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The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P< 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P< 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant.

O presente estudo objetivou avaliar a eficácia de um protocolo one-step de congelamento do sêmen suíno utilizando dimetilacetamida (DMA) e etilenoglicol como crioprotetores. Durante 10 semanas, a fração rica dos ejaculados de dois machos suínos foram coletados, uma vez por semana. Após a coleta, os ejaculados foram diluídos (1:1; v/v) no diluidor de resfriamento. Após a avaliação da concentração espermática, os ejaculados foram agrupados em um pool com o mesmo número de espermatozoides de cada macho e estabilizados a 20 °C por 120 min. Os criopropetores foram adicionados ao diluidor de congelamento a 20 °C, em diferentes concentrações, compondo seis tratamentos: glicerol (controle), 1,25% e 2,5%; etilenoglicol, 1,5% e 2,5%; e DMA, 2,5% e 5,0%. As amostras foram armazendadas em palhetas de 0,25 mL contendo 35 x 106 espermatozoides. Após 90 min a 20 °C as palhetas foram submetidas a uma curva de resfriamento até 5 °C (0,3 a 0,5 °C/mim) e mantidas a 5 °C por 60 min. O congelamento foi realizado a partir da colocação das palhetas horizontalmente a 5 cm acima do nitrogênio líquido por 10 min, com sua posterior imersão no nitrogênio líquido. Após o descongelamento a 37 °C por 30 segundos a qualidade espermática foi avaliada através de um sistema computadorizado e por citometria de fluxo. A motilidade espermática foi maior (P < 0,05) nos tratamentos com 5,0% e 2,5% DMA (22,2 ± 2,6% e 20,0 ± 2,8%, respectivamente) do que no tratamento com 2,5% etilenoglicol (8,2 ± 1,0%). A integridade da membrana plasmática (P = 0,08) e potencial de membrana mitocondrial (P = 0,27) foi similar entre os tratamentos. O tratamento com 2,5% de etilenoglicol foi menos eficiente em manter membrana acrossomal intacta (P< 0,01). Alguns parâmetros de cinética espermática (DAP, DCL, DSL, VAP, VCL, VSL e ALH) foram afetados positivamente pelo uso de DMA a 5.0%. O protocolo simplificado para congelamento de sêmen suíno resultou em motilidade espermática insatifatória após o descongelamento, independente do crioprotetor utilizado.

Resumo

The aim of the present study was to evaluate semen cryopreservation with ACP-Lact® diluent, which consists of coconut water powder (ACP) added to goat milk powder. After thawing, the samples were evaluated for sperm kinetics, membrane evaluation and in vivo insemination. For cryopreservation, a pool was made with the ejaculate of six goats, diluted in four equal aliquots for the respective treatments: T1 (ACP-Lact®); T2 (ACP-Lact® 50%); T3 (ACP + 2.5% egg yolk) and T4 (Tris + 2.5% egg yolk). After dilution of the treatments, the samples were placed in 0.5 ml straws and chilled at a rate of -1.07°C/min. After reaching 4°C and stabilizing for one hour, the straws were placed in nitrogen vapour at -60°C for 15 minutes and then immersed in liquid nitrogen (-196ºC). The straws were thawed in a 37°C water bath and kinetic assessments were performed immediately using a computerized semen analysis program (CSA), viability (EN), membrane functionality (HOST), mitochondrial activity (DAB) and DNA integrity assessment of spermatozoa. For the in vivo experiment, ten goats were inseminated, divided into two groups of five goats each, G1 inseminated with ACP-Lact® and G2 with ACP, by fixed-time artificial insemination (FTAI). Regarding the kinetic parameters, the ACP-Lact® treatment showed higher progressive motility (PM) and sperm velocity than the other treatments (36.77%). In the VSL parameter the ACP-Lact diluent was superior to ACP and Tris. In viability the treatment with ACP-Lact® was superior to the treatment with Tris, 95% and 83% respectively. In FTAI two goats were born out of the 5 goats inseminated with ACP-Lact®. It was concluded that the use of ACP-Lact® for cryopreservation of caprine semen is efficient in maintaining seminal parameters during thawing in vitro and in vivo and proved to be a good alternative extender for the caprine species.(AU)

Resumo

ABSTRACT: The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

RESUMO: A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.

Resumo

The domestic pig breeds are in danger of extinction whereas the erosion of their gene pool is a serious concern because they significantly contribute to the rich biodiversity. Overall aim of this study was to determine the protocol for preserving the semen of the Windsnyer boars for conservation. A total of 18 ejaculates (6 replications/boar) were collected from three Windsnyer boars of proven fertility with the use of hand-gloved approach method, twice per week. Boars semen were pooled and extended with Beltsville Thawing Solution [(BTS) IMV Technologies, France], held at 18°C for 3 hours and centrifuged. The sperm pellet was re-suspended with Fraction A (20% egg yolk + BTS) and cooled at 5°C for 1 hour. Following cooling, semen was divided and diluted into different cryoprotectants (ethylene glycol, glycerol, propanediol, ethylene glycol + glycerol + propanediol) at equal contribution to make the total concentrations [4, 8, 12 and 16% and the 0% (control; without cryoprotectant)] and loaded into 0.25 mL straws. Two cryopreservation methods (liquid nitrogen vapour and controlled rated) were used to cryopreserve the semen straws. Semen straws were thawed at different temperatures (5, 18, 37 and 40°C) and evaluated for sperm motility, viability, and morphology traits. Post-thawed sperm total motility (36.0±5.3) and live normal sperm (49.5±8.3) percentages were recorded to be higher in the treatment supplemented with 16% glycerol (P<0.05). The highest sperm total motility percentage was recorded at 40°C (26.8±3.2) thawing temperature for liquid nitrogen vapour treatment (P<0.05). In conclusion, 16% glycerol was found to be the suitable cryoprotectant concentration for semen cryopreserved with liquid nitrogen vapour method and thawed at 40°C.(AU)

Resumo

Agricultural practices such as the use of cover crops inoculated with microorganisms aim to transform productive systems into sustainable ecosystems, since it reduces negative impacts on the environment and production costs and at the same time, increases the productivity of the main crops by improving soil attributes. This study aimed to determine the effect of the application of beneficial rhizobacteria in cover crops grown in the off-season, the accumulation of nutrients by these plants and weed control. The experiments were conducted in an experimental area of Embrapa Arroz e Feijão, in the 2020, 2021 and 2022 seasons, with a randomized block design, in the 2x8 factorial scheme and four replications. The treatments consisted of the combination of 8 vegetable covers ((1. fallow/weeds), 2. Corn, 3. Mix 1 (White Lupin, Buckwheat, White Oats, Black Oats, C. ochroleuca, C. Juncea, Forage Turnip, Coram Cane); 4. 2 (Buckwheat, C. spectabiliis, turnip, black oat); 5. Mix 3 (Millet, C. ochroleuca, black aveite, white oat, buckwheat, Coracan grass); 6. Mix 4 (C. spectabilis, buckwheat, millet and C. breviflora); 7. Mix (Oats, Buckwheat, Millet, Piatã and C. Ocholeuca); and 8. Mix 6 (Oats black, Turnip Forage, White Lupine, Coracan Grass, Buckwheat)) with or without microorganisms (rhizobacteria Serratia marcencens (BRM 32114) + Bacillus sp. (BRM 63573)). The evaluations consisted of the content of nutrients and dry biomass of the aerial part, using the LSD statistical test (p <0.05). Vegetable toppings, especially corn, mix 4 and mix 5, treated with beneficial rhizobacteria increased the amount of phytomass at 60 days after planting, accumulation of macronutrients N, P, K, Ca and Mg and micronutrients Cu, Fe and Mn compared to the fallow treatment. Higher S content was found in the straws of mix 2 and mix 4 and Zn in corn straws, mix 4 and mix 5, regardless of microbial treatment. In addition, plant covers provided reductions in weed incidence. Therefore, plant cover and multifunctional microorganisms stand out as sustainable technologies, since the improvement of nutritional conditions and the amount of dry biomass of cover crops can directly reflect the availability of nutrients for the successor crop and greater protection and organic matter content for the soil.(AU)

Práticas agrícolas, como o uso de plantas de cobertura inoculadas com microrganismos, objetivam transformar os sistemas produtivos em ecossistemas sustentáveis, uma vez que reduzem os impactos negativos ao ambiente e os custos de produção e, ao mesmo tempo, incrementam a produtividade das culturas principais por meio da melhoria nos atributos do solo. Este estudo objetivou determinar o efeito da aplicação de rizobactérias benéficas em plantas de cobertura cultivadas na entressafra, no acúmulo de nutrientes por essas plantas e no controle de plantas daninhas. Os experimentos foram conduzidos em área experimental em Santo Antônio de Goiás, Goiás, nas safras 2020, 2021 e 2022, com delineamento em blocos casualizados, no esquema fatorial 2x8 e quatro repetições. Os tratamentos consistiram na combinação de 8 coberturas vegetais ((1. pousio/plantas daninhas), 2. Milho, 3. Mix 1 (Tremoço Branco, Trigo Mourisco, Aveia Branca, Aveia Preta, C. ochroleuca, C. Juncea, Nabo Forrageiro, Capim coracana); 4. Mix 2 (trigo Mourisco, C. spectabiliis, nabo forrageiro, aveia preta); 5. Mix 3 (Milheto, C. ochroleuca, aveita preta, aveia branca, trigo mourisco, Capim coracana); 6. Mix 4 (C. spectabilis, trigo mourisco, milheto e C. breviflora); 7. Mix 5 (Aveia, Trigo Mourisco, Milheto, Piatã e C. Ocholeuca); e 8. Mix 6 (Aveia preta, Nabo Forrageiro, Tremoço Branco, Capim coracana, Trigo Mourisco)) com ou sem microrganismos (rizobactérias Serratia marcencens (BRM 32114) + Bacillus sp. (BRM 63573)). As avaliações consistiram no teor de nutrientes e biomassa seca da parte aérea, utilizando o teste estatístico LSD (p<0,05). Coberturas vegetais, com destaque para milho, mix 4 e mix 5, tratadas com rizobactérias benéficas aumentaram a quantidade de fitomassa, aos 60 dias após o plantio, acúmulo de macronutrientes N, P, K, Ca e Mg e micronutrientes Cu, Fe e Mn em comparação com o tratamento pousio. Maior teor de S foi encontrado nas palhadas do mix 2 e mix 4 e de Zn nas palhadas de milho, mix 4 e mix 5, independentemente do tratamento microbiano. Além disso, as coberturas vegetais proporcionaram reduções na incidência de plantas daninhas. Portanto, cobertura vegetal e microrganismos multifuncionais se destacam como tecnologias sustentáveis, uma vez que a melhoria das condições nutricionais e da quantidade de biomassa seca das plantas de cobertura podem refletir diretamente na disponibilidade de nutrientes para a cultura sucessora e maior proteção e conteúdo de matéria orgânica para o solo(AU)

Resumo

The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.

Resumo

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

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Searching for improvements in semen cryopreservation, natural substances are commonly studied focusing to improve the sperm quality. The aim of this study were evaluated the effect of adding orange, pineapple, and beet juices in different concentrations and combinations to the ram semen cryopreservation extender. Five ejaculates from five adult rams were used. The semen pool was diluted in egg yolk-based extender and mixed with the following 15 treatments (at a final concentration of 400.106 sptz/mL): orange 10% (O10) and 15% (O15); pineapple 10% (P10) and 15% (P15); beet 10% (B10) and 15% (B15); pineapple + orange 10% (PO10) and 15% (PO15); pineapple + beet 10% (PB10) and 15% (PB15); beet + orange 10% (BO10) and 15% (BO15); pineapple + beet + orange 10% (PBO10) and 15% (PBO15); and the control group (CON). Post-thaw in 0.25 mL straws semen quality analysis of cryopreserved semen was performed by CASA and flow cytometry. Analysis of variance (PROC GLM) was carried out and the averages were compared using the SNK test. Pearson's correlation test was also performed. No effect was noted in the addition of juices to the semen extender prior to cryopreservation. Post-thawed, although, statistically similar to the control group, the total motility of the B10 group reached acceptable standards of total motility. In addition, B10 group showed the highest values (p< 0.05) of progressive motility than control group or other treatments. The addition of 10% beet juice to the ram semen extender can improve the cryopreservation of sperm motility.

Em busca de melhorias na criopreservação do sêmen, substâncias naturais são comumente estudadas com o objetivo de melhorar a qualidade do sêmen. O objetivo deste estudo foi avaliar o efeito da adição de sucos de laranja, abacaxi e beterraba em diferentes concentrações e combinações ao diluidor de criopreservação de sêmen ovino. Foram utilizados cinco ejaculados de cinco carneiros adultos. O pool de sêmen foi diluído em diluente à base de gema de ovo e misturado com os seguintes 15 tratamentos (na concentração final de 400x106 sptz/ml): laranja 10% (O10) e 15% (O15); abacaxi 10% (P10) e 15% (P15); beterraba 10% (B10) e 15% (B15); abacaxi + laranja 10% (PO10) e 15% (PO15); abacaxi + beterraba 10% (PB10) e 15% (PB15); beterraba + laranja 10% (BO10) e 15% (BO15); abacaxi + beterraba + laranja 10% (PBO10) e 15% (PBO15); e o grupo controle (CON). Pós-descongelação em palhetas de 0,25 ml a análise da qualidade do sêmen criopreservado foi realizada pelo CASA e citometria de fluxo. A análise de variância foi realizada e as médias comparadas pelo teste SNK. O teste de correlação de Pearson também foi realizado. Nenhum efeito foi observado na adição de sucos ao diluidor de sêmen antes da criopreservação. Após o descongelamento, embora estatisticamente semelhante ao grupo controle, a motilidade total do grupo B10 atingiu padrões aceitáveis de motilidade total. Além disso, o grupo B10 apresentou os maiores valores (p<0,05) de motilidade progressiva que o grupo controle ou os outros tratamentos. A adição de 10% de suco de beterraba ao diluente de sêmen ovino pode melhorar a criopreservação da motilidade espermática.

Resumo

Abstract Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

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Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.(AU)

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ABSTRACT: The management of white mold (Sclerotinia sclerotiorum(Lib.) De Bary) has been one of the main production limitations faced by soybean (Glycine max L.) producers. Considering the complex management of this disease and resistance structure of the pathogen, the present study was conducted in the municipalities of Guarapuava and Palmas in Paraná with the objective of managing the white mold of soybean using straws of winter cereals, such as oat, rye, and triticale. Initially, the three winter cereals were cultivated simultaneously in both the study areas. Straw production, plant height, and shoot fresh and dry weight were evaluated. Subsequently, BMX Apollo soybean was cultivated on cereal straws, and the incidence and severity of white mold were evaluated. In the in vitro experiment, 20 sclerotia covered by a layer of cereal (oat, rye, and triticale) straws were added and carpogenic germination was observed only in the control treatment after 98 days. Regardless of the study site, rye presented greater height and fresh weight than the remaining two cereals. Soybean cultivation on winter cereal straw reduced the incidence and severity of white mold. Cultivation on rye straw reduced mold incidence by 77.7% and 76.6% in Palmas and Guarapuava, respectively.

RESUMO: O manejo do mofo branco (Sclerotinia sclerotiorum) tem sido um dos principais limitantes de produção enfrentados por produtores de soja. Tendo em vista a complexidade do manejo desta doença e da estrutura de resistência do patógeno, o presente trabalho foi conduzido nos municípios de Guarapuava e Palmas - Paraná visando o manejo do mofo branco na cultura da soja utilizando palhadas de cereais de inverno, aveia, centeio e triticale. Inicialmente, cultivou-se os diferentes cereais de inverno, simultaneamente, nas duas áreas de estudo (Guarapuava e Palmas - PR). Avaliou-se a produção de palhada, a altura de plantas, a massa verde e seca da parte aérea. Posteriormente, semeou-se soja 'BMX Apolo' sobre as palhadas de cereais e avaliou-se a incidência e a severidade do mofo branco. No experimento in vitro, adicionou-se 20 escleródios cobertos por uma camada de palhas (aveia, centeio e triticale) e após 98 dias, observou-se germinação carpogênica somente no tratamento testemunha. Independentemente do local de estudo, o centeio destacou-se com maiores altura e massa verde. O cultivo sobre palhada de cereais de inverno reduziu a incidência e severidade de mofo branco com destaque para a palhada de centeio, reduzindo a incidência da doença em 77,7% e 76,6%, em Palmas e Guarapuava, respectivamente.

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Abstract The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/109) compared with the control (22.66±4.26 nmol/109). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.

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A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

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Devido à alta sensibilidade do espermatozoide suíno à criopreservação, torna-se importante o estudo acerca dos danos provocados à célula, pela agressão térmica ocasionada durante este processo. Sendo assim, avaliou-se neste trabalho, a influência de diferentes embalagens, utilizadas para o armazenamento de sêmen suíno, sobre a qualidade do espermatozoide criopreservado. Foram utilizados animais híbridos, a coleta do sêmen foi feita pela técnica da mão enluvada. Foram analisados o vigor (0 a 5), a motilidade (0 a 100%), a taxa de degradação da motilidade e a integridade acrossomal. O sêmen foi congelado em palhetas de 0,5mL, criotubos de 2,0mL e macrotubos de 4 e 5mL. As amostras de sêmen suíno congelado em palhetas apresentaram os melhores resultados de vigor espermático (2,3), de motilidade (45,4%), de taxa de degradação da motilidade (56,5%) e de integridade acrossomal (60,6%), quando comparadas às amostras criopreservadas nos demais tipos de embalagens avaliadas (p<0,05). Somente para o parâmetro taxa de degradação da motilidade, o sêmen conservado em palhetas apresentou resultados similares ao conservado em macrotubo de 4mL (54,1%). Os resultados pós-descongelação indicaram que o sêmen envasado nas palhetas foi o que apresentou características condizentes com a possibilidade de serem utilizados nos protocolos de inseminação artificial, com possibilidades de bons resultados de fertilidade. Concluiu-se que embalagens que permitam uma maior velocidade de trocas de temperatura entre as células encontradas no bordo e no centro, favorecem a uma melhor qualidade do sêmen descongelado.

Due to high sensitivity of the swine spermatozoa to cryopreservation, it is important to study the damage caused to the cell by thermal aggression during this process. Thus, this study evaluated the influence of different packages used for the storage of swine semen on the quality of cryopreserved spermatozoa. Hybrid animals were used, and semen collection was made by the gloved hand technique. The vigor (0 the 5), motility (0 the 100%), rate of motility degradation and acrosome integrity were analyzed. The semen was frozen in straws of 0.5mL, cryotubes of 2.0mL and macrotubes of 4 and 5mL. The swine semen samples frozen in straws showed the best results in sperm vigor (2.3), motility (45.4%), motility degradation rate (56.5%) and acrosomal integrity (60.6%), when compared to the cryopreserved samples in the other types of packaging evaluated (p<0.05). Only for the motility degradation rate parameter, the semen preserved in straws showed results similar to the one conserved in a 4 mL macrotube (54.1%). The results after thawing indicated that the semen packed in straws was the one that presented consistent characteristics with the possibility of being used in artificial insemination protocols with possibilities of good fertility results. It was concluded that packages that allow a higher speed of temperature changes between the cells found on the edge and in the center of them, favor a better quality of the thawed semen.

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A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

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The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

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

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

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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)

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Objetivou-se avaliar o efeito da adição de diferentes concentrações do colesterol carregado por ciclodextrina (CCC) sobre os espermatozoides congelados de ovinos. Foram coletados dois ejaculados de 10 carneiros (n=20) e diluídos em Tris-Gema de ovo até a concentração final de 200 x106 sptz/mL e mantidos em banho maria a 32 °C. O CCC foi adicionado: controle (0,0mg), 1,5mg, 3,0mg e 6,0mg de CCC/120 x106 sptz/mL. Após adição, o sêmen foi resfriado a 5 °C por duas horas, após esse período, envasado em palhetas de 0,5mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida/15 minutos 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 à análise de variância e médias comparadas pelo teste de Tukey a 5% de probabilidade. O maior percentual de integridade da membrana plasmática, acrossomal e a maior atividade mitocondrial foram obtidos utilizando 6,0mg de CCC. A adição de3,0mg de CCC manteve o percentual de motilidade espermática após a criopreservação, quando comparado aos demais tratamentos e controle. A adição de 1,5 e 3,0mg de CCC mantiveram o percentual de viabilidade espermática após a criopreservação acima de 65%. O número de espermatozoides com capacidade de ligação a membrana perivitelina da gema de ovo foi maior (p<0,05) no tratamento com 3,0mg de CCC. Concluiu se que a adição de CCC ao sêmen diluído, nas concentrações avaliadas, melhora a qualidade espermática após descongelação.

The objective was to evaluate the effect of adding different concentrations of cholesterol carried by cyclodextrin (CCC) on frozen ovine sperm. Two ejaculates were collected from 10 rams (n=20) and diluted in Tris-Yolk until the final concentration of 200 x 106 sptz/mL was reached and kept in a water bath at 32 °C. The CCC was added: control (0,0mg), 1.5mg, 3.0mg, and 6.0mg of CCC/120 x 106 sptz/mL. After the addition, the semen was cooled at 5 °C for two hours, after that period, filled in 0.5 mL straws, and then conditioned under liquid nitrogen vapor (N2L), at 8 cm of the liquid/15 minutes, and then immersed in N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity, and binding test. The variables were subjected to analysis of variance and means compared by Tukey's test at 5% probability. The highest percentage of plasma membrane integrity and the highest mitochondrial activity were obtained using 6.0mg of CCC. The addition of 3.0mg of CCC maintained the percentage of sperm motility after cryopreservation, when compared to other treatments and control. The addition of 1.5 and 3.0mg of CCC maintained the percentage of sperm viability after cryopreservation, above 65%. The count of sperm with ability to bind to the egg yolk perivitelline membrane was higher (p<0.05) with 3.0mg of CCC. It is concluded that the addition of CCC to the diluted semen, in the evaluated concentrations, improves the sperm quality after thawing.

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Prochilodus brevis is a fish species with an important ecological and economic role. Therefore, its captive breeding, together with sperm cryopreservation, come as an alternative to improve its availability. To perform sperm cryopreservation with quality results, it is necessary to develop studies on cryomedia composition, such as non-permeable cryoprotectants. The aim of the study was to test the efficacy of two non-permeable cryoprotectants on post-thawed kinetic parameters of Prochilodus brevis sperm. Twenty reproductive mature P. brevis males were selected and induced to sperm with carp pituitary extract. The sperm was collected, and five pools were made. Pools were diluted in 5% glucose and 10% DMSO added or not with three different concentrations of egg yolk (5; 10 or 12%). The samples were placed on French straws, frozen in dry shipper and stored in liquid nitrogen. After thawing, the kinetic analyses of computer assisted sperm analysis (CASA) were performed. There were no significant differences between the total motility of the control (60.48%) and treatments (49.0; 47.8 and 51.68%, respectively). The other kinetic parameters also showed no statistical difference. Thus, there is no need to use egg yolk in the freezing of P. brevis sperm when using a glucose and DMSO extender.