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The effect of antioxidants on the in vitro quality of fresh bull sperm stored in an egg yolk based diluent
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Abstracts / Animal Reproduction Science 169 (2016) 99–135
apolipoprotein, apoA-I, in the relevant molecular weight band on the gels. This proteomic approach confirmed the presence of apoA-I in fractions 7–10 of follicular fluid and fractions 8–10 of oviductal fluid. This indicates that HDLs in ovine follicular and oviductal fluid can be isolated using iodixanol and density ultracentrifugation. The establishment of this protocol in sheep provides an opportunity to investigate the function of HDLs as potential cholesterol acceptors from ram spermatozoa during in vitro capacitation, which will be the focus of future studies. http://dx.doi.org/10.1016/j.anireprosci.2016.03.064 P46 The effect of antioxidants on the in vitro quality of fresh bull sperm stored in an egg yolk based diluent S.A. Holden 1 , P. Lonergan 2 , S. Fair 1,∗ 1 Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland 2 School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland E-mail address: [email protected] (S. Fair).
This study assessed the effect of antioxidants on the in vitro quality of fresh bull sperm. Experiment 1: caprogen diluent was prepared containing a range of antioxidants; l-carnitine (0, 2.5, 10, 20 mM), Crocin (0, 0.5, 1.5, 2 mM), ␣-Tocopherol (0, 0.075, 0.4, 1 mM), Quercetin (0, 50, 175, 250 M) and Catalase (0, 150, 250, 500 IU). Ejaculates (n = 3) were diluted to 12 × 106 sperm/mL in each respective diluent, incubated at 37 ◦ C and assessed for superoxide production and acrosome integrity using flow cytometry at 0, 1, 2 and 4 h. Experiment 2: Caprogen diluent was prepared containing the antioxidants l-carnitine (0, 0.5, 2.5, 5, 10, 20 mM), Crocin (0, 0.25, 0.5, 1, 1.5, 2 mM), ␣-Tocopherol(0, 0.025, 0.075, 0.15 0.4, 0.8, 1 mM) and Quercetin (0, 20, 50, 100, 175, 250 M). Ejaculates (n = 9) were diluted to 12 × 106 sperm/mL in respective diluents, packaged in 0.25 mL straws and stored at 16–18 ◦ C. Motility and kinematic parameters were assessed using computer assisted sperm analysis while membrane fluidity and lipid peroxidation were assessed using flow-cytometry on days 0, 1, 3 and 5 post collection. Data were analysed using the repeated-measures analysis of variance in the Statistical Package for the Social Sciences (SPSS; version 22.0, IBM, Chicago, IL). Experiment 1: there was no treatment or time × treatment interaction on superoxide production or acrosome integrity (P > 0.05). However, there was an effect of time with regard to catalase as all treatments at 4 h incubation had greater acrosome intact populations compared to the control (P < 0.05). Similarly, with Quercetin treatments, superoxide production was lowest at 2 h (P < 0.05) but increased thereafter. Experiment 2: There was no effect of any of the antioxidants on the in vitro parameters assessed when compared to the control (P > 0.05). There
was an effect of day regarding most of the parameters assessed (excluding membrane fluidity of Crocin treatments) as motility declined and membrane fluidity peaked on day 1. In conclusion, this study found modest benefits in the inclusion of antioxidants in egg-yolk based diluents used for the fresh storage of bull sperm. http://dx.doi.org/10.1016/j.anireprosci.2016.03.065 P47 Role of EDTA as a chelating agent in canine semen cryopreservation C. Braud 1,∗ , L.C.F. Bergo 2 , G.R. Araújo 2 , T. Deco-Souza 2 , A.C. Csermak Jr 2 , L.R.B. Carazo 2 , L.C. Silva 2 , T.A.R. Paula 2 1
INP-Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31300 Toulouse, France 2 REPAAS Laboratório de Biotecnologia da Reproduc¸ão em Pequenos Animais e Animais Silvestres Vic¸osa, MG 36570-900, Brazil E-mail address: c.braud [email protected] (C. Braud). Increased capacitation of spermatozoa and premature acrosome reaction reduce chances of fertilization. Since calcium influx into sperm cells is involved in the acrosome reaction, it has been suggested that high calcium concentrations found in sperm freezing media could increase the risk of premature reactions. The objective of this study was to evaluate dog sperm viability in freezing media of various calcium concentrations, with EDTA as a calcium chelator. Three ejaculates each of three mixed breed dogs of good fertility were collected by manual stimulation. Immediately after collection, volume, sperm concentration and motility as well as the percentage of live and abnormal sperm cells were determined in the sperm rich fraction. The initial concentration of calcium was assayed by spectrophotometry (E-225D-Celm® – 370 nm; InVitro® ) in the two fractions. Fresh semen was divided into three aliquots and suspended in the same volume of tris-citrate egg yolk extender (Nutricell-SP-Brasil). Thereafter, a second extender containing 12% glycerol and 1% Equex (NutricellSP-Brasil) was added to obtain a final concentration of 100 × 106 spermatozoa/mL. EDTA solution was added at different concentrations for the three aliquots: no EDTA; 0.1% EDTA; 0.25% EDTA). After thawing, calcium concentration, sperm motility, viability and morphological defects were immediately evaluated. Sperm plasma membrane integrity and acrosomal membrane integrity were also assessed with a fluorescent staining technique using a combination of 2 L of PI (Sigma P4170), 100 L FITCPSA (Sigma L0770) and 200 L Hoëscht 33342 (Molecular Probes H1399). In samples without EDTA, calcium concentrations were 14 times higher (21.23 ± 2.41 mg/dL) than in fresh semen (1.52 ± 0.62 mg/dL). The addition of EDTA significantly reduced the extracellular calcium concentrations in both treatments (0.1% EDTA: 13.91 ± 1.48 mg/dL; 0.25% EDTA: 3.98 ± 1.59 mg/dL) but values still remained high compared with in fresh semen. No significant difference
Abstracts / Animal Reproduction Science 169 (2016) 99–135
apolipoprotein, apoA-I, in the relevant molecular weight band on the gels. This proteomic approach confirmed the presence of apoA-I in fractions 7–10 of follicular fluid and fractions 8–10 of oviductal fluid. This indicates that HDLs in ovine follicular and oviductal fluid can be isolated using iodixanol and density ultracentrifugation. The establishment of this protocol in sheep provides an opportunity to investigate the function of HDLs as potential cholesterol acceptors from ram spermatozoa during in vitro capacitation, which will be the focus of future studies. http://dx.doi.org/10.1016/j.anireprosci.2016.03.064 P46 The effect of antioxidants on the in vitro quality of fresh bull sperm stored in an egg yolk based diluent S.A. Holden 1 , P. Lonergan 2 , S. Fair 1,∗ 1 Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland 2 School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland E-mail address: [email protected] (S. Fair).
This study assessed the effect of antioxidants on the in vitro quality of fresh bull sperm. Experiment 1: caprogen diluent was prepared containing a range of antioxidants; l-carnitine (0, 2.5, 10, 20 mM), Crocin (0, 0.5, 1.5, 2 mM), ␣-Tocopherol (0, 0.075, 0.4, 1 mM), Quercetin (0, 50, 175, 250 M) and Catalase (0, 150, 250, 500 IU). Ejaculates (n = 3) were diluted to 12 × 106 sperm/mL in each respective diluent, incubated at 37 ◦ C and assessed for superoxide production and acrosome integrity using flow cytometry at 0, 1, 2 and 4 h. Experiment 2: Caprogen diluent was prepared containing the antioxidants l-carnitine (0, 0.5, 2.5, 5, 10, 20 mM), Crocin (0, 0.25, 0.5, 1, 1.5, 2 mM), ␣-Tocopherol(0, 0.025, 0.075, 0.15 0.4, 0.8, 1 mM) and Quercetin (0, 20, 50, 100, 175, 250 M). Ejaculates (n = 9) were diluted to 12 × 106 sperm/mL in respective diluents, packaged in 0.25 mL straws and stored at 16–18 ◦ C. Motility and kinematic parameters were assessed using computer assisted sperm analysis while membrane fluidity and lipid peroxidation were assessed using flow-cytometry on days 0, 1, 3 and 5 post collection. Data were analysed using the repeated-measures analysis of variance in the Statistical Package for the Social Sciences (SPSS; version 22.0, IBM, Chicago, IL). Experiment 1: there was no treatment or time × treatment interaction on superoxide production or acrosome integrity (P > 0.05). However, there was an effect of time with regard to catalase as all treatments at 4 h incubation had greater acrosome intact populations compared to the control (P < 0.05). Similarly, with Quercetin treatments, superoxide production was lowest at 2 h (P < 0.05) but increased thereafter. Experiment 2: There was no effect of any of the antioxidants on the in vitro parameters assessed when compared to the control (P > 0.05). There
was an effect of day regarding most of the parameters assessed (excluding membrane fluidity of Crocin treatments) as motility declined and membrane fluidity peaked on day 1. In conclusion, this study found modest benefits in the inclusion of antioxidants in egg-yolk based diluents used for the fresh storage of bull sperm. http://dx.doi.org/10.1016/j.anireprosci.2016.03.065 P47 Role of EDTA as a chelating agent in canine semen cryopreservation C. Braud 1,∗ , L.C.F. Bergo 2 , G.R. Araújo 2 , T. Deco-Souza 2 , A.C. Csermak Jr 2 , L.R.B. Carazo 2 , L.C. Silva 2 , T.A.R. Paula 2 1
INP-Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31300 Toulouse, France 2 REPAAS Laboratório de Biotecnologia da Reproduc¸ão em Pequenos Animais e Animais Silvestres Vic¸osa, MG 36570-900, Brazil E-mail address: c.braud [email protected] (C. Braud). Increased capacitation of spermatozoa and premature acrosome reaction reduce chances of fertilization. Since calcium influx into sperm cells is involved in the acrosome reaction, it has been suggested that high calcium concentrations found in sperm freezing media could increase the risk of premature reactions. The objective of this study was to evaluate dog sperm viability in freezing media of various calcium concentrations, with EDTA as a calcium chelator. Three ejaculates each of three mixed breed dogs of good fertility were collected by manual stimulation. Immediately after collection, volume, sperm concentration and motility as well as the percentage of live and abnormal sperm cells were determined in the sperm rich fraction. The initial concentration of calcium was assayed by spectrophotometry (E-225D-Celm® – 370 nm; InVitro® ) in the two fractions. Fresh semen was divided into three aliquots and suspended in the same volume of tris-citrate egg yolk extender (Nutricell-SP-Brasil). Thereafter, a second extender containing 12% glycerol and 1% Equex (NutricellSP-Brasil) was added to obtain a final concentration of 100 × 106 spermatozoa/mL. EDTA solution was added at different concentrations for the three aliquots: no EDTA; 0.1% EDTA; 0.25% EDTA). After thawing, calcium concentration, sperm motility, viability and morphological defects were immediately evaluated. Sperm plasma membrane integrity and acrosomal membrane integrity were also assessed with a fluorescent staining technique using a combination of 2 L of PI (Sigma P4170), 100 L FITCPSA (Sigma L0770) and 200 L Hoëscht 33342 (Molecular Probes H1399). In samples without EDTA, calcium concentrations were 14 times higher (21.23 ± 2.41 mg/dL) than in fresh semen (1.52 ± 0.62 mg/dL). The addition of EDTA significantly reduced the extracellular calcium concentrations in both treatments (0.1% EDTA: 13.91 ± 1.48 mg/dL; 0.25% EDTA: 3.98 ± 1.59 mg/dL) but values still remained high compared with in fresh semen. No significant difference