Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants

The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.(AU)

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.

Evaluation of biochemical and electrolytic components of semen from ram supplemented with different concentrations of selenium and its correlation with sperm quality / Avaliação de componentes bioquímicos e eletrolíticos do sêmen de ovinos suplementados com diferentes concentrações de selênio e sua correlação com a qualidade espermática

The aim of the study was to evaluate whether supplementation with different levels of selenium (Se) can change the biochemical and electrolytic components of semen, causing an improvement in seminal quality in rams. Thirty animals were kept in an intensive pen system, fed with hay and commercial ration, allocated into five groups (six animals/pen) and supplemented with a mineral mixture containing Omg (G1), 5mg (G2), 10mg (G3), 15mg (G4) and 20mg (G5) of Se/kg. Each group received a different treatment every 56 days and treatments were rotated between groups following a dynamic sequence. Semen samples were collected by electroejaculation after the end of each treatment to evaluate the levels of fructose, citric acid, potassium (K), sodium (Na), calcium (Ca), Se, zinc (Zn), manganese (Mn), sulfur (S) and lead (Pb). The statistical design was a 5x5 Latin square. The different levels of Se supplementation evaluated maintained the concentrations of electrolytes and minerals in the semen at the required levels and did not change the sperm quality, concluding that higher intakes of Se do not cause antagonistic effects on the absorption and subsequent action of other essential minerals supplied to the animals and still maintains electrolyte balance.

O objetivo do estudo foi avaliar se a suplementação com diferentes níveis de selênio (Se) pode alterar os componentes bioquímicos e eletrolíticos do sêmen, ocasionando, por conseguinte, uma melhoria na qualidade seminal em carneiros. Foram utilizados 30 animais, mantidos em sistema intensivo de baias, alimentados com feno e ração comercial, sendo alocados em cinco grupos (seis animais/baia) e suplementados com uma mistura mineral contendo 0mg (G1), 5mg (G2), 10mg (G3), 15mg (G4) e 20mg (G5) de Se/kg. Cada grupo recebeu um tratamento diferente a cada 56 dias, e foi realizado um rodízio de tratamentos entre os grupos seguindo uma sequência dinâmica. Amostras de sêmen foram colhidas por eletroejaculação após o fim de cada tratamento, a fim de se avaliarem os níveis de frutose, ácido cítrico, potássio (K), sódio (Na), cálcio (Ca), Se, zinco (Zn), manganês (Mn), enxofre (S) e chumbo (Pb). O desenho estatístico foi um quadrado latino 5x5. Os diferentes níveis de suplementação de Se avaliados mantiveram as concentrações de eletrólitos e de minerais do sêmen nos níveis exigidos e não modificaram a qualidade espermática, concluindo-se que maiores ingestões de Se não causam efeitos antagônicos na absorção e subsequente ação de outros minerais essenciais fornecidos aos animais e ainda mantêm o equilíbrio eletrolítico.

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)

Effects of insulin on sperm cell quality in ram semen cooled at 5°C / Efeitos da insulina sobre a qualidade das células espermáticas no sêmen ovino refrigerado a 5oC

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (µm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.(AU)

A insulina está presente no plasma seminal e participa de atividades espermáticas, como a motilidade e capacitação. Entretanto, os efeitos da insulina sobre a viabilidade do espermatozoide ovino resfriado ainda não estão elucidadas. Desta forma, o objetivo do presente estudo foi avaliar os efeitos da adição de insulina sobre o espermatozoide ovino durante o tempo de armazenamento à 5º C. Amostras espermáticas de seis carneiros da raça Santa Inês foram utilizadas. Os ejaculados foram divididos em duas aliquotas, com (grupo insulina) ou sem (grupo controle) adição de insulina (3 UI mL-1) no diluidor seminal e, posteriormente, resfriados até 5oC e mantidos armazenados por 48 horas. Em seguida, os espermatozoides foram avaliados quanto a motilidade e cinética utilizando um Sistema Computadorizado de Análise de Sêmen (CASA). Adicionalmente, as amostras espermáticas foram analizadas quanto a integridade acrosomal por meio de sondas fluorescentes (FITC-PNA) e, funcionalidade de membrana pelo teste hiposmótico. As análises seminais foram realizadas após 24 ou 48 horas de resfriamento. Foram verificados aumentos de espermatozoides com motilidade progressiva (%), retilinearidade (%), linearidade (%) e frequência de batimento caudal (BCF) (Hz) no grupo insulina após 24 ou 48 horas de resfriamento (p < 0.05). Entretanto, não houve efeito da adição de insulina sobre a porcentagem de espermatozoides moveis (%) e das velocidades espermáticas (VSL, VAP e VCL) (µm seg-1), integridade acrossomal e funcionalidade de membrana durante o resfriamento (p > 0.05). Conclui-se que adição de insulina (3 UI mL-1) no diluidor seminal melhora a qualidade da motilidade espermática durante o resfriamento.(AU)

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

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

Selenium supplementation prevents DNA damage in ram spermatozoa / Suplementação com selênio previne danos ao DNA espermático de ovinos

In the present study, we aimed to evaluate the effects of different concentrations of selenium (Se) ovine nutritional supplementation on spermatozoa DNA integrity. Thirty male ovines (age: 10 months) were used. They were fed with hay and ram food in an intensive system, which was divided into stalls (5 m long and 3 m wide) with feeding troughs, and had ad libitum access to food and water. Ovines in group 1 (G1, the negative control) received mineral salt supplementation without Se; ovines in G2 received the same mineral salt mixed with 5 mg Se (as sodium selenite)/kg mineral supplement;ovines in G3 received 10 mg Se/kg mineral supplement; ovines in G4 received 15 mg Se/kg mineral supplement; and ovines in G5 received 20 mg Se/kg mineral supplement. Ovines in all groups remained untreated for 14 days, followed by a treatment period of 56 days. Semen samples were obtained by electroejaculation. The DNA damage in semen samples was evaluated using the comet assay. The experimental design was implemented using a 5 × 5 Latin Square, i.e., five treatments and five experimental periods. The mean differences were compared using Tukeys test at a significance level of 5%. The control group (G1) showed a high percentage of DNA damage compared to the Se-treated groups (G2-G5). Therefore, Se supplementation could decrease the basal level of DNA damage in sperm cells, suggesting that Se might exert protective effects on sperm DNA.(AU)

O presente estudo teve por objetivo avaliar os efeitos da suplementação mineral com diferentes concentrações de selênio (Se) sobre a integridade de DNA espermático de ovinos. Utilizaram-se 30 machos, com 10 meses de idade. Eles foram mantidos em sistema intensivo, sendo alimentados com feno e ração própria para ovinos, divididos em baias (5 m x 3 m), com cochos e água ad libitum. Os ovinos do grupo 1 (G1=controle negativo) receberam suplementação de sal mineral sem a adição de Se, os animais do G2 receberam a mesma mistura mineral, porém com 5 mg de Se (selenito de sódio)/kg mistura mineral, os ovinos do G3 receberam 10 mg Se/kg mistura, os animais do G4 receberam 15 mg Se/kg mistura, os do G5 receberam 20 mg Se/kg mistura. Os ovinos de todos os grupos passaram por um período de adaptação de 14 dias, seguido por um período de tratamento de 56 dias. O sêmen foi colhido por meio de eletroejaculação. A integridade do DNA espermático foi avaliada por meio do teste de cometa. O modelo experimental utilizado foi Quadrado Latino 5 x 5, com cinco grupos e cinco períodos experimentais. A diferença entre as médias foi analisada pelo teste de Tukey, com 5% de nível de significância. O grupo controle (G1) apresentou elevada porcentagem de danos quando comparada aos demais grupos de tratamentos (G2 a G5). Portanto, a suplementação de Se diminui o nível de danos ao DNA espermáticos, sugerindo que o Se pode exercer efeitos protetores sobre o DNA dos espermatozoides de ovinos.(AU)

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

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

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

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

Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14)

The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a GelatinSepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.(AU)

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

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

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.

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.

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...(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)

Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)