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

Use of N-acetyl-L-cysteine on the cooled equine semen

Este estudo teve como objetivo avaliar o efeito da N-acetil-L-cisteína (NAC), adicionada ao diluente composto de leite em pó desnatado, sobre a viabilidade espermática e o estresse oxidativo do sêmen equino resfriado a 5°C. Ejaculados de 8 pôneis da raça Brasileira foram coletados em triplicata resultando em 24 ejaculados. O sêmen foi distribuído em 4 grupos: Equidil® + 0,00 mM (controle), 0,5 mM, 1,0 mM ou 2,5 mM de NAC. As amostras foram armazenadas em tubos de 15mL e mantidas em caixas de transporte de sêmen BotuFLEX® (Botupharma, Botucatu-SP, Brasil). Parâmetros como motilidade total (MT), motilidade progressiva (MP), vigor, pH, resposta ao teste hiposmótico (HOST) e atividade mitocondrial (MTT) foram avaliados nas 24 e 48 h, bem como no sêmen fresco, após a diluição. O vigor, MT e MP foram também avaliados após teste de termorresistência (TTR), na ausência e presença de peróxido. A MT, a MP, o vigor espermático e a MTT foram similares (P > 0.05) entre as concentrações de NAC, nas 24 e 48 h. A resposta ao HOST foi semelhante entre as concentrações de NAC (P > 0,05) nas 24 h de resfriamento, porém nas 48 h ocorreu diminuição da funcionalidade da membrana no grupo NAC 2,5 mM em comparação ao grupo EQUIDIL, sem adição de NAC. A MT, a MP e vigor, das amostras resfriadas por 24 h e submetidas ao TTR, diferiu entre sem e com peróxido (P 0.05) between NAC concentrations at 24 and 48 h. The response to HOST was similar between NAC concentrations (P > 0.05) at 24 h cooling, but at 48 h there was a decreased in membrane functionality in 2.5mM NAC group compared to the EQUIDIL group. TM, PM, and vigor of the samples cooled by 24 h and submitted to TTR differed between without and with peroxide (P< 0.05) in the EQUIDIL, 0.5mM and 1.0mM groups, but was similar in 2.5mM NAC. After cooling for 48 h, there was difference in vigor and TM between samples with and without peroxide (P < 0.05) in all groups tested, but the PM was similar between samples with and without peroxide at concentration 2.5mM of NAC. The pH of the EQUIDIL extender was higher and the EQUIDIL + 0.5mM and 1.0mM groups had intermediate values, while the 2.5mM NAC concentration generated lower values in the three evaluated periods (P < 0.05). There was no significant variation of pH between 0 and 24 h (P=0.7075) and between 0 and 48 h (P=0.4617) in all groups tested. The concentrations of NAC tested did not improve motility, plasma membrane integrity, mitochondrial activity and response to HOST equine spermatozoa cooled to 5°C and stored for 48 h. After TTR, the concentrations of NAC tested did not prevent the decrease of motility and sperm vigor in the presence of peroxide

Use of N-acetyl-L-cysteine on the cooled equine semen

Este estudo teve como objetivo avaliar o efeito da N-acetil-L-cisteína (NAC), adicionada ao diluente composto de leite em pó desnatado, sobre a viabilidade espermática e o estresse oxidativo do sêmen equino resfriado a 5°C. Ejaculados de 8 pôneis da raça Brasileira foram coletados em triplicata resultando em 24 ejaculados. O sêmen foi distribuído em 4 grupos: Equidil® + 0,00 mM (controle), 0,5 mM, 1,0 mM ou 2,5 mM de NAC. As amostras foram armazenadas em tubos de 15mL e mantidas em caixas de transporte de sêmen BotuFLEX® (Botupharma, Botucatu-SP, Brasil). Parâmetros como motilidade total (MT), motilidade progressiva (MP), vigor, pH, resposta ao teste hiposmótico (HOST) e atividade mitocondrial (MTT) foram avaliados nas 24 e 48 h, bem como no sêmen fresco, após a diluição. O vigor, MT e MP foram também avaliados após teste de termorresistência (TTR), na ausência e presença de peróxido. A MT, a MP, o vigor espermático e a MTT foram similares (P > 0.05) entre as concentrações de NAC, nas 24 e 48 h. A resposta ao HOST foi semelhante entre as concentrações de NAC (P > 0,05) nas 24 h de resfriamento, porém nas 48 h ocorreu diminuição da funcionalidade da membrana no grupo NAC 2,5 mM em comparação ao grupo EQUIDIL, sem adição de NAC. A MT, a MP e vigor, das amostras resfriadas por 24 h e submetidas ao TTR, diferiu entre sem e com peróxido (P 0.05) between NAC concentrations at 24 and 48 h. The response to HOST was similar between NAC concentrations (P > 0.05) at 24 h cooling, but at 48 h there was a decreased in membrane functionality in 2.5mM NAC group compared to the EQUIDIL group. TM, PM, and vigor of the samples cooled by 24 h and submitted to TTR differed between without and with peroxide (P< 0.05) in the EQUIDIL, 0.5mM and 1.0mM groups, but was similar in 2.5mM NAC. After cooling for 48 h, there was difference in vigor and TM between samples with and without peroxide (P < 0.05) in all groups tested, but the PM was similar between samples with and without peroxide at concentration 2.5mM of NAC. The pH of the EQUIDIL extender was higher and the EQUIDIL + 0.5mM and 1.0mM groups had intermediate values, while the 2.5mM NAC concentration generated lower values in the three evaluated periods (P < 0.05). There was no significant variation of pH between 0 and 24 h (P=0.7075) and between 0 and 48 h (P=0.4617) in all groups tested. The concentrations of NAC tested did not improve motility, plasma membrane integrity, mitochondrial activity and response to HOST equine spermatozoa cooled to 5°C and stored for 48 h. After TTR, the concentrations of NAC tested did not prevent the decrease of motility and sperm vigor in the presence of peroxide

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