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Effect of addition of acetyl-cysteine and glutathione peroxidase in freezing extender of stallion semen
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6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
Effect of addition of acetyl-cysteine and glutathione peroxidase in freezing extender of stallion semen
Gender influences expression of IGF1 (Insulin like growth factor 1) in preimplantative equine embryos
L.O. Barros 1, S.V. Silva 1, F.C. Almeida 1, E.C.B. Silva 1, 2, G.F. Carneiro 3, and M.M.P. Guerra 1 1 Laboratório de Andrologia (ANDROLAB), Departamento de Medicina Veterinária - UFRPE. Av. Dom Manoel de Medeiros, s/n. Dois Irmãos, Recife – PE. CEP: 52171-900, Brazil, 2 Curso de Doutorado da Rede Nordeste de Biotecnologia – RENORBIO, 3 Universidade Federal Rural de Pernambuco – UFRPE, Unidade Acadêmica de Garanhuns – UAG, Brazil
J. Beckelmann, S. Budik, and C. Aurich Center for Artificial Insemination and Embryo Transfer, University of Veterinary Sciences, 1210 Vienna, Austria
The objective of this study was to analyze the effect of addition of different concentrations of the antioxidants glutathione peroxidase (GPx) and cysteine in frozen semen medium of stallions. Five semen samples were collected from each of five Quarter Horse stallions of proven fertility using an artificial vagina. Semen was diluted in BotucrioÒ medium (BotupharmaÒ, Botucatu, Brazil) and divided into 5 parts for the following experimental groups: G1 ¼ Control (without antioxidants); G2 ¼ 0.5 mM of N-AcetylCysteine; G3 ¼ 1 mM of N-Acetyl-Cysteine; G4 ¼ 1 IU of GPx and G5 ¼ 5 IU of GPx. The semen aliquots were frozen in 0.5-mL straws with a concentration of 300 x 106 of viable spermatozoa/mL using an automated freezer. The straws were thawed at 37 C for 30 seconds and incubated at 36 C for 60 minutes; the samples were analyzed immediately after thawing (T0) and after 60 minutes of incubation (T60). Sperm cells were analyzed for motility using computer assisted sperm motility analyzer (Sperm Class AnalyserÒ SCA, Microptic S.L., “version 3.2.0, Spain”); fluorescent probes were used to analyze plasma membrane (CFDA-PI), acrosome integrity (FITC-PSA), and potential of mitochondrial membrane (JC-1) by epifluorescence microscopy. The motility parameters assessed were total motility (MT) (%), progressive motility (MP) (%), average path velocity (VAP) (mm/s), straight linear velocity (VSL) (mm/s), and population of rapid spermatozoa (ER) (%). Sperm kinetics exhibited higher values of VCL and VAP (P < 0.05) in G1 and at T0 as compared to T60. Stallion 4 showed increased values of all parameters as compared with other stallions (P < 0.05) demonstrating individuality of stallions. No difference was observed in motility either after addition of cysteine or GPx compared with control. Semen samples supplemented with 5 UI of GPx or with 0.5 mM of cysteine had a higher (P < 0.05) percentage of intact acrosomes at T0 than at T60, an effect due to incubation time. Semen samples supplemented with 1 mM of cysteine presented a higher (P < 0.05) percentage of spermatozoa with intact acrosomes at T60 than GPx (1 and 5 UI) and cysteine (0.5 mM) demonstrating a protection effect to the acrosomes. Spermatozoa with a higher percentage of mitochondrial membrane potential were found at T60 (P < 0.05) in stallion 5, higher than in stallions 1 and 2. We conclude that the addition of antioxidants had no effect on kinetic parameters. The addition of cysteine (1mM) in frozen semen medium improved acrosome integrity after incubation. Further studies are necessary to evaluate these results, and a future fertility trial is planned.
In several species growth factors including IGF1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. Information on the role of growth factors in the regulation of equine embryo development is sparse. IGF1 (Willmann et al. 2011, Theriogenology 76, 61-73) and IGF binding protein 3 (Herrler et al. 2000, Biol of Reprod 62, 1804–1811) are expressed in equine conceptuses around day 10 of pregnancy. Nutritional status is closely related to the IGF system and is known to affect sex ratio of offspring in red deer (Flint et al. 1997 Gen Comp Endocrinol 106, 374-383; LunaEstrada et al. 2006 Small Ruminant Res 65, 154-160). Body condition of mares is also suggested to influence gender ratio in foals. We tested the hypothesis that gender of equine preimplantative embryos influences their IGF1 expression. In the study, IGF1 RNA expression of in vivo produced equine embryos at different times of pregnancy (day 8 n ¼ 5; day 10 n ¼ 5; day 12 n ¼ 14) was analysed. IGF1 RNA expression was evaluated by quantitative RT-PCR in comparison to b-actin. The sex of the embryos was determined by detection of X inactivating specific transcript (Xist) RNA in female embryos. From 24 embryos tested, 14 showed positive Xist expression and were thus classified as female (58%). Xist-negative embryos were always detected to be positive for eSRY DNA confirming them to be male. IGF1 RNA expression was influenced both by gender and day of pregnancy (relative expression of IGF1 in relation to b-actin, day 10: male 13.7 6.9, female 63.3 3.3, day 12: male 11.9 3.9, female 30.9 5.4; P < 0.05 for day of pregnancy, P < 0.001 for gender). Results suggest an increased expression of IGF1 in female equine embryos. Gender-related influences on expression of the IGF system are probably based on epigenetic effects. They may account for unbalanced birth of offspring of one gender that has been reported to occur under specific conditions (Flint et al. 1997; Luna-Estrada et al. 2006; Almeida Camargo et al. 2010 Anim Reprod Sci 120, 10-15).
Heart rate, heart rate variability and cortisol release in breeding stallions in response to semen collection M. Becker-Birck 1, M. Wulf 1, M. Mohr 3, R. Erber 1, 2, N. Ille 2, and C. Aurich 1, 2 1 Graf-Lehndorff Institute for Equine Science, Vienna University of Veterinary Sciences, Neustadt (Dosse), Germany, 2 Centre for Artificial Insemination and Embryo Transfer, Vienna University of Veterinary Sciences, Vienna, Austria, 3 Brandenburg State Stud, Neustadt (Dosse), Germany While stress of horses in equestrian sports is well-known, the stress response of breeding stallions to semen
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
Effect of addition of acetyl-cysteine and glutathione peroxidase in freezing extender of stallion semen
Gender influences expression of IGF1 (Insulin like growth factor 1) in preimplantative equine embryos
L.O. Barros 1, S.V. Silva 1, F.C. Almeida 1, E.C.B. Silva 1, 2, G.F. Carneiro 3, and M.M.P. Guerra 1 1 Laboratório de Andrologia (ANDROLAB), Departamento de Medicina Veterinária - UFRPE. Av. Dom Manoel de Medeiros, s/n. Dois Irmãos, Recife – PE. CEP: 52171-900, Brazil, 2 Curso de Doutorado da Rede Nordeste de Biotecnologia – RENORBIO, 3 Universidade Federal Rural de Pernambuco – UFRPE, Unidade Acadêmica de Garanhuns – UAG, Brazil
J. Beckelmann, S. Budik, and C. Aurich Center for Artificial Insemination and Embryo Transfer, University of Veterinary Sciences, 1210 Vienna, Austria
The objective of this study was to analyze the effect of addition of different concentrations of the antioxidants glutathione peroxidase (GPx) and cysteine in frozen semen medium of stallions. Five semen samples were collected from each of five Quarter Horse stallions of proven fertility using an artificial vagina. Semen was diluted in BotucrioÒ medium (BotupharmaÒ, Botucatu, Brazil) and divided into 5 parts for the following experimental groups: G1 ¼ Control (without antioxidants); G2 ¼ 0.5 mM of N-AcetylCysteine; G3 ¼ 1 mM of N-Acetyl-Cysteine; G4 ¼ 1 IU of GPx and G5 ¼ 5 IU of GPx. The semen aliquots were frozen in 0.5-mL straws with a concentration of 300 x 106 of viable spermatozoa/mL using an automated freezer. The straws were thawed at 37 C for 30 seconds and incubated at 36 C for 60 minutes; the samples were analyzed immediately after thawing (T0) and after 60 minutes of incubation (T60). Sperm cells were analyzed for motility using computer assisted sperm motility analyzer (Sperm Class AnalyserÒ SCA, Microptic S.L., “version 3.2.0, Spain”); fluorescent probes were used to analyze plasma membrane (CFDA-PI), acrosome integrity (FITC-PSA), and potential of mitochondrial membrane (JC-1) by epifluorescence microscopy. The motility parameters assessed were total motility (MT) (%), progressive motility (MP) (%), average path velocity (VAP) (mm/s), straight linear velocity (VSL) (mm/s), and population of rapid spermatozoa (ER) (%). Sperm kinetics exhibited higher values of VCL and VAP (P < 0.05) in G1 and at T0 as compared to T60. Stallion 4 showed increased values of all parameters as compared with other stallions (P < 0.05) demonstrating individuality of stallions. No difference was observed in motility either after addition of cysteine or GPx compared with control. Semen samples supplemented with 5 UI of GPx or with 0.5 mM of cysteine had a higher (P < 0.05) percentage of intact acrosomes at T0 than at T60, an effect due to incubation time. Semen samples supplemented with 1 mM of cysteine presented a higher (P < 0.05) percentage of spermatozoa with intact acrosomes at T60 than GPx (1 and 5 UI) and cysteine (0.5 mM) demonstrating a protection effect to the acrosomes. Spermatozoa with a higher percentage of mitochondrial membrane potential were found at T60 (P < 0.05) in stallion 5, higher than in stallions 1 and 2. We conclude that the addition of antioxidants had no effect on kinetic parameters. The addition of cysteine (1mM) in frozen semen medium improved acrosome integrity after incubation. Further studies are necessary to evaluate these results, and a future fertility trial is planned.
In several species growth factors including IGF1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. Information on the role of growth factors in the regulation of equine embryo development is sparse. IGF1 (Willmann et al. 2011, Theriogenology 76, 61-73) and IGF binding protein 3 (Herrler et al. 2000, Biol of Reprod 62, 1804–1811) are expressed in equine conceptuses around day 10 of pregnancy. Nutritional status is closely related to the IGF system and is known to affect sex ratio of offspring in red deer (Flint et al. 1997 Gen Comp Endocrinol 106, 374-383; LunaEstrada et al. 2006 Small Ruminant Res 65, 154-160). Body condition of mares is also suggested to influence gender ratio in foals. We tested the hypothesis that gender of equine preimplantative embryos influences their IGF1 expression. In the study, IGF1 RNA expression of in vivo produced equine embryos at different times of pregnancy (day 8 n ¼ 5; day 10 n ¼ 5; day 12 n ¼ 14) was analysed. IGF1 RNA expression was evaluated by quantitative RT-PCR in comparison to b-actin. The sex of the embryos was determined by detection of X inactivating specific transcript (Xist) RNA in female embryos. From 24 embryos tested, 14 showed positive Xist expression and were thus classified as female (58%). Xist-negative embryos were always detected to be positive for eSRY DNA confirming them to be male. IGF1 RNA expression was influenced both by gender and day of pregnancy (relative expression of IGF1 in relation to b-actin, day 10: male 13.7 6.9, female 63.3 3.3, day 12: male 11.9 3.9, female 30.9 5.4; P < 0.05 for day of pregnancy, P < 0.001 for gender). Results suggest an increased expression of IGF1 in female equine embryos. Gender-related influences on expression of the IGF system are probably based on epigenetic effects. They may account for unbalanced birth of offspring of one gender that has been reported to occur under specific conditions (Flint et al. 1997; Luna-Estrada et al. 2006; Almeida Camargo et al. 2010 Anim Reprod Sci 120, 10-15).
Heart rate, heart rate variability and cortisol release in breeding stallions in response to semen collection M. Becker-Birck 1, M. Wulf 1, M. Mohr 3, R. Erber 1, 2, N. Ille 2, and C. Aurich 1, 2 1 Graf-Lehndorff Institute for Equine Science, Vienna University of Veterinary Sciences, Neustadt (Dosse), Germany, 2 Centre for Artificial Insemination and Embryo Transfer, Vienna University of Veterinary Sciences, Vienna, Austria, 3 Brandenburg State Stud, Neustadt (Dosse), Germany While stress of horses in equestrian sports is well-known, the stress response of breeding stallions to semen