Cooling of porcine semen in an extender supplemented with carvacrol.

The addition of antioxidants in boar semen is an alternative to mitigate the reduction of sperm quality during preservation. To evaluate the effect of carvacrol on cooling of boar semen. Fifteen ejaculates from five boars were extended in MR-A® with 0, 5, 10, 15, 15, 20, 25 and 30 µM of carvacrol (C) and were cooled for 5 days at 16°C. Sperm motility and kinetics were evaluated with computer-assisted semen analysis (CASA). At 0 and 96 h, membrane functionality was determined by hypoosmotic test; reactive oxygen species (ROS) production and total antioxidant capacity (TAC) by spectrofluorimetry and mitochondrial membrane potential (Δ¥M) by flow cytometry. Linear models, regression analysis and comparison of means by Duncan test, were conducted. The addition of carvacrol did not influence sperm motility, but at low concentrations decreased ROS production, whereas 30 µM C reduced the membrane functionality and 25 µM C decreased Δ¥M. In addition, regression coefficients showed that C produced a lower rate of decrease in different parameters of sperm motility and kinetics. During cooling there is a reduction in sperm quality due to the excessive production of ROS, generating oxidative stress and affecting cell permeability and functionality. In this study, it was possible to demonstrate the protective activity of C as a molecule capable of neutralizing free radicals. In addition, it has been proposed that C is also capable of reducing peroxyl radicals, superoxide radicals, hydrogen peroxide and nitric oxide. Carvacrol can mitigate the reduction of boar semen quality during the storage period under cooling conditions. Likewise, it can reduce ROS production and modulate the mitochondrial activity of porcine sperm.

Estimation of sperm concentration limits to produce intrauterine insemination doses in swine.

This study evaluated the effect of sperm concentration of boar semen doses, for intrauterine artificial insemination (IUAI), on semen quality and established concentration limits for their production. Twenty ejaculates from four crossbred mature PIC® boars were collected to produce 50 mL semen doses in a split sample, reaching the following sperm concentrations: ~20, 30, 60, and 100 × 106 cells/mL. Doses were produced using Androstar® Plus, stored at 17°C, and evaluated until 120 h of storage. There was a linear decrease in sperm motility as the sperm concentration increased (p linear < .01). The concentration which no longer affected the total and progressive motility was 59 and 55 × 106 cells/mL, respectively (corresponding to 71% and 62%, respectively). The pH linearly decreased as the sperm concentration increased (p < .01); yet, at 72 and 120 h, the parameter dramatically reduced in boar semen doses with 60 and 100 × 106 cells/mL. The percentage of cells with intact plasma and acrosomal membranes or with high mitochondrial membrane potential was not influenced by the sperm concentration (p ≥ .15). In conclusion, sperm motility was negatively affected in highly (60 and 100 × 106 cells/mL) concentrated doses. To achieve suitable sperm motility, boar semen doses may not surpass the sperm concentration of 55 × 106 cells/mL. The effect of low-concentrated boar semen doses on sperm quality still needs to be better evaluated, mainly considering the influence of extender type and thermo-resistance conditions.

Bacteria and Boar Semen Storage: Progress and Challenges.

Porcine breeding today is based on artificial insemination with chilled semen. This is stored at 5 °C with antibiotic supplementation to avoid bacteriospermia. There are many negative consequences on sperm quality and functionality as a result of bacterial contamination, as well as on the health of the sow. Nowadays, various techniques are being developed to reduce the indiscriminate use of antibiotics and thus avoid the generation of antibiotic resistance genes. This review aims to inform about the bacterial contamination consequences of storing liquid semen from boar and to provide an update on current methods and alternatives to antibiotic use in cold storage.

Boar semen cryopreserved with trehalose-containing liposomes: disaccharide determination and rheological behaviour.

This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing-thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.

A 5-color flow cytometry panel to assess plasma membrane integrity, acrosomal status, membrane lipid organization and mitochondrial activity of boar and stallion spermatozoa following liquid semen storage.

For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rhodamine 123, Merocyanine 540, Propidium Iodide, PNA-Alexa Fluor 647, and Hoechst 33342, respectively. The sixth aliquot was stained with all the five fluorochromes simultaneously, whereas spectral overlap was corrected by a compensation matrix. Strong correlations were found between the single and 5-color staining assays for boar sperm (A: 0.99, B: 0.96, C: 0.93, D: 0.98, E: 0.99; P < 0.01). Furthermore, moderate and substantial Concordance Correlation Coefficients (CCC) were presented by all these parameters (0.99, 0.96, 0.92, 0.98, and 0.99, respectively). For stallion sperm, the correlation coefficients between the assays were also strong (A: 0.99, B: 0.98, C: 0.99, D: 0.99, E: 0.95; P < 0.01) and substantial CCC were observed for all of them (0.99, 0.97, 0.99, 0.99, and 0.90, respectively). For both species, the mean difference between the methods (d̅) did not overtake 0.84. The results confirmed that this 5-color panel could be successfully implemented for analyzing boar and stallion sperm quality in a single, practical and quick FC assay.

Addition of chlorogenic acid and caffeine during the processing of cooled boar semen / Adição de ácido clorogênico e cafeína durante o processamento do sêmen suíno resfriado

A study was conducted to evaluate the effect of chlorogenic acid (ChA) added pre-cooling and its combination with caffeine added during warming on cooled-stored boar semen parameters. Ten ejaculates were diluted in commercial extender with or without 4.5mg/ml ChA and stored at 15°C. After 0, 24 and 72 hours of storage, aliquots of these doses were taken and incubated at 37°C in the presence or absence of 8.0mM caffeine. Semen quality was evaluated after 10 and 120 minutes of incubation. The ChA increased (P <0.01) the sperm motility, viability, acrosomal integrity and the percentage of spermatozoa with high mitochondrial activity (PMHA), however, decreased (P <0.01) the malondialdehyde (MDA) concentration. Caffeine increased (P<0.05) the sperm motility, viability, PMHA and the MDA concentration and reduced (P <0.05) the acrosome integrity. When associated (ChA+caffeine), there was an increase (P <0.05) in sperm motility and viability, PMHA and acrosome integrity. The addition of ChA to the dilution medium improves the quality of the swine inseminating doses. The addition of caffeine during re-warming is only recommended when the semen is stored for prolonged periods (72h), and the inseminating dose should be used immediately after its addition.(AU)

O objetivo deste estudo foi avaliar os efeitos da adição de ácido clorogênico (ChA) antes do resfriamento e sua combinação com cafeína adicionada durante o reaquecimento sobre a qualidade do sêmen suíno resfriado. Dez ejaculados foram diluídos em diluidor comercial com adição ou não de 4,5mg/mL de ChA e armazenados a 15°C. Após zero, 24 e 72 horas de armazenamento, 10mL foram retirados e incubados a 37°C na presença ou ausência de 8,0mM de cafeína. A qualidade seminal foi avaliada após 10 e 120 minutos de incubação. O ChA aumentou (P<0,01) a motilidade, a viabilidade, a integridade acrosomal e a porcentagem de espermatozoides com alta atividade mitocondrial (PMHA), entretanto diminuiu (P<0,01) a concentração de malondialdeído (MDA). A cafeína aumentou (P<0,05) a motilidade, a viabilidade, a PMHA e a concentração de MDA e reduziu a integridade acrossomal. Quando associados (ChA+cafeína), houve aumento (P<0,05) na motilidade, na PMHA, na viabilidade e na integridade acrossomal. Conclui-se que a adição de ChA ao meio de diluição melhora a qualidade das doses inseminantes de suínos. A adição de cafeína durante o reaquecimento só é recomendada ao sêmen adicionado de ChA quando esse for armazenado por períodos prolongados (72h), devendo a dose inseminante ser utilizada imediatamente após sua adição.(AU)

Addition of chlorogenic acid and caffeine during the processing of cooled boar semen / Adição de ácido clorogênico e cafeína durante o processamento do sêmen suíno resfriado

A study was conducted to evaluate the effect of chlorogenic acid (ChA) added pre-cooling and its combination with caffeine added during warming on cooled-stored boar semen parameters. Ten ejaculates were diluted in commercial extender with or without 4.5mg/ml ChA and stored at 15°C. After 0, 24 and 72 hours of storage, aliquots of these doses were taken and incubated at 37°C in the presence or absence of 8.0mM caffeine. Semen quality was evaluated after 10 and 120 minutes of incubation. The ChA increased (P <0.01) the sperm motility, viability, acrosomal integrity and the percentage of spermatozoa with high mitochondrial activity (PMHA), however, decreased (P <0.01) the malondialdehyde (MDA) concentration. Caffeine increased (P<0.05) the sperm motility, viability, PMHA and the MDA concentration and reduced (P <0.05) the acrosome integrity. When associated (ChA+caffeine), there was an increase (P <0.05) in sperm motility and viability, PMHA and acrosome integrity. The addition of ChA to the dilution medium improves the quality of the swine inseminating doses. The addition of caffeine during re-warming is only recommended when the semen is stored for prolonged periods (72h), and the inseminating dose should be used immediately after its addition.(AU)

O objetivo deste estudo foi avaliar os efeitos da adição de ácido clorogênico (ChA) antes do resfriamento e sua combinação com cafeína adicionada durante o reaquecimento sobre a qualidade do sêmen suíno resfriado. Dez ejaculados foram diluídos em diluidor comercial com adição ou não de 4,5mg/mL de ChA e armazenados a 15°C. Após zero, 24 e 72 horas de armazenamento, 10mL foram retirados e incubados a 37°C na presença ou ausência de 8,0mM de cafeína. A qualidade seminal foi avaliada após 10 e 120 minutos de incubação. O ChA aumentou (P<0,01) a motilidade, a viabilidade, a integridade acrosomal e a porcentagem de espermatozoides com alta atividade mitocondrial (PMHA), entretanto diminuiu (P<0,01) a concentração de malondialdeído (MDA). A cafeína aumentou (P<0,05) a motilidade, a viabilidade, a PMHA e a concentração de MDA e reduziu a integridade acrossomal. Quando associados (ChA+cafeína), houve aumento (P<0,05) na motilidade, na PMHA, na viabilidade e na integridade acrossomal. Conclui-se que a adição de ChA ao meio de diluição melhora a qualidade das doses inseminantes de suínos. A adição de cafeína durante o reaquecimento só é recomendada ao sêmen adicionado de ChA quando esse for armazenado por períodos prolongados (72h), devendo a dose inseminante ser utilizada imediatamente após sua adição.(AU)

Uso de diluentes alternativos e do ácido fítico adicionado ao sêmen suíno conservado / Use of alternative extenders and the phytic acid added to stored boar semen

Antioxidantes são utilizados na conservação do sêmen de diversas espécies domésticas. Diante disso, o presente estudo teve como objetivo verificar o melhor diluente para adicionar diferentes concentrações de ácido fítico e verificar os efeitos sobre os espermatozoides suínos conservados. Na etapa I, 60 ejaculados e três diluentes foram utilizados: BTS; LPD e ACP-103®. Já na etapa 2, O BTS como melhor diluente da I etapa, foi acrescido de três concentrações de ácido fítico (n=60): (T1) BTS (controle); BTS+175µM de ácido fítico (T2); BTS+350µM (T3); e BTS+525µM (T4). Foram realizadas análises diárias de vigor e motilidade, e em D0 e D4 foram realizados os testes hiposmótico e de vitalidade e integridade acrossomal. A motilidade espermática no T4 foi superior (p<0,05) em relação ao T1. Em relação ao percentual de espermatozoides vivos, não houve diferença significativa em nenhum dos tratamentos testados, porém, tratando-se da integridade da membrana, o T4 em D0 e D4 obteve significativamente melhor resultado (p<0,05) em relação ao T1. A adição do ácido fítico no BTS promoveu efeitos positivos em determinadas análises, porém, mais estudos devem ser conduzidos para elucidação dos efeitos do ácido fítico sobre o metabolismo espermático.(AU)

Antioxidants are used for the preservation of semen from several domestic species. Diante disso, the present study aimed to determine the best diluent for adding different concentrations of phytic acid and verify the effects on boar sperm kept. In the step I, 60 ejaculated and three extenders were used: BTS; LPD and ACP-103®. In the step II, the BTS as best diluent of step I, has received three concentrations of phytic acid: (T1) BTS (control); BTS + 175mM of phytic acid (T2); BTS + 350mM (T3) and BTS + 525mM (T4). Daily analyzes of vigour and motility were performed, and in D0 and D4 hyposmotic and vitality and acrosomal integrity. The T4 on sperm motility was higher (p<0.05) compared to T1. Regarding the percentage of live sperm, no significant difference was found in any of the treatments tested, however, in the case of membrane integrity, T4 in D0 and D4 achieved significantly better results (p<0.05) in comparison with T1. The addition of phytic acid in BTS promoted positive effects in some analyzes, but more studies must be conducted to elucidate the effects of phytic acid on sperm metabolism. (AU)

Uso de diluentes alternativos e do ácido fítico adicionado ao sêmen suíno conservado / Use of alternative extenders and the phytic acid added to stored boar semen

Antioxidantes são utilizados na conservação do sêmen de diversas espécies domésticas. Diante disso, o presente estudo teve como objetivo verificar o melhor diluente para adicionar diferentes concentrações de ácido fítico e verificar os efeitos sobre os espermatozoides suínos conservados. Na etapa I, 60 ejaculados e três diluentes foram utilizados: BTS; LPD e ACP-103®. Já na etapa 2, O BTS como melhor diluente da I etapa, foi acrescido de três concentrações de ácido fítico (n=60): (T1) BTS (controle); BTS+175µM de ácido fítico (T2); BTS+350µM (T3); e BTS+525µM (T4). Foram realizadas análises diárias de vigor e motilidade, e em D0 e D4 foram realizados os testes hiposmótico e de vitalidade e integridade acrossomal. A motilidade espermática no T4 foi superior (p<0,05) em relação ao T1. Em relação ao percentual de espermatozoides vivos, não houve diferença significativa em nenhum dos tratamentos testados, porém, tratando-se da integridade da membrana, o T4 em D0 e D4 obteve significativamente melhor resultado (p<0,05) em relação ao T1. A adição do ácido fítico no BTS promoveu efeitos positivos em determinadas análises, porém, mais estudos devem ser conduzidos para elucidação dos efeitos do ácido fítico sobre o metabolismo espermático.

Antioxidants are used for the preservation of semen from several domestic species. Diante disso, the present study aimed to determine the best diluent for adding different concentrations of phytic acid and verify the effects on boar sperm kept. In the step I, 60 ejaculated and three extenders were used: BTS; LPD and ACP-103®. In the step II, the BTS as best diluent of step I, has received three concentrations of phytic acid: (T1) BTS (control); BTS + 175mM of phytic acid (T2); BTS + 350mM (T3) and BTS + 525mM (T4). Daily analyzes of vigour and motility were performed, and in D0 and D4 hyposmotic and vitality and acrosomal integrity. The T4 on sperm motility was higher (p<0.05) compared to T1. Regarding the percentage of live sperm, no significant difference was found in any of the treatments tested, however, in the case of membrane integrity, T4 in D0 and D4 achieved significantly better results (p<0.05) in comparison with T1. The addition of phytic acid in BTS promoted positive effects in some analyzes, but more studies must be conducted to elucidate the effects of phytic acid on sperm metabolism.

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Bacterial Contaminants and Antimicrobial Susceptibility Profile of Boar Semen in Southern Brazil Studs / Contaminantes bacterianos y perfil de susceptibilidad del semen porcino en centros de recogida em Brasil

Abstract Objective. To assess the microbiological profile in seven Boar Studs (BS) in Southern Brazil, as well as evaluate antimicrobial susceptibility response to most commonly found microorganisms in BS. Material and methods. Bacteriologic analysis was carried out in samples from the water purification system, semen extender, raw and stored semen, lab benches, and other working surfaces. Results. Growth of a mixed bacterial population was observed in water samples from all but one BS. Approximately 85% of the BS had significant contamination on their working surfaces with at least one bacterial contaminant. A total of 86% of raw semen samples were contaminated with one or more different bacteria, while 100% of the boar studs provided contaminated samples. Bacterial susceptibility to antimicrobial agents varied from over 80% for gentamycin, neomycin and ceftiofur to 40% or less for penicillin and lincomycin. Conclusions. The identification of the critical points provides necessary support to devise better strategies to minimize contamination in BS. Also, assessing the level of antimicrobial drug resistance offers accurate information to formulate more efficient antibacterial protocols that closely observe the rational use of antibiotics.

Resumen Objectivo. Evaluar el perfil microbiológico de siete centros de recogida de semen porcino (BS) en el sur de Brasil, así como evaluar la susceptibilidad antimicrobiana a la mayoría de los microorganismos encontrados en los BS. Material y métodos. El análisis bacteriológico se realizó en muestras del sistema de purificación de agua, diluyentes de semen, semen crudo y almacenado, bancos de laboratorio y otras superficies de trabajo. Resultados. El crecimiento de una población bacteriana mixta se observó en muestras de agua de todas las BS excepto una. Aproximadamente el 85% de la BS tenía contaminación significativa en sus superficies de trabajo con al menos un contaminante bacteriano. Un total de 86% de muestras de semen crudo fueron contaminadas con una o más bacterias diferentes, mientras que el 100% de BS proporcionaron muestras contaminadas. La susceptibilidad bacteriana a agentes antimicrobianos varió en más del 80% para gentamicina, neomicina y ceftiofur a 40% o menos para penicilina y lincomicina. Conclusiones. La identificación de los puntos críticos proporciona el apoyo necesario para idear mejores estrategias para minimizar la contaminación en CRSP. Además, la evaluación del nivel de resistencia a los antimicrobianos ofrece información precisa para formular protocolos antibacterianos más eficientes que tengan en cuenta el uso racional de los antibióticos.

Uso de diferentes íons de cálcio adicionados ao diluente de sêmen suíno resfriado / Use of different calcium ions added in the extender of swine cooled semen

Estudos mostram que o íon cálcio é muito importante para regulação da motilidade espermática. Assim, objetivou-se avaliar a qualidade dos espermatozoides suínos resfriados sob o efeito de diferentes tipos e concentrações de íons cálcio no diluente BTS. O sêmen de varrões foi coletado e analisado, quanto aos seus parâmetros. O experimento contou com a utilização de diferentes tipos de cálcio, diluídos em BTS: Carbonato de cálcio, Hidróxido de cálcio, Fostato dibásico anidro de cálcio, Sulfato hidratado de cálcio e Sulfato anidro de cálcio, adicionados ao diluente BTS em diferentes concentrações: 0 mM (controle); 0,4mM; 4mM; 40Mm e 400mM. Após a coleta e avaliação seminal, as amostras foram diluídas earmazenadas a 17°C. Para a avaliação do efeito do cálcio, foi retirado uma alíquota de cada amostra, submetidaà temperatura de 37° C para a avaliação do vigor, motilidade e vitalidade espermática, realizadasno D0, D2 e D4. A adição dos sais de cálcio permite hiperativação aos espermatozoides quanto à membrana espermática e a membrana acrossômica, estes sais, em seus níveis já mencionados, não exerceram efeito negativo.(AU)

Studies show that calcium ion is very important for the regulation of sperm motility. Thus the objective was to assess the quality of sperm swine flu under the effect of different types and concentrations of calcium ions in the BTS. The semen of boars was collected andanalyzed for their parameters. The experiment included the use of different types of calcium diluted in BTS: calcium carbonate, calcium hydroxide, calcium phosphate dibasic anhydrous, hydrated calcium sulphate, anhydrous calcium sulphate, added to the BTS extender in different concentrations: 0 mM (control); 0.4 mM; 4 mM; 40mm and 400 mM. After collecting and seminal review, the samples were diluted and stored at 17°C. To evaluate the effect of calcium was removed from an aliquot of each sample, which wassubjected to a temperature of 37 °C for vigor, vitality and sperm motility, performed on D0, D2 and D4. The addition of calcium salts provides the sperm hyperactivation, and as a sperm membrane and acrosome membrane, these salts in their levels mentioned above did not exert a negative effect.(AU)

Uso de diferentes íons de cálcio adicionados ao diluente de sêmen suíno resfriado / Use of different calcium ions added in the extender of swine cooled semen

Estudos mostram que o íon cálcio é muito importante para regulação da motilidade espermática. Assim, objetivou-se avaliar a qualidade dos espermatozoides suínos resfriados sob o efeito de diferentes tipos e concentrações de íons cálcio no diluente BTS. O sêmen de varrões foi coletado e analisado, quanto aos seus parâmetros. O experimento contou com a utilização de diferentes tipos de cálcio, diluídos em BTS: Carbonato de cálcio, Hidróxido de cálcio, Fostato dibásico anidro de cálcio, Sulfato hidratado de cálcio e Sulfato anidro de cálcio, adicionados ao diluente BTS em diferentes concentrações: 0 mM (controle); 0,4mM; 4mM; 40Mm e 400mM. Após a coleta e avaliação seminal, as amostras foram diluídas earmazenadas a 17°C. Para a avaliação do efeito do cálcio, foi retirado uma alíquota de cada amostra, submetidaà temperatura de 37° C para a avaliação do vigor, motilidade e vitalidade espermática, realizadasno D0, D2 e D4. A adição dos sais de cálcio permite hiperativação aos espermatozoides quanto à membrana espermática e a membrana acrossômica, estes sais, em seus níveis já mencionados, não exerceram efeito negativo.

Studies show that calcium ion is very important for the regulation of sperm motility. Thus the objective was to assess the quality of sperm swine flu under the effect of different types and concentrations of calcium ions in the BTS. The semen of boars was collected andanalyzed for their parameters. The experiment included the use of different types of calcium diluted in BTS: calcium carbonate, calcium hydroxide, calcium phosphate dibasic anhydrous, hydrated calcium sulphate, anhydrous calcium sulphate, added to the BTS extender in different concentrations: 0 mM (control); 0.4 mM; 4 mM; 40mm and 400 mM. After collecting and seminal review, the samples were diluted and stored at 17°C. To evaluate the effect of calcium was removed from an aliquot of each sample, which wassubjected to a temperature of 37 °C for vigor, vitality and sperm motility, performed on D0, D2 and D4. The addition of calcium salts provides the sperm hyperactivation, and as a sperm membrane and acrosome membrane, these salts in their levels mentioned above did not exert a negative effect.

State-of-the-art of boar sperm preservation in liquid and frozen state

Pig breeding is mainly conducted through Artificial Insemination (AI) in Western and developing countries. Apart from requiring specific catheters and trained staff, preserving boar semen in proper conditions is needed to ensure high reproductive performances. Although, at present, boar sperm may be preserved in liquid (15-17ºC) or frozen states, more than 95% of AIs are conducted using liquid semen. The present work reviews the state-the-art of these two preservation technologies. Thus, the composition and types of extenders for liquid-stored semen are discussed, together with the specific requirements for boar sperm, which are stored at 15-17ºC. Commercial extenders for liquid semen are compared and the effects of storage on sperm quality are also summarised. In the second part of the manuscript, the main features of boar sperm cryopreservation are described and reference to cryodamage is also made. These cryoinjuries mainly affect sperm motility, membrane permeability and chromatin integrity. Furthermore, the individual variability in the sperm resilience to withstand cryopreservation procedures is reviewed and a brief summary about freezability markers is also included. Final sections briefly discuss the improvement of freezing extenders with additives, such as seminal plasma and antioxidants, and highlight the relevance of using a proper AI technique to avoid dramatic drops in reproductive performance.(AU)

Adição de Trolox ao diluente leite em pó desnatado, durante a conservação do sêmen suíno a 10 °C / Addition of Trolox into skimmed milk powder extender during the boar semen storage at 10 °C

Os antioxidantes são capazes de proteger as células do estresse oxidativo, podendo ser utilizados em diluentes de refrigeração, a fim de aumentar a viabilidade do sêmen. O presente estudo teve como objetivo verificar os efeitos da adição de diferentes concentrações de trolox sobre espermatozoides suínos diluídos e conservados em leite em pó desnatado (LPD) a 10 °C. Foram utilizados 63 ejaculados, submetidos a 4 tratamentos: T1 = LPD (controle); T2 = LPD+25.000μg de trolox/100mL; T3 =LPD+50.000μg de trolox/100mL; e T4 = LPD+75.000μg de trolox/100mL. Foram realizadas análises diárias (D0 a D4) de vigor e motilidade e nos D0 e D4 foi realizado o teste hiposmótico, vitalidade espermática e integridade acrossomal. As médias de vigor e motilidade do T1 foram superiores em relação aos demais tratamentos, embora não diferentes do T2 e T3 (D0 a D2). O T4 apresentou as piores médias de vigor e motilidade, não diferindo do T3. O percentual de espermatozoides vivos e integridade de membranas plasmática e acrossomal, não foram diferentes entre os tratamentos, tanto no D0 quanto no D4. A adição de trolox ao diluente LPD não promoveu efeitos benéficos sobre a conservação do sêmen. Elevadas concentrações de trolox prejudicaram a qualidade espermática, não sendo aplicáveis na preparação de doses inseminantes. A ausência de efeitos positivos sugere uma possível interferência do diluente LPD sobre a ação antioxidante do trolox. Maiores estudos são necessários, a fim de se poder evidenciar a ação do trolox; bem como sua possível interação com o diluente do sêmen.(AU)

Antioxidants are able to protect cells from oxidative stress and may be used in refrigeration extenders to increase the sperm viability. The present study aimed to determine the effects of adding different concentrations of trolox on boar spermatozoa diluted and stored in skimmed milk powder (LPD) at 10 °C. In 63 ejaculates were used and subjected to four treatments: T1 = LPD (control); T2 = LPD+25.000μg of trolox/100mL; T3 = LPD+50.000μg of trolox/100mL; and T4 = LPD+75.000μg of trolox/100mL. Analyzes of vigor and sperm motility were performed daily (D0 to D4). At D0 and D4 hiposmotic, sperm vitality and acrosomal integrity tests were performed. The mean vigour and motility Of T1 were higher compared to the other treatments, although not different from T2 and T3 (D0 toD2). The T4 had the worst average vigour and motility, not differing from T3. The percentages of live sperm and integrity of plasma and acrosomal membranes were not different between treatments, both D0 and in D4. The addition of the trolox in LPD diluent did not promote beneficial effects on the preservation of semen. High concentrations of trolox impaired sperm quality, not being an applicable option in the preparation of insemination doses. The absence of positive effects suggests a possible interference of LPD on the antioxidant activity of trolox. Further studies are necessary to demonstrate the antioxidant activity of trolox, as well its possible interaction with the semen extender.Keywords: boar semen, skimmed milk powder, trolox, antioxidante.(AU)

Head morphometry and chromatin instability in normal boar spermatozoa and in spermatozoa with cytoplasmic droplets

In boar studs, morphological analyses are used to evaluate sperm quality and there by categorize ejaculates as either approved or rejected. Normally, morphological characteristics correlate with chromatin disorders, but studies to date have only considered the average of abnormalities; cells were not segregated as normal or abnormal. The aim of this study was to assess whether the presence of cytoplasmic droplets was associated with morphometric characteristics and chromatin instability of spermatozoa heads. Morphological analyses were performed on semen from 11 boars using phase contrast microscopy (200 cells per sample). Normal cells were differentiated from those with cytoplasmic droplets and both types were evaluated separately. Photomicrographs were acquired ofnormal spermatozoa (Group NOR, N = 1,207) as well as spermatozoa with proximal and distal cytoplasmic droplets (Group DROP, N = 725). Sperm-head morphometry and chromatin structure were evaluated using the toluidine blue technique. Spermatozoa heads in the DROP group were longer (8.37 ± 0.60 × 8.31 ± 0.53; P = 0.025), narrower (4.16 ± 0.21 × 4.19 ± 0.19; P = 0.03), And more symmetric on the sides (0.973 ± 0.012 × 0.971 ± 0.011; P = 0.007) than were spermatozoa heads of the NOR group. The DROP group also had a greater average ellipticity (0.335 ± 0.034 × 0.329 ± 0.031; P= 0.0004),a greater percentage of decondensed chromatin (2.71 ± 3.87 × 2.28 ± 1.38; P < 0.0008), and a greater chromatin heterogeneity (4.66 ± 1.40 × 4.40 ± 1.42;P < 0.0001). A greater frequency of semen collection results in a shorter period of cell maturation and this probably affected the degree of chromatin condensation and the cytoplasmic droplet migration, with concomitant effect onthe head morphometry measurements.(AU)

Adição de Trolox ao diluente leite em pó desnatado, durante a conservação do sêmen suíno a 10 °C / Addition of Trolox into skimmed milk powder extender during the boar semen storage at 10 °C

Os antioxidantes são capazes de proteger as células do estresse oxidativo, podendo ser utilizados em diluentes de refrigeração, a fim de aumentar a viabilidade do sêmen. O presente estudo teve como objetivo verificar os efeitos da adição de diferentes concentrações de trolox sobre espermatozoides suínos diluídos e conservados em leite em pó desnatado (LPD) a 10 °C. Foram utilizados 63 ejaculados, submetidos a 4 tratamentos: T1 = LPD (controle); T2 = LPD+25.000μg de trolox/100mL; T3 =LPD+50.000μg de trolox/100mL; e T4 = LPD+75.000μg de trolox/100mL. Foram realizadas análises diárias (D0 a D4) de vigor e motilidade e nos D0 e D4 foi realizado o teste hiposmótico, vitalidade espermática e integridade acrossomal. As médias de vigor e motilidade do T1 foram superiores em relação aos demais tratamentos, embora não diferentes do T2 e T3 (D0 a D2). O T4 apresentou as piores médias de vigor e motilidade, não diferindo do T3. O percentual de espermatozoides vivos e integridade de membranas plasmática e acrossomal, não foram diferentes entre os tratamentos, tanto no D0 quanto no D4. A adição de trolox ao diluente LPD não promoveu efeitos benéficos sobre a conservação do sêmen. Elevadas concentrações de trolox prejudicaram a qualidade espermática, não sendo aplicáveis na preparação de doses inseminantes. A ausência de efeitos positivos sugere uma possível interferência do diluente LPD sobre a ação antioxidante do trolox. Maiores estudos são necessários, a fim de se poder evidenciar a ação do trolox; bem como sua possível interação com o diluente do sêmen.

Antioxidants are able to protect cells from oxidative stress and may be used in refrigeration extenders to increase the sperm viability. The present study aimed to determine the effects of adding different concentrations of trolox on boar spermatozoa diluted and stored in skimmed milk powder (LPD) at 10 °C. In 63 ejaculates were used and subjected to four treatments: T1 = LPD (control); T2 = LPD+25.000μg of trolox/100mL; T3 = LPD+50.000μg of trolox/100mL; and T4 = LPD+75.000μg of trolox/100mL. Analyzes of vigor and sperm motility were performed daily (D0 to D4). At D0 and D4 hiposmotic, sperm vitality and acrosomal integrity tests were performed. The mean vigour and motility Of T1 were higher compared to the other treatments, although not different from T2 and T3 (D0 toD2). The T4 had the worst average vigour and motility, not differing from T3. The percentages of live sperm and integrity of plasma and acrosomal membranes were not different between treatments, both D0 and in D4. The addition of the trolox in LPD diluent did not promote beneficial effects on the preservation of semen. High concentrations of trolox impaired sperm quality, not being an applicable option in the preparation of insemination doses. The absence of positive effects suggests a possible interference of LPD on the antioxidant activity of trolox. Further studies are necessary to demonstrate the antioxidant activity of trolox, as well its possible interaction with the semen extender.Keywords: boar semen, skimmed milk powder, trolox, antioxidante.

State-of-the-art of boar sperm preservation in liquid and frozen state

Pig breeding is mainly conducted through Artificial Insemination (AI) in Western and developing countries. Apart from requiring specific catheters and trained staff, preserving boar semen in proper conditions is needed to ensure high reproductive performances. Although, at present, boar sperm may be preserved in liquid (15-17ºC) or frozen states, more than 95% of AIs are conducted using liquid semen. The present work reviews the state-the-art of these two preservation technologies. Thus, the composition and types of extenders for liquid-stored semen are discussed, together with the specific requirements for boar sperm, which are stored at 15-17ºC. Commercial extenders for liquid semen are compared and the effects of storage on sperm quality are also summarised. In the second part of the manuscript, the main features of boar sperm cryopreservation are described and reference to cryodamage is also made. These cryoinjuries mainly affect sperm motility, membrane permeability and chromatin integrity. Furthermore, the individual variability in the sperm resilience to withstand cryopreservation procedures is reviewed and a brief summary about freezability markers is also included. Final sections briefly discuss the improvement of freezing extenders with additives, such as seminal plasma and antioxidants, and highlight the relevance of using a proper AI technique to avoid dramatic drops in reproductive performance.

Head morphometry and chromatin instability in normal boar spermatozoa and in spermatozoa with cytoplasmic droplets

In boar studs, morphological analyses are used to evaluate sperm quality and there by categorize ejaculates as either approved or rejected. Normally, morphological characteristics correlate with chromatin disorders, but studies to date have only considered the average of abnormalities; cells were not segregated as normal or abnormal. The aim of this study was to assess whether the presence of cytoplasmic droplets was associated with morphometric characteristics and chromatin instability of spermatozoa heads. Morphological analyses were performed on semen from 11 boars using phase contrast microscopy (200 cells per sample). Normal cells were differentiated from those with cytoplasmic droplets and both types were evaluated separately. Photomicrographs were acquired ofnormal spermatozoa (Group NOR, N = 1,207) as well as spermatozoa with proximal and distal cytoplasmic droplets (Group DROP, N = 725). Sperm-head morphometry and chromatin structure were evaluated using the toluidine blue technique. Spermatozoa heads in the DROP group were longer (8.37 ± 0.60 × 8.31 ± 0.53; P = 0.025), narrower (4.16 ± 0.21 × 4.19 ± 0.19; P = 0.03), And more symmetric on the sides (0.973 ± 0.012 × 0.971 ± 0.011; P = 0.007) than were spermatozoa heads of the NOR group. The DROP group also had a greater average ellipticity (0.335 ± 0.034 × 0.329 ± 0.031; P= 0.0004),a greater percentage of decondensed chromatin (2.71 ± 3.87 × 2.28 ± 1.38; P < 0.0008), and a greater chromatin heterogeneity (4.66 ± 1.40 × 4.40 ± 1.42;P < 0.0001). A greater frequency of semen collection results in a shorter period of cell maturation and this probably affected the degree of chromatin condensation and the cytoplasmic droplet migration, with concomitant effect onthe head morphometry measurements.