Scrotal circumference in Karagouniko and Chios sheep in relation to the aerial environment

Local breeds are a valuable asset in sheep farming systems in Greece and other Mediterranean countries. Among these, Karagouniko and Chios sheep hold a special position, participating in numerous genetic improvement programs, but a research gap exists on their scrotal circumference (SC) in relation to the environmental parameters photoperiod (PHOTO), air temperature, sunshine and rainfall. Therefore, four seasons were examined: April-June and July-September (Long days, increasing and decreasing photoperiod length, respectively), and October-December and January-March (Short days, decreasing and increasing photoperiod length, respectively). Repeated measures analysis of variance and linear correlation analysis were used to investigate the effect of the period on SC and the relationships of SC and each of the environmental parameters above, respectively, in both breeds. Also, independent t-tests were used to investigate SC differences between Karagouniko and Chios. A progressive increase of SC in both breeds coincided with the lowering of PHOTO from April-June to October-December. A significant correlation was confirmed between the SC of each breed and PHOTO, shedding light on the importance of this parameter on SC. Chios sheep had significantly larger (P < 0.05) SC (35.23 cm) than the Karagouniko one (34.59 cm). Better exploitation of these sheep breeds could be derived from the current study's findings.

Effect of seasonally changing aerial environment on testosterone in Karagouniko and Chios sheep

A well-established activity in Greece and other Mediterranean countries is sheep farming. Karagouniko and Chios sheep are promising breeds and targets for the reproductive physiologist's attention. Under this context, part of the reproductive physiology of Karagouniko and Chios was investigated, taking into account the environment. Specifically, four time periods were considered; April-June (Long days, increasing photoperiod length), July-September (Long days, decreasing photoperiod length), October-December (Short days, decreasing photoperiod length), and January-March (Short days, increasing photoperiod length). The effect of the period on the concentration of the testosterone (main male hormone) in rams of the breeds above was studied, by using repeated measures analysis of variance, also taking into account the environmental variables air temperature, relative humidity, sunshine, rainfall, and photoperiod, in the Artificial Insemination Center of Karditsa (39°21'18''N, 21°54'19''E), Greece. The lowering of photoperiod from April-June to July-September (long days) and afterward, even more to October-December (short days) coincided with a progressive increase of testosterone in both breeds. Photoperiod seems to have a major role in the concentration of Karagouniko and Chios breeds' testosterone compared to the other environmental variables. When considering the whole experimental period, Chios sheep had significantly higher (P < 0.05) testosterone (1.95 ng/ml) than the Karagouniko one (1.55 ng/ml). Our findings could contribute towards the more rational exploitation of the examined sheep breeds.(AU)

Effect of seasonally changing aerial environment on testosterone in Karagouniko and Chios sheep

A well-established activity in Greece and other Mediterranean countries is sheep farming. Karagouniko and Chios sheep are promising breeds and targets for the reproductive physiologist's attention. Under this context, part of the reproductive physiology of Karagouniko and Chios was investigated, taking into account the environment. Specifically, four time periods were considered; April-June (Long days, increasing photoperiod length), July-September (Long days, decreasing photoperiod length), October-December (Short days, decreasing photoperiod length), and January-March (Short days, increasing photoperiod length). The effect of the period on the concentration of the testosterone (main male hormone) in rams of the breeds above was studied, by using repeated measures analysis of variance, also taking into account the environmental variables air temperature, relative humidity, sunshine, rainfall, and photoperiod, in the Artificial Insemination Center of Karditsa (39°21’18’’N, 21054’19’’E), Greece. The lowering of photoperiod from April-June to July-September (long days) and afterward, even more to October-December (short days) coincided with a progressive increase of testosterone in both breeds. Photoperiod seems to have a major role in the concentration of Karagouniko and Chios breeds' testosterone compared to the other environmental variables. When considering the whole experimental period, Chios sheep had significantly higher (P < 0.05) testosterone (1.95 ng/ml) than the Karagouniko one (1.55 ng/ml). Our findings could contribute towards the more rational exploitation of the examined sheep breeds.

DNA methylation level of gene SIRT1 in ram spermatozoa and relationship with fertilizing ability according to breed and age

Background: Effect of the epigenetic factors on the male fertility is well proofed. Sperm acts as a carrier of genetic material, and its DNA methylome can affect maternal pregnancy rate and offspring phenotype. However, the research on the DNA methylation in the spermatozoids of livestock males, in particular rams, is still limited. To best of our knowledge the data about as a global as well as gene specific DNA methylation in ram spermatozoa from different breeds and ages are missed in the scientific literature. The present study was designed to analyze the relationship between methylation levels of the important for spermatogenesis gene SIRT1 in spermatozoa and fertilizing ability of sperm in rams from different breeds and ages. Materials, Methods & Results: The ejaculates of 16 rams from Lacaune, East Friesian and Assaf breeds at age between 18 to 96 months were evaluated. The kinematic parameters of 2 semen samples from each animal were estimated by CASA. The separated spermatozoa were used for DNA extraction followed by bisulfite conversion. The DNA methylation of SIRT1 was detected through quantitative methylation-specific PCR using 2 sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. The breed and age effect on the gene SIRT1 methylation by ANOVA was estimated. Experimental females included 393 clinically healthy milk ewes (Lacaune, n = 131; East Friesian sheep, n = 100 and Assaf, n = 162) in breeding season. Reproductive performances (conception rate at lambing, lambing percentage and fecundity) of ewes, inseminated by sperm of the investigated rams, were statistically processed. ANOVA showed that the animal breed influences significantly on the level of DNA methylation of gene SIRT1 in ram spermatozoa (P = 0.002) An average value of DNA methylation of SIRT1 in ram sperm from Lacaune breed was significantly higher than in Assaf and East Friesian (81.21 ± 15.1% vs 36.7 ± 14.2% and 38.3 ± 18.6 respectively, P < 0.01). The highest percent of SIRT1 methylation was observed in old animals compared to the young and middle-age. Moderate and strong correlations (r from 0.44 to 0.71, P < 0.05) between the methylation level of the SIRT1 gene in rams' sperm and reproductive parameters of inseminated ewes in all breeds were established. Discussion: Our data are the first message about the effect of breed on the specificity of DNA methylation of gene SIRT1 in ram spermatozoa. These results demonstrated an existence of the sheep breeds with high and low level of DNA methylation of gene SIRT1 in ram sperm. Although the effect of age on the methylation level in sperm is still discussable, our results showed a moderate correlation between age and methylation level of SIRT1 in spermatozoa of rams. Taking into account that DNA methylation in sperm is stabilized with puberty onset and is a heritable epigenetic modification, it can be a promising marker of sperm quality in animal breeding. In all investigated breeds the rams with relatively high level of DNA methylation of gene SIRT1 in spermatozoa (50-68%) demonstrated a high conception rate at lambing (> 70%). In conclusion, the DNA methylation level of the SIRT1 gene in ram spermatozoa is determined by both the breed and the age of the animals and correlates with fertilizing ability of sperm.

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

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.

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

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.(AU)

Sheep urinary tract architecture is not affected by acute urethral obstruction / A arquitetura do trato urinário de ovinos não é afetada pela obstrução uretral aguda

Urolithiasis affects the urinary tract of small ruminants, thereby requiring the animal to prematurely terminate breeding. Morphometric study of organs can be used as a diagnostic method. Thus, this study aimed to describe the macroscopic, histopathological, and histomorphometric changes in the urinary tract of sheep with urolithiasis. For this purpose, 14 healthy male Santa Inês sheep, approximately 90 days old, were studied and fed an experimental diet. After the development of urolithiasis, the animals were reorganized into two groups, D1 (without urolithiasis) and D2 (with urolithiasis) for comparative data analysis. Sheep were necropsied to evaluate the pathological changes, followed by macroscopic morphometric analysis, and the histopathological and histomorphometric characteristics were described. Urethral necrosis and a full urinary bladder were observed in all animals that developed the disease. The comparison between sheep with and without urolithiasis showed no significant difference (P < 0.05) in the evaluated macroscopic morphometric variables, except for the right ureter width. Regarding the histopathological evaluation, multifocal areas of mild to moderate congestion within the glomerular tufts and protein in the tubular lumen of the kidneys were observed. In the liver, mild to moderate fatty degeneration was noted in the centrolobular regions, and an ulcerated focal area in the bladder mucosa was observed in only one animal. The present study demonstrated that the formulated diet was effective in inducing clinical disease. In acute obstructive urolithiasis in sheep tissue, lesions in the liver and urinary tract were observed, although there were no significant histomorphometric changes.(AU)

Urolitíase acomete o trato urinário de pequenos ruminantes causando a saída prematura de animais destinados à reprodução. O estudo morfométrico dos órgãos pode ser empregado como método de auxílio diagnóstico. Assim, este estudo objetivou descrever as alterações macroscópicas, histopatológicas e histomorfométricas do trato urinário de ovinos com urolitíase. Com esse fim, foram utilizados 14 ovinos hígidos, machos, da raça Santa Inês com idade aproximada de 90 dias, que receberam dieta experimental. Após o desenvolvimento da urolitíase os animais foram reorganizados em dois grupos experimentais distintos D1 (sem urolitíase) e D2 (com urolitíase) para a análise comparada dos dados. Os ovinos foram necropsiados para descrição das alterações patológicas, seguindo-se a análise morfométrica macroscópica e descrição quanto as características histopatológicas e histomorfométricas. Necrose de processo uretral e bexiga urinária repleta foram observados em todos os animais que desenvolveram a doença. Na comparação entre os ovinos com e sem urolitíase não houve diferença significativa (P < 0,05) nas variáveis morfométricas macroscópicas avaliadas, a exceção da largura do ureter direito. Quanto à avaliação histopatológica, foram observadas áreas multifocais de discreta a moderada congestão dos tufos glomerulares e proteína no lúmen tubular nos rins. No fígado, observou-se nas regiões centrolobulares, discreta a moderada degeneração gordurosa e apenas em um animal foi observada, macro e microscopicamente, área focal ulcerada na mucosa da bexiga. Os achados da presente pesquisa demonstraram que a dieta formulada foi eficaz na indução da doença clínica. Na urolitíase obstrutiva aguda em ovinos, lesões teciduais em fígado e trato urinário são observadas, mas não há alterações histomorfométricas significativas.(AU)

Estresse calórico e reprodução em ovinos: fundamentos e perspectivas tecnológicas / Heat stress on sheep reproduction: fundamentals and technological perspectives

A ovinocultura é uma importante atividade que produz proteínas de alto valor biológico, mas seus ganhos podem ser reduzidos em função do estresse ambiental. Isso reforça a importância de se estudar a relação entre o ambiente térmico e o animal, identificando animais mais adaptados e férteis, e melhores práticas de manejo. O ovino (Ovis aries) é um animal homeotérmico, que mantem sua temperatura corporal em equilíbrio dinâmico. Quando em estresse calórico, os ovinos usam mecanismos sensíveis e latentes para dissipar o calor acumulado, com destaque para o redirecionamento do fluxo sanguíneo, a ofegação e a sudorese. O escroto desempenha importante crucial na termorregulação dos testículos, os quais precisam funcionar sob em até 6,0oC abaixo da temperatura interna corpórea. A hipertermia testicular compromete a espermatogênese, reduz a concentração seminal, a motilidade progressiva e a viabilidade espermática. Ainda, leva a aumento dos defeitos morfológicos espermáticos, na produção de espécies reativas de oxigênio e na fragmentação do DNA espermático, diminuindo a capacidade fecundante. Tecnologias disruptivas para monitoramento do ambiente de produção, da termorregulação e do bem-estar dos animais já são realidade e se encontram em expansão, favorecendo a tomada de decisões em tempo real e o desempenho reprodutivo dos ovinos.

Sheep farming is a relevant activity that provides proteins of high biological value, but its gains can be reduced due to environmental stress. This reinforces the importance of studying the relationship between the thermal environment and the animal, identifying more adapted and fertile animals, and better management practices. Sheep (Ovis aries) are homeothermic animals and thus maintain their body temperature in a state of dynamic balance. Under heat stress, sheep dissipate accumulated heat through sensitive and latent mechanisms, primarily using redirection of blood flow, panting and sweating. The scrotum plays a crucial role in the thermoregulation of the testicles, which need to be maintained up to 6.0oC below the body core temperature. Testicular hyperthermia impairs spermatogenesis, reduces seminal concentration, progressive motility and sperm viability. Furthermore, it leads to an increase in sperm morphological defects, in the production of reactive oxygen species, and in sperm DNA fragmentation, reducing the fertilizing capacity. Disruptive technologies for monitoring the production systems, animal thermoregulation and welfare are already a reality and are expanding, favoring realtime decision making and the reproductive performance of sheep.

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

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

Efeito da adição de nanopartículas de Óxido de Zinco ao diluente de congelação do sêmen ovino / Effect of adding zinc oxide nanoparticles to the freezing extender of ram semen

The aim of this study was to evaluate the effect of adding zinc oxide nanoparticles to the ram semen cryopreservation diluter. Semen pools (n=6), from three Santa Inês breeders, were diluted in Tris-yolk (5% glycerol), supplemented with zinc oxide nanoparticles (0, 25, 75 and 150μg/mL) at a concentration of 200x10 6 sperm/mL. The samples were frozen in an automated system and stored in liquid nitrogen (-196 °C). At the timeof analysis, the semen samples were thawed (37 °C/30s) and evaluated for sperm kinetics, plasma and acrosomal membrane integrity and mitochondrial membrane potential. There was no significant difference (p>0.05) between the experimental groups in the parameters of kinetics, plasma membrane and acrosome integrity. As for the potential of mitochondrial membrane, all treated groups were significantly (p≤0.05) larger than the control. It was concluded that the zinc oxide nanoparticles increase the mitochondrial membrane potential of ram sperm submitted to the freezing/thawing process.

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

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

Efeito da adição de nanopartículas de Óxido de Zinco ao diluente de congelação do sêmen ovino / Effect of adding zinc oxide nanoparticles to the freezing extender of ram semen

The aim of this study was to evaluate the effect of adding zinc oxide nanoparticles to the ram semen cryopreservation diluter. Semen pools (n=6), from three Santa Inês breeders, were diluted in Tris-yolk (5% glycerol), supplemented with zinc oxide nanoparticles (0, 25, 75 and 150μg/mL) at a concentration of 200x10 6 sperm/mL. The samples were frozen in an automated system and stored in liquid nitrogen (-196 °C). At the timeof analysis, the semen samples were thawed (37 °C/30s) and evaluated for sperm kinetics, plasma and acrosomal membrane integrity and mitochondrial membrane potential. There was no significant difference (p>0.05) between the experimental groups in the parameters of kinetics, plasma membrane and acrosome integrity. As for the potential of mitochondrial membrane, all treated groups were significantly (p≤0.05) larger than the control. It was concluded that the zinc oxide nanoparticles increase the mitochondrial membrane potential of ram sperm submitted to the freezing/thawing process.(AU)

Trehalose addition to a Tris-fructose egg yolk extender on quality of ram sperm preserved at 0 °C

The present study investigated the effects of various concentrations of trehalose in Tris-fructose egg yolk diluent on ram semen preservation at 0 ℃. Semen was collected by artificial vagina ejaculation from six rams of proven fertility. High-quality ejaculates were diluted with 0 (control), 5, 10, 15, and 20 mM trehalose of Tris-fructose egg yolk extender and control (tris-fructose egg yolk extender without trehalose), respectively. Then, the ejaculates were diluted to a concentration of 5×108 sperm/mL, cooled to 0 ℃ for 90 min, and maintained at that temperature for twelve days. The diluted semen samples were examined, and their sperm progressive motility, membrane functionality, and acrosome integrity recorded at 0, 24, 72, 144, 216, and 288 h. Two hundred ninety-six ewes were transcervically inseminated with the 216-h control (without trehalose) or the optimal trehalose concentration group semen, and the pregnancy and lambing rates were measured. No significant differences were established in the sperm progressive motility and membrane functionality among the control and 5, 10, 15, and 20 mM groups. The sperm samples of trehalose addition groups had no significant difference in the acrosome integrity of sperm, but they were, nonetheless, significantly higher than those in the control. No significant difference was detected in the lambing and pregnancy rates between the 5 mM and control groups. These results suggest that ram sperm is capable of fertilization after cooling and preservation at 0 ℃ by the use of 5 mM trehalose for Tris-fructose egg yolk diluent. Under these conditions, ram sperm can be more effectively preserved than under other four concentrations of diluents.(AU)

Addition of synthetic amino acids in sheep semen improves its quality after cryopreservation / Adição de aminoácidos sintéticos no sêmen ovino melhora a qualidade espermática após a criopreservação do sêmen

Some amino acids can protect mammalian sperm cells against oxidation during thermal stress caused by freezing/thawing. Thus, the objective was to evaluate the protective action of the association of the amino acids L-proline (Pro) and L-glutamine (Glu) against the cryoinjury caused to sheep sperm after cryopreservation. Eight ejaculates were collected from four sheep (n=32) and diluted in Tris-Egg Yolk-Glycerol until the final concentration of 200 x106 sptz/mL and kept in a water bath at 32 °C. The amino acids were added as follows: control (without adding amino acids), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µMPro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) and Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Afterwards, the semen was cooled to 5 °C for 2 h, after that period, filled in 0.5 mL straws and then placed under liquid nitrogen vapor (N2L), 8 cm from the liquid sheet for 15min, and then immersed on the N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity and binding test. The variables were subjected to the normality tests (Lilliefors test) and homoscedasticity tests (Cochran and Bartlett test), afterwards the variables of normal distribution were subjected to analysis of variance and the means compared by the Tukey test with a significance level of 5%. The Pro+Glu 3 group exhibited sperm with a greater (P<0.05) motility after thawing. In addition, the highest percentage of plasma and acrosomal membrane integrity were obtained using Pro+Glu 1, Pro+Glu 2 and Pro+Glu 3; and Pro+Glu 2 and Pro+Glu 3, respectively. Amino acids also kept mitochondrial activity high compared to the control, with Pro+Glu 3 resulting in greater activity (P<0.05). Sperm viability was higher (P<0.05) with the use of Pro+Glu 2 and Pro+Glu 3 than in the control. The number of sperm that showed the ability to bind to the egg yolk perivitelline membrane was higher (P<0.05) in semen treated with amino acids. It is concluded that the addition of synthetic amino acids in the semen of sheep before cryopreservation improves sperm quality and fertilization potential and can thus be added in cryopreservation protocols.

Alguns aminoácidos podem proteger as células espermáticas de mamíferos contra a oxidação durante o estresse térmico causado na congelação/descongelação. Dessa forma, objetivou-se avaliar a ação protetora da associação dos aminoácidos L-prolina (Pro) e L-glutamina (Glu) contra as crioinjúrias causadas aos espermatozoides de ovino após a criopreservação. Foram coletados oito ejaculados de quatro carneiros (n=32) e diluídos em Tris-Gema de ovo-Glicerol até a concentração final de 200 x106 sptz/mL e, mantidos em banho maria a 32 °C. Os aminoácidos foram adicionados da seguinte forma: controle (sem adição de aminoácidos), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µM Pro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) e Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Depois, o sêmen foi resfriado a 5 °C por 2 h, após esse período, envasado em palhetas de 0,5 mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida por 15 min, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas aos testes de normalidade (Teste de Lilliefors) e homocedacidade (Teste de Cochran e Bartlett), posteriormente as variáveis de distribuição normal foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey com nível de significância de 5%. O grupo Pro+Glu 3 exibiu espermatozoides com uma maior (P<0,05) motilidade após o descongelamento. Além disso o maior percentual de integridade da membrana plasmatica e acrossomal foram obtidos utilizando Pro+Glu 1, Pro+Glu 2 e Pro+Glu 3; e Pro+Glu 2 e Pro+Glu 3, respectivamente. Os aminoácidos também mantiveram alta a atividade mitocondrial em comparação com o controle, com Pro+Glu 3 resultando numa maior atividade (P<0,05). A viabilidade dos espermatozoides foi maior (P<0,05) com o uso de Pro+Glu 2 e Pro+Glu 3 do que no controle. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com aminoácidos. Conclui-se que, a adição dos aminoácidos sintéticos no sêmen de ovinos antes da criopreservação melhora a qualidade espermática e o potencial fecundante, podendo assim serem adicionados em protocolos de criopreservação.

Addition of synthetic amino acids in sheep semen improves its quality after cryopreservation / Adição de aminoácidos sintéticos no sêmen ovino melhora a qualidade espermática após a criopreservação do sêmen

Some amino acids can protect mammalian sperm cells against oxidation during thermal stress caused by freezing/thawing. Thus, the objective was to evaluate the protective action of the association of the amino acids L-proline (Pro) and L-glutamine (Glu) against the cryoinjury caused to sheep sperm after cryopreservation. Eight ejaculates were collected from four sheep (n=32) and diluted in Tris-Egg Yolk-Glycerol until the final concentration of 200 x106 sptz/mL and kept in a water bath at 32 °C. The amino acids were added as follows: control (without adding amino acids), Pro+Glu 1 (100 M Pro + 500 M Glu), Pro+Glu 2 (300 M Pro + 1000 M Glu), Pro+Glu 3 (500 M Pro + 1500 M Glu) and Pro+Glu 4 (700 M Pro + 2000 M Glu). Afterwards, the semen was cooled to 5 °C for 2 h, after that period, filled in 0.5 mL straws and then placed under liquid nitrogen vapor (N2L), 8 cm from the liquid sheet for 15 min, and then immersed on the N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity and binding test. The variables were subjected to the normality tests (Lilliefors test) and homoscedasticity tests (Cochran and Bartlett test), afterwards the variables of normal distribution were subjected to analysis of variance and the means compared by the Tukey test with a significance level of 5%. The Pro+Glu 3 group exhibited sp

Alguns aminoácidos podem proteger as células espermáticas de mamíferos contra a oxidação durante o estresse térmico causado na congelação/descongelação. Dessa forma, objetivou-se avaliar a ação protetora da associação dos aminoácidos L-prolina (Pro) e L-glutamina (Glu) contra as crioinjúrias causadas aos espermatozoides de ovino após a criopreservação. Foram coletados oito ejaculados de quatro carneiros (n=32) e diluídos em Tris-Gema de ovo-Glicerol até a concentração final de 200 x106 sptz/mL e, mantidos em banho maria a 32 °C. Os aminoácidos foram adicionados da seguinte forma: controle (sem adição de aminoácidos), Pro+Glu 1 (100 μM Pro + 500 μM Glu), Pro+Glu 2 (300 μM Pro + 1000 μM Glu), Pro+Glu 3 (500 μM Pro + 1500 μM Glu) e Pro+Glu 4 (700 μM Pro + 2000 μM Glu). Depois, o sêmen foi resfriado a 5 °C por 2 h, após esse período, envasado em palhetas de 0,5 mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida por 15 min, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas aos testes de normalidade (Teste de Lilliefors) e homocedacidade (Teste de Cochran e Bartlett), posteriormente as variáveis de distribuição normal foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey com nível de significância de 5%. O grupo Pro+Glu 3 exibiu espermatozoides com uma maior (P<0,05) motilidade após o descongelamento. Além disso o maior percentual de integridade da membrana plasmatica e acrossomal foram obtidos utilizando Pro+Glu 1, Pro+Glu 2 e Pro+Glu 3; e Pro+Glu 2 e Pro+Glu 3, respectivamente. Os aminoácidos também mantiveram alta a atividade mitocondrial em comparação com o controle, com Pro+Glu 3 resultando numa maior atividade (P<0,05). A viabilidade dos espermatozoides foi maior (P<0,05) com o uso de Pro+Glu 2 e Pro+Glu 3 do que no controle. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com aminoácidos.(AU)

Addition of synthetic amino acids in sheep semen improves its quality after cryopreservation / Adição de aminoácidos sintéticos no sêmen ovino melhora a qualidade espermática após a criopreservação do sêmen

Some amino acids can protect mammalian sperm cells against oxidation during thermal stress caused by freezing/thawing. Thus, the objective was to evaluate the protective action of the association of the amino acids L-proline (Pro) and L-glutamine (Glu) against the cryoinjury caused to sheep sperm after cryopreservation. Eight ejaculates were collected from four sheep (n=32) and diluted in Tris-Egg Yolk-Glycerol until the final concentration of 200 x106 sptz/mL and kept in a water bath at 32 °C. The amino acids were added as follows: control (without adding amino acids), Pro+Glu 1 (100 M Pro + 500 M Glu), Pro+Glu 2 (300 M Pro + 1000 M Glu), Pro+Glu 3 (500 M Pro + 1500 M Glu) and Pro+Glu 4 (700 M Pro + 2000 M Glu). Afterwards, the semen was cooled to 5 °C for 2 h, after that period, filled in 0.5 mL straws and then placed under liquid nitrogen vapor (N2L), 8 cm from the liquid sheet for 15 min, and then immersed on the N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity and binding test. The variables were subjected to the normality tests (Lilliefors test) and homoscedasticity tests (Cochran and Bartlett test), afterwards the variables of normal distribution were subjected to analysis of variance and the means compared by the Tukey test with a significance level of 5%. The Pro+Glu 3 group exhibited sp

Alguns aminoácidos podem proteger as células espermáticas de mamíferos contra a oxidação durante o estresse térmico causado na congelação/descongelação. Dessa forma, objetivou-se avaliar a ação protetora da associação dos aminoácidos L-prolina (Pro) e L-glutamina (Glu) contra as crioinjúrias causadas aos espermatozoides de ovino após a criopreservação. Foram coletados oito ejaculados de quatro carneiros (n=32) e diluídos em Tris-Gema de ovo-Glicerol até a concentração final de 200 x106 sptz/mL e, mantidos em banho maria a 32 °C. Os aminoácidos foram adicionados da seguinte forma: controle (sem adição de aminoácidos), Pro+Glu 1 (100 μM Pro + 500 μM Glu), Pro+Glu 2 (300 μM Pro + 1000 μM Glu), Pro+Glu 3 (500 μM Pro + 1500 μM Glu) e Pro+Glu 4 (700 μM Pro + 2000 μM Glu). Depois, o sêmen foi resfriado a 5 °C por 2 h, após esse período, envasado em palhetas de 0,5 mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida por 15 min, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas aos testes de normalidade (Teste de Lilliefors) e homocedacidade (Teste de Cochran e Bartlett), posteriormente as variáveis de distribuição normal foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey com nível de significância de 5%. O grupo Pro+Glu 3 exibiu espermatozoides com uma maior (P<0,05) motilidade após o descongelamento. Além disso o maior percentual de integridade da membrana plasmatica e acrossomal foram obtidos utilizando Pro+Glu 1, Pro+Glu 2 e Pro+Glu 3; e Pro+Glu 2 e Pro+Glu 3, respectivamente. Os aminoácidos também mantiveram alta a atividade mitocondrial em comparação com o controle, com Pro+Glu 3 resultando numa maior atividade (P<0,05). A viabilidade dos espermatozoides foi maior (P<0,05) com o uso de Pro+Glu 2 e Pro+Glu 3 do que no controle. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com aminoácidos.

Seroprevalence of Corynebacterium pseudotuberculosis and Toxoplasma gondii in sheep in semi-arid region of Ceará state / Soroprevalência de Corynebacterium pseudotuberculosis de Toxoplasma gondii em ovinos de região semi-árida do estado do Ceará

The present study was conducted to investigate in 20 extensive sheep farms for the seroprevalence of Corynebacterium pseudotuberculosis (n=402) and Toxoplasma gondii (n=228). Enzyme-linked immunosorbent assay (ELISA) was used for the detection of antibodies to C. pseudotuberculosis/T. gondii. It was observed that C. pseudotuberculosis showed the highest prevalence in the region (34.07%) with statistically significant presence (p 0.05) in ewes. Antibodies to T. gondii was reported in 14.91% of the animals studied. About C. pseudotuberculosis/T. gondii coinfection the categories of rams showed significant (p 0.05) differences, suggesting that this gender could perpetuate the diseases in the flocks. It was concluded that the knowledge about the diseases in the region under study would facilitate the execution of prophylactic measures, especially against the diseases that pose risks to the public health and cause damages to the producer.(AU)

O presente estudo foi conduzido para investigar a soroprevalência em 20 fazendas de criação extensiva de ovinos quanto à presença de anticorpos para Corynebacterium pseudotuberculosis (n=402) e Toxoplasma gondii (n=228). Ensaio de imunoabsorção enzimático (ELISA) foi utilizado para a detecção de anticorpos contra C. pseudotuberculosis/T. gondii. Observou-se que C. pseudotuberculosis apresentou a maior prevalência na região (34,07%), com presença estatisticamente significante (p 0,05) nas categorias de ovelha. Anticorpos contra T. gondii foram encontrados em 14,91% dos animais estudados. Sobre a coinfecção de C. pseudotuberculosis/T. gondii, as categorias carneiro apresentaram diferenças significativas (p 0,05), sugerindo que esse gênero poderia perpetuar as doenças nos rebanhos. Concluiu-se que o conhecimento sobre as doenças na região em estudo facilitaria a execução de medidas profiláticas, principalmente contra as doenças que apresentam riscos à saúde pública e causam danos ao produtor.(AU)

Action of cholesterol-loaded cyclodextrin on viability of ram semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some casescan also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood,however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancingincreased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol fromthe plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ramnaturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%.The aim of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, inthe kinetics and viability of frozen and thawed ram spermatozoa.Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups:control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubatedat 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics,plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated.Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP,μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLCshowed decreased levels of superoxide anion formation (O2-), a free...

Action of cholesterol-loaded cyclodextrin on viability of ram semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some casescan also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood,however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancingincreased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol fromthe plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ramnaturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%.The aim of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, inthe kinetics and viability of frozen and thawed ram spermatozoa.Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups:control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubatedat 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics,plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated.Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP,μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLCshowed decreased levels of superoxide anion formation (O2-), a free...(AU)

Detection of Mycobacterium avium subsp. paratuberculosis in reproductive tissue and semen of naturally infected rams

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), disease that causes a syndrome of bad nutrient absorption, weight loss and eventually death. The intestine is the main target organ where the infection develops; however, there is evidence of infection by MAP in extra-intestine sites of sheep, including mesenteric nodes and semen. The aim of the study was to identify the presence of MAP in reproductive tissue and semen of infected Pelibuey rams in clinical state of PTB. Seven rams were used in clinical PTB state and a non-infected ram by MAP of the Pelibuey breed, confirmed by serology, nPCR and bacteriological culture, with average weight and age of 57.23 ± 1.73 kg and 2.91 ± 0.17 years, respectively. The presence of MAP was identified in different tissue samples: spleen (1/7, 14.3% and 2/7, 28.6%), small intestine (3/7, 42.9% and 4/7, 57.1%) and mesenteric lymph nodes (3/7, 42.9% and 3/7, 42.9%), with nPCR and culture, respectively. It was also identified in epididymis tissue (1/7, 14.3%), Cowper gland (2/7, 28.6%) and prostate (1/7, 14.3%), using nPCR, although without detection in culture. It was identified in testicular tissue in 42.8% (3/7; culture or nPCR technique), but in 28.6% (2/7) with both techniques. Finally, the presence of MAP was identified in 42.9% (3/7) of semen samples with nPCR; however, it was not detected through culture. In conclusion, the presence of MAP was identified in lymphatic, digestive tissue, and semen; the presence of MAP was reported for the first time in epididymis, Cowper gland, prostate and testicles of infected Pelibuey rams.