymis of rats such as seminiferous tubules degeneration, decreasing number of spermatogenic cells, edema, expansion of interstitial spaces, cell necrosis, and reducing the diameter of the epididymal tube compared to the control group. Thus, we strongly suggest that the mixture of pesticides causes damages to the male reproductive system.

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Arsenic exposure is a global health concern. This toxic metalloid is ubiquitous in the environment and contaminates food and drinking water. Once ingested, it undergoes a complex metabolic process within the body, which contributes to its accumulation and reactivity. Arsenic toxicity stems from the induction of oxidative stress, inhibition of thiol-containing proteins, and mimicry of inorganic phosphates. Arsenic poisoning is associated with the development of reproductive disorders. In males, arsenic causes a reduction in testicular weight and alterations in steroidogenesis and spermatogenesis. Moreover, it reduces the number and quality of spermatozoa harvested from the cauda epididymis. The mitochondria are targets of arsenic toxicity because of the production of free radicals and their high content of cysteine-rich proteins and fatty acids. Mitochondrial dysfunction may contribute to reproductive disorders because this organelle is crucial for controlling testicular and epididymal events related to sperm production and maturation. All of these alterations mediated by arsenic exposure contribute to the failure of male reproductive competence by reducing gamete viability. This review describes the potential mechanisms of arsenic toxicity, its detrimental effects on male reproductive organs, and consequences on sperm fertility.(AU)

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Abstract This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.

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Abstract ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.

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ABSTRACT Description and seasonal variation in epididymal histomorphometry of Dermanuracinerea (Chiroptera: Phyllostomidae) in a fragment of the Atlantic Forest in the Northeastern Brazil. This study aimed to evaluate seasonal patterns in the histomorphometry of the epididymis of Dermanura cinerea (Gervais, 1856) in a fragment of the Atlantic Forest in the Northeastern Brazil. Eighteen adult male specimens captured by mist net were used. The field-work occurred monthly over 18 months, during two consecutive nights. Meteorological data (precipitation) were obtained from the National Institute of Meteorology. After euthanasia, specimens had the epididymis collected, which were fixed and processed. The histological slides produced were stained by Hematoxylin - Eosin and analyzed by optical microscopy. The morphometric parameters analyzed were the tubular, lumen and epithelium areas, of the regions of the initial segment, caput, corpus and cauda of the epididymis. The histomorphometric data were submitted to the Mann-Whitney U test analyzes. The results showed that D. cinerea presented spermatozoa in all regions of the epididymis, except in the initial segment. The highest averages of the tubular, lumen and epithelial areas in the four regions were observed during the dry months. Therefore, D cinerea presented greater sensibility in the region of the cauda of the epididymis, during the months with low rainfall indices. This indicates that environmental conditions have considerable influence on the epididymal morphophysiology of this species, especially in relation to the storage of sperm in the tail of this organ, in area of Atlantic forest in northeastern Brazil.

RESUMO Esse estudo objetivou avaliar sazonalmente a histomorfometria do epidídimo de Dermanura cinerea (Gervais, 1856) em um fragmento de Mata Atlântica no nordeste do Brasil. Foram utilizados 18 espécimes machos adultos capturados por redes de neblina. As coletas ocorreram mensalmente ao longo de dezoito meses, durante duas noites consecutivas e os dados meteorológicos foram fornecidos pelo Instituto Nacional de Meteorologia. Depois de eutanasiados, os espécimes tiveram os epidídimos coletados e esses órgãos foram fixados e processados. As lâminas histológicas foram coradas por Hematoxilina - Eosina e analisadas em microscopia óptica. Os parâmetros morfométricos analisados foram as áreas do túbulo, do lúmen e do epitélio das regiões do segmento inicial, cabeça, corpo e cauda do epidídimo. Os dados histomorfométricos obtidos foram submetidos às análises no teste U de Mann-Whitney. Os resultados revelaram que D. cinerea apresentou espermatozoides em todas as regiões do epidídimo, exceto no segmento inicial. As maiores médias das áreas tubular, do lúmen e do epitélio nas quatro regiões, foram constatadas durante os meses secos. Portanto, D cinerea apresentou maior sensibilidade na região da cauda do epidídimo, ao longo dos meses com baixos índices pluviométricos. Isso indica que as condições ambientais apresentam considerável influência sobre a morfofisiologia epidídimária dessa espécie, sobretudo, em relação ao armazenamento de espermatozoides na cauda desse órgão, em área de Mata atlântica do nordeste brasileiro.

Resumo

Description and seasonal variation in epididymal histomorphometry of Dermanuracinerea (Chiroptera: Phyllostomidae) in a fragment of the Atlantic Forest in the Northeastern Brazil. This study aimed to evaluate seasonal patterns in the histomorphometry of the epididymis of Dermanura cinerea (Gervais, 1856) in a fragment of the Atlantic Forest in the Northeastern Brazil. Eighteen adult male specimens captured by mist net were used. The field-work occurred monthly over 18 months, during two consecutive nights. Meteorological data (precipitation) were obtained from the National Institute of Meteorology. After euthanasia, specimens had the epididymis collected, which were fixed and processed. The histological slides produced were stained by Hematoxylin - Eosin and analyzed by optical microscopy. The morphometric parameters analyzed were the tubular, lumen and epithelium areas, of the regions of the initial segment, caput, corpus and cauda of the epididymis. The histomorphometric data were submitted to the Mann-Whitney U test analyzes. The results showed that D. cinerea presented spermatozoa in all regions of the epididymis, except in the initial segment. The highest averages of the tubular, lumen and epithelial areas in the four regions were observed during the dry months. Therefore, D cinerea presented greater sensibility in the region of the cauda of the epididymis, during the months with low rainfall indices. This indicates that environmental conditions have considerable influence on the epididymal morphophysiology of this species, especially in relation to the storage of sperm in the tail of this organ, in area of Atlantic forest in northeastern Brazil.

Esse estudo objetivou avaliar sazonalmente a histomorfometria do epidídimo de Dermanura cinerea (Gervais, 1856) em um fragmento de Mata Atlântica no nordeste do Brasil. Foram utilizados 18 espécimes machos adultos capturados por redes de neblina. As coletas ocorreram mensalmente ao longo de dezoito meses, durante duas noites consecutivas e os dados meteorológicos foram fornecidos pelo Instituto Nacional de Meteorologia. Depois de eutanasiados, os espécimes tiveram os epidídimos coletados e esses órgãos foram fixados e processados. As lâminas histológicas foram coradas por Hematoxilina - Eosina e analisadas em microscopia óptica. Os parâmetros morfométricos analisados foram as áreas do túbulo, do lúmen e do epitélio das regiões do segmento inicial, cabeça, corpo e cauda do epidídimo. Os dados histomorfométricos obtidos foram submetidos às análises no teste U de Mann-Whitney. Os resultados revelaram que D. cinerea apresentou espermatozoides em todas as regiões do epidídimo, exceto no segmento inicial. As maiores médias das áreas tubular, do lúmen e do epitélio nas quatro regiões, foram constatadas durante os meses secos. Portanto, D cinerea apresentou maior sensibilidade na região da cauda do epidídimo, ao longo dos meses com baixos índices pluviométricos. Isso indica que as condições ambientais apresentam considerável influência sobre a morfofisiologia epidídimária dessa espécie, sobretudo, em relação ao armazenamento de espermatozoides na cauda desse órgão, em área de Mata atlântica do nordeste brasileiro.

Resumo

Técnicas de coleta de espermatozoides epididimários são utilizadas em diversas espécies animais, sendo ferramentas de biotecnologias importantes aplicadas em casos de animais que precisam ser castrados ou que vieram a óbito. O objetivo deste trabalho foi avaliar a cinética de espermatozoides criopreservados de cães obtidos por meio de técnicas de recuperação epididimária. Foram coletados 30 complexos testículo-epidídimos (CTE) de cães saudáveis, sendo que cada CTE de determinado animal foi destinado para cada técnica utilizada, 15 direcionados para a técnica de recuperação de espermatozoides epididimários por fluxo retrógrado (FR) e 15 por flutuação (FL). Os CTE foram acondicionados em sacos plásticos identificados, contendo solução salina e levados ao Laboratório de Biotecnologia da Reprodução Animal da Universidade Federal do Piauí (LBRA/UFPI) para a realização da diluição e criopreservação espermática após a recuperação epididimária. A congelação foi realizada por meio de uma rampa manual, com as amostras refrigeradas a 5 °C, mantidas em vapor de nitrogênio por 5 minutos e, posteriormente, colocadas no botijão. A análise computadorizada do sêmen (CASA) nas amostras descongeladas foi realizada na Universidade Estadual do Ceará (UECE), sendo avaliados dez parâmetros seminais. Foi empregada a análise de variância, aplicando o teste de Tukey. Nessas amostras, houve diferença significativa quanto à motilidade progressiva entre as técnicas testadas e a motilidade total apresentou valores superiores a 30%, não havendo influência das técnicas nos demais parâmetros seminais. Concluise que a cinética de espermatozoides de cães obtidos por recuperação epididimária avaliados pelo CASA apresentou resultados satisfatórios após a criopreservação.

Epididymal sperm collection techniques are used in several animal species and are important biotechnology tools applied to animals that needed to be castrated or that died. The objective of this study was to evaluate the kinetics of cryopreserved dog sperm obtained by epididymal recovery techniques. Thirty testicular-epididymal complexes (CTE) were collected from healthy dogs, with each CTE of a given animal being assigned to each technique used, 15 of them directed to the retrograde flow epididymal sperm recovery technique and 15 by fluctuation technique. The CTE were placed in identified plastic bags, containing saline solution and taken to the Animal Reproduction Biotechnology Laboratory of the Federal University of Piauí (LBRA/UFPI) for sperm dilution and cryopreservation after epididymal recovery. The freezing was carried out using a manual ramp, with the samples refrigerated at 5 °C, maintained in nitrogen vapor exposition five minutes and, later, placed in the container. Computer Assisted Sperm Analysis (CASA) was performed in the thawed samples at the State University of Ceará (UECE) and ten seminal parameters were evaluated. Analysis of variance was applied by the Tukey test. In thawed samples, there was a significant difference in progressive motility between the tested techniques and total motility presented values greater than 30%, with no influence of the recovery technique on remaining parameters. It was concluded that the sperm kinetics of dogs obtained by epididymal recovery evaluated by CASA presented satisfactory results after cryopreservation.

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O objetivo deste trabalho foi avaliar a recuperação de espermatozoides epididimários de cães castrados, utilizando as técnicas de fluxo retrógrado (FR) e flutuação (FL) em diluidor Tris-gema, antes e após a criopreservação. Foram coletados 30 complexos testículo-epididímos (CTE), sendo 15 para FR e 15 para FL, e, logo após a recuperação dos espermatozoides, foram analisadas as alterações morfológicas nessas células espermáticas. Após a adição do diluidor, foram avaliados os parâmetros de motilidade total (MOT) e vigor (V) espermáticos. O sêmen pós-criopreservado foi submetido ao teste de termorresistência nos tempos T0, T30, T60 e T90 minutos, além da avaliação das membranas plasmática e acrossomal por sondas fluorescentes. Não houve diferença estatística entre as técnicas quanto à MOT e ao vigor no sêmen diluído (FR-MOT: 82,3% e V: 3,4; FL-MOT: 79,6% e V: 3,2) e pós-criopreservado (FR-MOT: 34% e V: 2,8; FL-MOT: 30% e V: 2,7). A partir do T30, houve diferença significativa quanto à MOT e ao vigor nas técnicas utilizadas, e o tempo também prejudicou o acrossoma espermático a partir do T30. Conclui-se que as técnicas de recuperação de espermatozoides epididimários de cães castrados, testadas neste trabalho, podem ser utilizadas para refrigeração e criopreservação de sêmen.(AU)

The objective of this work was to evaluate the recovery of epididymal spermatozoa from castrated dogs using retrograde flow (FL) and flotation (FL) techniques in Tris-egg yolk diluent, before and after cryopreservation. Thirty testicle-epididymal complexes (CTE) were collected, 15 for FR and 15 for FL and soon after spermatozoid recovery, morphological changes in these spermatic cells were analyzed. After addition of the diluent, the parameters of total motility (MOT) and vigor (V) were evaluated. The post-cryopreserved semen was submitted to thermoresistance (TTR) test at T0, T30, T60 and T90 minutes, as well as the plasma and acrosomal membrane evaluation by fluorescent probes. There was no statistically significant difference between techniques tested for MOT and vigor in the diluted semen (FR-MOT: 82.3% and V: 3.4, FL-MOT: 79.6% and V: 3.2) and post-cryopreserved (FR-MOT: 34% and V: 2.8, FL-MOT: 30% and V: 2.7). From the T30 there was a significant difference regarding MOT and vigor in the used techniques, and the time also damaged the spermatic acrosome from the T30. It is concluded that the epididymal spermatozoa recovering techniques from castrated dogs, tested in this study, can be used for semen refrigeration and cryopreservation.(AU)

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This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P0.05) during freezing; however, after thawing, it decreased (P0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

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The objective of this study was to evaluate the morphology, morphometry, and membrane integrity of epididymal spermatozoa of spotted pacas using spermatic cells collected from the epididymal tails of five animals. The flotation method using the ACP-123® and Botusemen Special® extenders was performed, and samples were stained in Diff-Quick and eosin-nigrosine. Descriptive statistics of data were obtained and Students t-test was performed. The morphology of 200 Diff-Quick-stained spermatozoa showed that they had an oval head with three vesicles in the acrosomal region, a midpiece, an elongated tail; moreover, 27% of the spermatozoa exhibited cellular defects. The morphometry of 100 sperm cells (analyzed with an optical microscope and the EZ Leica LAS software for Windows) presented the following measurements (mean ± SD): total length 43.87 ± 4.91 μm, head 7.54 ± 0.82 μm, midpiece 5.35 ± 0.83 μm, tail 30.72 ± 2.55 μm, and head width 5.30 ± 0.68 μm. Of the 2,000 cells stained with eosin-nigrosine for membrane integrity evaluation, 83.8% diluted in ACP-123® and 72.9% diluted in Botusemen® had intact membranes. The results of this study suggest that epididymal spermatozoa of pacas can be used in assisted reproduction programs; moreover, our study adds knowledge to the reproductive biology of wild animals, and encourages further research on the role of the three acrosomal vesicles present in this species.

Com o objetivo de avaliar a morfologia, a morfometria e a integridade de membrana dos espermatozoides epididimários de paca, células espermáticas oriundas da cauda do epidídimo foram obtidas de cinco animais pelo método de flutuação, utilizando os diluidores ACP-123 ® e Botusemen Special®, coradas em Panótico rápido e Eosina-Nigrosina. Os dados foram analisados por estatística descritiva e teste t de Student. A morfologia de 200 espermatozoides corados em Panótico rápido evidenciou que os mesmos possuíam: cabeça ovalada com três vesículas na região acrossomal, peça intermediária, cauda alongada, sendo os defeitos celulares de 27%. A morfometria de 100 células espermáticas (efetuada com microscópio óptico e softwares EZ Leica LAS de aquisição de imagens para sistemas operacionais Windows) apresentou as seguintes medidas (média ± d.p): comprimento total 43,87 ± 4,91 µm, cabeça 7,54 ± 0,82 µm, peça intermediária 5,35 ± 0,83 µm, cauda 30,72 ± 2,55 µm e largura da cabeça 5,30 ± 0,68 µm. Das 2.000 células coradas em Eosina-Nigrosina para avaliação da integridade da membrana, 83,8 % das diluídas em ACP 123 ® e 72,9 % das diluídas em Botusemen® estavam com as membranas intactas. Os resultados sugerem que espermatozoides epididimários de pacas podem ser utilizados em técnicas de reprodução assistida, agregam conhecimentos a esta área e suscitam novos estudos sobre o papel das três vesículas acrossomais características nesta espécie.

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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)

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A gota citoplasmática é uma protuberância de citoplasma geralmente posicionada na peça intermediária dos espermatozoides. É considerada uma organela transitória, uma vez que migra pelo espermatozoide durante o trânsito epididimário, quando é removida dessas células na maioria dos mamíferos. Dúvidas sobre se a presença desta gota está relacionada de forma positiva ou negativa com a função e fertilidade espermática são frequentes, já que sua presença no ejaculado pode afetar a fertilidade espermática e o uso do sêmen em biotécnicas da reprodução. Porém, mesmo não sendo totalmente compreendida a nível molecular e funcional, sabe-se que a gota citoplasmática é necessária ao espermatozoide durante seu processo de maturação no epidídimo. Por isso, o objetivo desta revisão foi abordar pontos sobre a fisiologia e funções das gotas citoplasmáticas, bem como relacionar sua presença com a maturação e fertilidade dos espermatozoides.

The cytoplasmic droplet is a protuberance of cytoplasm usually positioned in the sperm midpiece. It is considered a transient organelle that migrates through the sperm cell during its epididymal transit when it is finally removed from the cell in most mammals. Doubts over whether the presence of cytoplasmic droplets is positively or negatively related to sperm functions and fertility are common, as its presence in the ejaculate may affect sperm fertility and the use of semen in assisted reproductive technology. Although the understanding of cytoplasmic droplets function is not fully clarified at molecular and functional levels, it is known that cytoplasmic droplet is necessary for spermatozoa, especially during their maturation in the epididymal duct. Therefore, the aim of this review was to discuss aspects of the physiology and function of cytoplasmic droplets, as well as to relate their presence to sperm maturation and their consequent fertility.

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The objectives of this twelve-week feeding trial were to determine the effects of dietary Saccharomyces cerevisiae and/or zinc oxide on epididymal sperm characteristics, testicular morphometric traits, and growth parameters of bucks. 16 (New Zealand White) bucks 16-wk-old, weighing 2.8kg were randomly allotted to one of 4 treatment groups. Each treatment was replicated four times, with 1 buck per replicate, in a completely randomized design (CRD). Each treatment group was randomly assigned to one of the four commercial experimental diets designated thus: TA = control diet with no additives, TB = 0.12g/kg Saccharomyces cerevisiae, TC = 150mg/kg zinc oxide and TD = 0.12g/kg Saccharomyces cerevisiae +150 mg/kg zinc oxide. Although treatment had no effect (p>0.05) on final body weight, average daily weight gain and feed conversion ratio, epididymal sperm characteristics and testicular morphometric traits differed significantly (p 0.05). Bucks on TB(Saccharomyces cerevisiae-based diet) had improved (p 0.05) sperm concentration, motility and live sperm, tubule diameter, epididymal volume, volume fraction of duct, and total duct volume, but decreased testicular volume. Bucks fed TA (control diet) had improved volume fraction of tubule but recorded the highest incidence of head and tail sperm abnormality. Though, TC(zinc oxide-based diet) enhanced (p 0.05) seminal vesicle volume, sperm pH was better among bucks fed TD (Saccharomyces cerevisiae + zinc oxide-based diet). It can be concluded that dietary inclusion of Saccharomyces cerevisiae at 0.12g/kg of feed improved epididymal sperm characteristics and testicular morphometric traits of rabbit bucks.

Resumo

Objetivou-se com o estudo investigar o efeito de diferentes osmolaridades do diluidor Triscitrato em espermatozóides epididimários de gatos domésticos (Feliscatus) e a congelação com glicerol ou etilenoglicol. Foram realizados dois experimentos, com 10 gatos. No Experimento 1, avaliou-se a manutenção dos parâmetros espermáticos em diluidor tris-citrato com osmolaridades 275, 325, 375, 425, 475 e 525mOsm, nos tempos (T0= 0, T1= 30 e T2= 60 min). No Experimento 2 a congelação foi realizada utilizando as osmolaridades 325 e 375 do glicerol a 4% ou etilenoglicol a 3%, 6%. Dentre as osmolaridades, quanto à motilidade a 325 mOsm não diferiu estatisticamente com o fluido epidídimal (controle) nos três tempos e a 375 mOsm no T0 e T1, e ambas não apresentaram diferenças estatísticas entre si e entre os tempos em todos os parâmetros espermáticos. O uso de 4% de glicerol em diluidor com 375 mOsm foi superior, apresentando motilidade de 25% ± 6, vigor 4, integridade de membrana plasmática de 48% ± 9, sem diferenças estatísticas com o resfriamento e na morfologia não foram encontradas diferenças estatísticas entre as duas osmolaridades. Portanto, o Tris-citrato com 325 e 375 mOsm entre as osmolaridades testadas e pós congelação com 375 mOsm e glicerol 4% manteve os parâmetros espermáticos.

The aim of this study was to investigate the effect of different osmotic potentials of Tris-citrate extender in epididymal spermatozoa of domestic cats (Felis catus), frozen with glycerol or ethyleneglycol. Two experiments were carried out, with ten cats. In the first experiment, the influence of extender with the osmolarityof 275, 325, 375, 425, 475 and 525 mOsm on sperm parameters were evaluated. In the second experiment, slow freezing was performed using glycerol at 4% or ethyleneglycolat 3% and 6% added to extender with 325and 375 mOsm. Among the osmolarities, the motilityat 325 mOsm did not differ statistically with epididymal fluid (control) at all times evaluated and at 375 mOsmat T0 and T1, and both showed no statistical differences between each other and between the times in all sperm parameters. Glycerol 4% added to extender with 475 mOsm was superior, presenting motilityof 25% ± 6, vigor 4, plasma membrane integrity of 48% ± 9, without statistical differences with cooling and in morphology, no statistical differences were found between the two osmolarities. Therefore, 325 and 375 mOsm Triscitrate between the osmolarities tested and after freezing with 375 mOsm and 4%, glycerol maintained the sperm parameters.

Resumo

O objetivo do presente estudo foi avaliar o efeito da adição de plasma seminal de garanhões de alta e baixa fertilidade sobre a congelabilidade e a viabilidade de espermatozoides do ejaculado (EJ) e do epidídimo (EP) de garanhões subférteis. Foram utilizados seis garanhões com histórico de subfertilidade. Após coleta, espermatozoides do ejaculado foram divididos em três alíquotas: BotuSêmen® (EJ-CT); plasma seminal de alta qualidade espermática (EJ-PS1); e plasma seminal de baixa qualidade espermática (EJ-PS2). O mesmo protocolo foi realizado com espermatozoides da cauda do epidídimo após orquiectomia (EP-CT; EP-PS1; EP-PS2). Foram realizadas avaliações da cinética espermática pelo CASA e análises de integridade de membrana, acrossoma, fragmentação de DNA, capacitação espermática e peroxidação espermática por citometria de fluxo. Não foram observadas diferenças na cinética espermática entre EJ e EP, logo após a descongelação. Porém, foi observada maior (P<0,05) porcentagem de células com membranas plasmática e acrossomal íntegras nos grupos EP (EP-CT:31,7±7,5b; EP-PS1:35,2±7,0b; EP-PS2:33,9±7,2b) em comparação aos grupos EJ (EJ-CT:15,1±4,9a; EJ-PS1:11,7±4,5a; EJ-PS2:13,1±5,2a). Adicionalmente, foram observadas diferenças no índice de fragmentação de DNA (EJ-CT:2,6±0,6a; EJ-PS1:2,4±0,8a; EJ-PS2:3,0±0,8a; EP-CT:1,4±0,4b; EP-PS1:1,2±0,3b; EP-PS2:1,3±0,2b). Concluiu-se que a adição de 20% de plasma seminal, oriundo de animais férteis ou subférteis, previamente à congelação de espermatozoides epidídimários de animais subférteis não interfere na qualidade espermática.(AU)

The aim of this study was to compare the effect of the addition of seminal plasma from high and low fertility stallions on sperm viability of frozen-thawed sperm cells from ejaculate and from epididymal tail of subfertile stallions. Six stallions with a history of subfertility were used. After collection, ejaculate spermatozoa were divided into three aliquots: Botu-Semen® (EJ-CT); High-quality seminal plasma (EJ-PS1); Low-quality seminal plasma (EJ-PS2). The same was done with sperm cells from epididymis tail after orchiectomy (EP-CT; EP-PS1; EP-PS2). Evaluations of sperm kinetics were assessed by CASA and membrane and acrosome integrity, DNA fragmentation, sperm capacitation and sperm peroxidation were assessed by flow cytometry. After thawing, no differences were observed between ejaculated sperm (EJ) and epididymal sperm (EP) in any CASA evaluations. However, higher (P< 0.05) percentage of cells with intact plasma and acrossomal membranes was observed in EP groups (EP-CT:31.7±7.5b; EP-PS1:35.2±7.0b; EP-PS2:33.9±7.2b) compared to EJ groups (EJ-CT:15.1±4.9a, EJ-PS1:11.7±4.5a, EJ-PS2:13.1±5,2a). In addition, differences in DNA fragmentation index were observed (EJ-CT:2.6±0.6a; EJ-PS1:2.4±0.8a; EJ-PS2:3.0±0.8a; CT:1.4±0.4b; EP-PS1:1.2±0.3b; EP-PS2:1.3±0.2b). It was concluded that the addition of 20% seminal plasma from fertile or subfertile animals prior to the freezing of epididymal spermatozoa from subfertile animals does not interfere in sperm quality.(AU)

Resumo

Objetivou-se testar concentrações de Aloe vera para produção de diluidor vegetal na refrigeração espermática. Amostras obtidas de epidídimos bovinos foram recuperadas e homogeneizadas para formação do pool. Sete grupos experimentais foram formados: GC= Grupo TRIS-Gema; G5= AV5%+1,488g frutose; G10= AV10%+1,488g frutose; G20= AV20%+1,488g frutose; GF5= AV5%+2,976g frutose; GF10= AV10%+2,976g frutose; GF20= AV20%+2,976g frutose. Os grupos foram submetidos às avaliações de quantificação de açúcares redutores; reologia; potencial hidrogeniônico (pH) a 5 °C e 37 ºC; motilidade subjetiva, integridade e funcionalidade da membrana plasmática nos períodos T0 (momento diluição) e T2 (ao atingir 5 ºC); crescimento microbiológico após 48h e 72h de refrigeração. Os G20 e GF20 apresentaram maior (p0,05) na integridade e funcionalidade da membrana plasmática entre grupos e tempo de avaliação. No crescimento microbiológico, após 72h, o G20 apresentou efeito antimicrobiano. Conclui-se que concentrações de extrato de Aloe vera interferem negativamente na motilidade de espermatozoides epididimários bovino, porém preservam a integridade e funcionalidade da membrana plasmática e apresentam efeito antimicrobiano. Estudos para determinação da viscosidade ideal do extrato e aprimoramento da confecção deste possível diluidor vegetal devem ser encorajados.

The current study aimed to test concentrations of Aloe vera for production of plant origin extender to sperm cooling. Samples obtained from bovine epididyms were recovered and homogenized for pool formation. Seven groups were performed: GC= TRIS-egg yolk Group; G5= AV5%+1.488g fructose; G10= AV10%+1.488g fructose; G20= AV20%+1.488g fructose; GF5= AV5%+2.976g fructose; GF10= AV10%+2.976g fructose; GF20= AV20%+2.976g fructose. The groups they were submitted to the quantification of sugar reduction; rheology; hydrogenation potential (pH) at 5 °C and 37 °C; subjective motility, integrity and functionality of the plasma membrane at T0 (dilution moment) and T2 (reaching 5 ºC) periods; microbiological growth after 48h and 72h by refrigeration. The G20 and GF20 presented higher (p0.05) were observed in the integrity and functionality of the plasma membrane among groups and times of evaluation. In the microbiological growth, after 72h, the G20 presented antimicrobial effect. Therefore, concentrations of Aloe vera extract negatively interfere with bovine epididymal sperm motility, but preserve integrity and functionality of spermatozoa plasma membrane and present antimicrobial effect. Studies to determine the ideal viscosity of the extract and improvement of the preparation of this possible plant origin extender should be encouraged.

Resumo

A produção animal depende da reprodução de machos e fêmeas domésticas. Em reprodutores ruminantes, o estresse térmico é a principal causa de degeneração testicular e, nesta, ocorrem desde mudanças anatômicas na gônada masculina até alterações microscópicas, do epitélio seminífero aos espermatozoides em trânsito no trato reprodutor masculino. Essas alterações microscópicas são simultâneas a diversas mudanças na composição proteica do plasma seminal dos reprodutores, o que está ligado a defeitos espermáticos de ordem maior (de origem primariamente testicular) ou menor (que surgem durante o trânsito epididimário) e, consequentemente, aos parâmetros de cinética espermática e à fecundidade do ejaculado. Assim, esta revisão objetiva descrever as alterações que ocorrem quando reprodutores ruminantes domésticos são acometidos de degeneração testicular por falha na termorregulação, correlacionando-as às mudanças encontradas no plasma seminal, na morfologia espermática e, paralelamente, aos parâmetros de motilidade espermática.

Animal production depends on the reproduction of domestic males and females. In ruminant breeding, heat stress is the main reason of testicular degeneration, causing anatomical changes in the male gonad even microscopic changes, from the seminiferous epithelium until the spermatozoa in transit in the male reproductive tract. These microscopic changes occur simultaneously to several changes in the protein composition of the seminal plasma of the reproductive tract, which is related to higher sperm defects (of primarily testicular origin) or minor defects (that arise during epididymal transit) and, consequently, to the parameters of sperm kinetics and ejaculate fecundity. Thus, this review aims to describe the changes that occur when domestic ruminants are affected by testicular degeneration due to failure in thermoregulation, correlating them to the changes found in seminal plasma, sperm morphology in parallel to sperm motility parameters.