Viability of Pony Stallion Semen in Different Temperature and Dilution

Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.Materials, Methods & Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P < 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P < 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P > 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P > 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender

PREVALÊNCIA DE PARASITISMO EM CÃES DOMICILIADOS NUM BAIRRO DE SANTA MARIA - RS

O presente estudo foi objetivado a identificar os endoparasitas presentes em cães domiciliados e assim conhecer aszoonoses de risco para população. A avaliação dos animais, machos e fêmeas de diferentes faixas etárias, foi realizada nacasa dos proprietários de um bairro carente no município de Santa Maria. Foram visitadas 178 residências sendo avaliados240 cães, destes 87,9% apresentavam um ou mais gênero de endoparasita, sendo encontrados os gêneros Ancylostoma sp.parasitando 167 (69,6%) animais, além dos gêneros Trichuris sp. (11,25%), Dipylidium  sp. (3,75%), Toxocara sp. (15%),Giardia sp. (12,08%), Isospora  sp. (7,08%) e Cryptosporidium  sp. (8,75%). Os resultados mostraram que os proprietáriosestão em constante risco epidemiológico, devido ao  elevado parasitismo dos cães

Sperm separation for cooling of equine sperm

As biotécnicas da reprodução na espécie equina avançaram na última década, tanto em conhecimento agregado por pesquisas como também pela demanda do mercado. No entanto, na espécie equina machos com subfertilidade são diagnosticados frequentemente com elevado número de espermatozoides com alterações morfológica e/ou imóveis. A utilização apenas de células viáveis para realizar o processo de resfriamento busca evitar perda de material (diluente) e produção de metabólitos tóxicos aos espermatozoides viáveis. O objetivo deste estudo foi utilizar a separação espermática (lã de vidro e centrifugação com Androcoll®) pré-resfriamento para incrementar a viabilidade do sêmen de garanhões pôneis refrigerados a 5ºC durante 48h. Os parâmetros motilidade, funcionalidade de membrana (HOST), viabilidade espermática (CFDA/PI), atividade mitocondrial e morfologia espermática foram avaliados no sêmen fresco e refrigerado (24 e 48h). A utilização da filtração por lã de vidro ou Androcoll® pré-resfriamento do sêmen equino selecionou espermatozoides com maior motilidade, funcionalidade de membrana, viabilidade espermática e atividade mitocondrial. Adicionalmente, a filtração por lã de vidro proporcionou refrigerar sêmen com elevado número de células morfologicamente normais sem perdas significativas de espermatozoides pelo processo de filtração. Tanto a técnica de lã de vidro como a centrifugação com Androcoll® mostraram-se eficientes em separar ejaculados com maior viabilidade para o resfriamento. Já a técnica de lã de vidro apresenta-se como uma técnica de baixo custo e de fácil execução para ser aplicada tanto para pequenos, ou grandes volumes de sêmen.The reproduction biotechnologies in equine species have advanced in the last decade both in aggregate knowledge by research as well as the market demand. However, it in the equine species often with male subfertility where the ejaculate has a high number of sperm with morphological and / or property changes. The use of only viable cells to perform the cooling process seeks to avoid loss of material (diluent) and production of toxic metabolites to viable sperm. The aim of this study was to use the sperm separation (glass and spin wool with Androcoll®) pre-cooling to increase the viability of semen chilled ponies stallions at 5 ° C for 48 hours. We evaluated the motility parameters, membrane functionality (HOST), sperm viability (CFDA / PI), mitochondrial activity and morphology in fresh and chilled semen (24 and 48h). The use of filtration glass wool or Androcoll® pre-cooling of equine semen selected sperm with higher motility, functionality membrane, sperm viability and mitochondrial activity. In addition to filtration through glass wool afforded cooling semen with a high number of morphologically normal cells without significant losses of spermatozoids the filtration process. Both glass wool technique as centrifugation with Androcoll® were efficient in separating ejaculated more viability for cooling. Already glass wool technique presents itself as a low cost and simple technique to be applied to both small or large volumes of semen

Sperm separation for cooling of equine sperm

As biotécnicas da reprodução na espécie equina avançaram na última década, tanto em conhecimento agregado por pesquisas como também pela demanda do mercado. No entanto, na espécie equina machos com subfertilidade são diagnosticados frequentemente com elevado número de espermatozoides com alterações morfológica e/ou imóveis. A utilização apenas de células viáveis para realizar o processo de resfriamento busca evitar perda de material (diluente) e produção de metabólitos tóxicos aos espermatozoides viáveis. O objetivo deste estudo foi utilizar a separação espermática (lã de vidro e centrifugação com Androcoll®) pré-resfriamento para incrementar a viabilidade do sêmen de garanhões pôneis refrigerados a 5ºC durante 48h. Os parâmetros motilidade, funcionalidade de membrana (HOST), viabilidade espermática (CFDA/PI), atividade mitocondrial e morfologia espermática foram avaliados no sêmen fresco e refrigerado (24 e 48h). A utilização da filtração por lã de vidro ou Androcoll® pré-resfriamento do sêmen equino selecionou espermatozoides com maior motilidade, funcionalidade de membrana, viabilidade espermática e atividade mitocondrial. Adicionalmente, a filtração por lã de vidro proporcionou refrigerar sêmen com elevado número de células morfologicamente normais sem perdas significativas de espermatozoides pelo processo de filtração. Tanto a técnica de lã de vidro como a centrifugação com Androcoll® mostraram-se eficientes em separar ejaculados com maior viabilidade para o resfriamento. Já a técnica de lã de vidro apresenta-se como uma técnica de baixo custo e de fácil execução para ser aplicada tanto para pequenos, ou grandes volumes de sêmen.The reproduction biotechnologies in equine species have advanced in the last decade both in aggregate knowledge by research as well as the market demand. However, it in the equine species often with male subfertility where the ejaculate has a high number of sperm with morphological and / or property changes. The use of only viable cells to perform the cooling process seeks to avoid loss of material (diluent) and production of toxic metabolites to viable sperm. The aim of this study was to use the sperm separation (glass and spin wool with Androcoll®) pre-cooling to increase the viability of semen chilled ponies stallions at 5 ° C for 48 hours. We evaluated the motility parameters, membrane functionality (HOST), sperm viability (CFDA / PI), mitochondrial activity and morphology in fresh and chilled semen (24 and 48h). The use of filtration glass wool or Androcoll® pre-cooling of equine semen selected sperm with higher motility, functionality membrane, sperm viability and mitochondrial activity. In addition to filtration through glass wool afforded cooling semen with a high number of morphologically normal cells without significant losses of spermatozoids the filtration process. Both glass wool technique as centrifugation with Androcoll® were efficient in separating ejaculated more viability for cooling. Already glass wool technique presents itself as a low cost and simple technique to be applied to both small or large volumes of semen

Decontamination of naturally contaminated liquid nitrogen storage tanks

The objective of this study was to evaluate the efficacy of cleaning and decontamination procedures in liquid nitrogen tanks. We evaluated 151 canisters and 133 bottoms from 133 nitrogen tanks of companies or farms for the presence of bacteria and fungi. Samples were collected from the canisters and the bottom of tanks containing liquid nitrogen. Tanks were divided into Group 1 (G1): tanks decontaminated with 2% glutaraldehyde - Glutaron® II (n = 16 canisters in 8 tanks); Group 2 (G2): decontamination with 70% ethanol (n = 20 canisters in 10 tanks); and Group 3 (G3): decontamination with 70% ethanol (n = 115 canisters in 115 tanks). Tanks in Groups 1 and 2 belonged to companies; Group 3 tanks belonged to farms. The culture of canisters showed twelve genera of bacteria and five genera of fungi. Bacillus cereuswas the most prevalent bacterial contaminant (42/133) in liquid nitrogen tanks (31.57%). Decontamination by 2% glutaraldehyde plus 70% ethanol was effective and no difference was found between the decontamination methods of Groups 1 and 2. In Group 3 the decontamination method was considered effective. Handling procedures with high hygienic standards should be recommended to avoid contamination of liquid nitrogen tanks on farms

Bicarbonato de sódio e HEPES como tampões para o resfrigeração de sêmen de pôneis

The composition of semen diluents can modify its viability during cooling. The buffering effects of HEPES and sodium bicarbonate were evaluated considering the pH and sperm viability. The semen of seven adult Brazilian ponies was evaluated before and after cooling at 5oC for 24 h and 48 h. A non-buffered skim milk powder extender (C) and the same extender buffered with sodium bicarbonate (SB) and HEPES (H) were used. After dilution, semen (three ejaculates/pony) was centrifuged and the seminal plasma discarded. Sperm was then diluted with SB, H or C and its concentration adjusted to 50 x 106 sptz/mL. Progressive motility evaluated after dilution showed similar results with all extenders (71.42% (SB), 74.28% (H), and 74.52% (C)). Sperm motility was evaluated 24 h and 48 h after cooling for SB (44.76% and 25.23%), H (51.42% and 38.09%) and C (54.05% and 41.66%, respectively). Plasma membrane integrity was similar after exposure to the three extenders (62.71% (SB), 68.76% (H), and 69.23% (C)). Mitochondrial activity was higher in SB immediately after dilution (SB= 1.05nm, H= 0.81nm, C= 0.79nm), and after 24 h (0.83nm (SB), 0.73nm (H) and 0.64nm (C)). After 48 h, the mitochondrial activity decreased to 0.72nm (SB), 0.69nm (H), and 0.63nm (C) (P > 0.05). The pH, osmolarity and pH after 48 h of cooling of the diluted semen were higher in SB (8; 382; 7.9), intermediate in H (7.5; 362; 7.32) and lower in C (7.16; 350; 7.07). Lipid peroxidation and its induction were similar in all groups. Data were analyzed by analysis of variance (ANOVA), and Duncan’s test was used to evaluate the significant differences (P 0.05). O pH, a osmolaridade e o pH do sêmen diluído após as 48 h de refrigeração foram maiores em SB (8; 382; 7,9), intermediário em H (7,5; 362; 7,32) e menor em C (7,16; 350; 7,07). A peroxidação lipídica e sua indução foram semelhantes em todos os grupos. As médias foram avaliadas através de análise de variância (ANOVA) e o Teste Duncan foi utilizado para analisar as diferenças significativas (P < 0.05). O bicarbonato de sódio reduziu a motilidade progressiva e aumentou o pH do sêmen. O diluente C foi considerado mais adequado para uso imediato na inseminação artificial. O diluente não tamponado e tampado com HEPES foram considerados apropriados para o resfriamento de sêmen de equino durante 48 h a 5°C

Viability of Pony Stallion Semen in Different Temperature and Dilution

Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.Materials, Methods &amp; Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P &lt; 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P &lt; 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P &gt; 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P &gt; 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender

Seminal Plasma: Effect on Motility, Membrane Functionality, and Spermatic Chromatin Dispersion of Equine Sperm Treated with N-acetyl-L-cysteine at 5°C

Background: N-acetyl-L-cysteine (NAC) is a low molecular weight thiol studied as an antioxidant for stallion semen preservation without changes on sperm viability. Equine seminal plasma is rich in sulfur proteins (cysteine residues) named CRISPS, which, when combined with sulfur-containing antioxidants, can enhance the appearance of DNA lesions. The aim of this study was to assess and compare the effect of different concentrations of NAC by evaluating motility, membrane function and sperm chromatin integrity of equine semen cooled at 5°C in 50% of seminal plasma.Materials, Methods & Results: Nine ejaculates from 9 stallions were divided into 4 aliquots, diluted and divided in nonsupplemented skim milk group (0.0 mM), or supplemented with 5.0, 2.5 and 0.5 mM NAC. Evaluations were made at 0 h, 24 h and 48 h of cooling, except for motility which was evaluated only up to 24 h. The 0.5 (59.7 μM2) and 5.0 mM NAC (55.5 μM2) groups showed similar areas of sperm chromatin dispersion among all groups. However, the area of chromatin dispersion between the non-supplemented group was higher = 65.3 μM2 than the group supplemented with 2.5 mM. The percentage of cells with a functional plasma membrane was similar between supplemented and non-supplemented (0.0 mM) groups, but higher (P < 0.05) in the 0.5 mM NAC (39.7 and 39.8%, respectively) than that of 2.5 mM (34.5%) and 5.0 mM (34.2%) concentrations. Progressive motility was similar among all groups supplemented with NAC. The 0.5 mM NAC group showed 35.2% motile cells while the non-supplemented group exhibited 36.2%. Although 50% seminal plasma was used, NAC did not affect sperm chromatin integrity.Discussion: Seminal plasma interfered more in the results of different concentrations of NAC. This statement is proven by the motility analysis where all NAC concentrations showed similar results. Plasma percentage higher than 20% in diluted semen causes deleterious effects on sperm, such as decreased motility and fertilizing capacity. The membrane analysis in our study was compromised because NAC (2.5 to 5.0 mM) showed high osmolarity. As this was not adjusted, it affected the result. The 2.5 mM NAC group showed a lower area of sperm chromatin dispersion than none-treated sperm, although showing similar results to the other treatments. In a study with semen of Mangalarga Marchador stallions, the 2.5 mM of NAC was able to protect sperm membrane integrity. However, in another study, where semen was kept cooled between 5 and 15°C, no change was observed on sperm quality over different concentrations of NAC. This reinforces that 2.5 mM of NAC provides adequate protection to semen exposed to harmful conditions.The high percentage of plasma associated with this sulfur antioxidant did not compromise DNA integrity, as NAC concentration used was 100 times less than the concentration needed to induce DNA lesions

Descrição de um novo foco endêmico de esquistossomose mansônica no Estado de São Paulo, Brasil The description of a new endemic focus of schistosomiasis mansoni in the state of S. Paulo, Brazil

Foi descrito novo foco endêmico de esquistossomose mansônica situado na Cidade de Bebedouro, Estado de São Paulo, Brasil. Foram analisados 221 casos diagnosticados da doença segundo a idade, sexo e origem (autóctone ou não), bem como foram discutidos os fatores envolvidos no aparecimento do foco, alertando-se para a necessidade de medidas de controle.<br>A new endemic focus of schistosomiasis mansoni in the town of Bebedouro, S. Paulo, Brazil, is described. Two hundred and twenty-one cases are analyzed according to sex, age, and origin (autochthonous or not), the factors involved in the emergence of the focus are discussed and the necessity for control measure is emphasized