Sodium bicarbonate and HEPES as buffer components for cooling the semen of pony stallions / 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 5o C 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). Sodium bicarbonate reduced the progressive motility and increased the semen pH. The extender C was considered more appropriate for immediate use in artificial insemination. The non-buffered and HEPES-buffered extenders were considered appropriate for the cooling of equine semen for 48 h at 5°C.

A composição dos diluentes de sêmen pode modificar sua viabilidade durante o processo de resfriamento. O efeito de tamponamento do HEPES e Bicarbonato de Sódio foi avaliado considerando o pH e a viabilidade espermática. Sete pôneis brasileiros adultos tiveram seu sêmen avaliado antes e após a refrigeração a 5°C durante 24 h e 48 h. Um diluente de leite em pó desnatado não tamponado (C) e um diluente tamponado com bicarbonato de sódio (SB) ou HEPES (H) foram utilizados. Após a diluição, o sêmen (três ejaculados/ pônei) foi centrifugado e o sobrenadante foi descartado. O sêmen foi então diluído com SB, H ou C e a concentração ajustada para 50 x 106 espermatozoides/mL. A motilidade progressiva avaliada após a diluição apresentou resultados similares para todos os diluentes (71,42% (SB), 74,28% (H), 74,52% (C)). A motilidade espermática foi avaliada 24 h e 48 h após o resfriamento, respectivamente, para SB (44,76%, 25,23%), H (51,42%, 38,09%) e C (54,05%, 41,66%). A integridade da membrana plasmática foi semelhante após a exposição aos três diluentes (62,71% (SB), 68,76% (H), 69,23% (C)). A atividade mitocondrial após a diluição foi maior em SB (SB = 1.05nm, H = 0.81nm, C = 0.79nm) e após 24 h foi 0.83nm (SB), 0.73nm (H) e 0.64nm (C). A atividade mitocondrial após 48 h diminuiu para 0.72nm (SB), 0.69nm (H) e 0.63nm (C) (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...

Sodium bicarbonate and HEPES as buffer components for cooling the semen of pony stallions / 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 5o C 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 Duncans test was used to evaluate the significant differences (P < 0.05). Sodium bicarbonate reduced the progressive motility and increased the semen pH. The extender C was considered more appropriate for immediate use in artificial insemination. The non-buffered and HEPES-buffered extenders were considered appropriate for the cooling of equine semen for 48 h at 5°C.(AU)

A composição dos diluentes de sêmen pode modificar sua viabilidade durante o processo de resfriamento. O efeito de tamponamento do HEPES e Bicarbonato de Sódio foi avaliado considerando o pH e a viabilidade espermática. Sete pôneis brasileiros adultos tiveram seu sêmen avaliado antes e após a refrigeração a 5°C durante 24 h e 48 h. Um diluente de leite em pó desnatado não tamponado (C) e um diluente tamponado com bicarbonato de sódio (SB) ou HEPES (H) foram utilizados. Após a diluição, o sêmen (três ejaculados/ pônei) foi centrifugado e o sobrenadante foi descartado. O sêmen foi então diluído com SB, H ou C e a concentração ajustada para 50 x 106 espermatozoides/mL. A motilidade progressiva avaliada após a diluição apresentou resultados similares para todos os diluentes (71,42% (SB), 74,28% (H), 74,52% (C)). A motilidade espermática foi avaliada 24 h e 48 h após o resfriamento, respectivamente, para SB (44,76%, 25,23%), H (51,42%, 38,09%) e C (54,05%, 41,66%). A integridade da membrana plasmática foi semelhante após a exposição aos três diluentes (62,71% (SB), 68,76% (H), 69,23% (C)). A atividade mitocondrial após a diluição foi maior em SB (SB = 1.05nm, H = 0.81nm, C = 0.79nm) e após 24 h foi 0.83nm (SB), 0.73nm (H) e 0.64nm (C). A atividade mitocondrial após 48 h diminuiu para 0.72nm (SB), 0.69nm (H) e 0.63nm (C) (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...(AU)

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 5o C 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 0.05) 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.[...]

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 5o C 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.[...](AU)

Seminal plasma: effect on motility, membrane functionality, and spermatic chromatin dispersion of equine sperm treated with N-acetyl-L-cysteine at 5C

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 5C 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 M2) and 5.0 mM NAC (55.5 M2) 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 M2 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 [...](AU)

Seminal plasma: effect on motility, membrane functionality, and spermatic chromatin dispersion of equine sperm treated with N-acetyl-L-cysteine at 5C

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 5C 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 M2) and 5.0 mM NAC (55.5 M2) 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 M2 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 [...]