- Email: [email protected]
Effect of diode laser on motility, plasma and acrosomal membrane integrity, and mitochondrial membrane potential of cryopreserved stallion spermatozoa
8
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
unlikely as lactoferrin, an iron-binding protein present in stallion seminal plasma, catalyzes the production of the hydroxyl radical in vitro in a Fenton type Haber–Weiss reaction. It should also be considered that other antioxidative enzymes than catalase might be more important for the protection of the condensed sperm chromatin structure. doi:10.1016/j.anireprosci.2008.05.085 7 Effect of diode laser on motility, plasma and acrosomal membrane integrity, and mitochondrial membrane potential of cryopreserved stallion spermatozoa A.C. Brand˜ao ∗ , R.P. Arruda, A.F.C. Andrade, F.G. Zaffalon, O.F.B. Tarrag´o, J.A. Visintin, M.E.O.A. Assumpc¸a˜ o Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of S˜ao Paulo, S˜ao Paulo, Brazil E-mail address: [email protected] (A.C. Brand˜ao). Sperm motility depends on energy consumption. In some species sperm mitochondria play an important role in the production of energy for tail activity. Low-level laser irradiation increases this production as a modulation tool. The objective of this study was to analyze the effect of a continuous 650-nm wave length diode laser irradiation with 6 J/cm2 for 120 s, in the motility, plasma and acrosomal membrane integrity and mitochondrial membrane potential in fresh and frozen equine spermatozoa. Five ejaculates were obtained from each of five stallions (n = 25). Semen was packaged into 0.5-mL straws with 200 × 106 cells/mL in a Botu-CrioTM (BiotechBotucatu-Ltda/ME, Botucatu, Brazil) and frozen by automated technique using a programmed machine (TK3000® , TK Tecnologia em Congelac¸a˜ o-Ltda, Uberaba, Brazil). Fresh samples were divided in two groups: spermatozoa treated with laser and Control 1—non-treated spermatozoa. Frozen samples were divided in three groups: spermatozoa treated with laser before freezing; spermatozoa treated with laser after thawing and Control 2—cryopreserved spermatozoa. Cryopreserved samples were analyzed immediately after thawing (time 0) and 2 h after thawing (time 2). Motility was evaluated by computer assisted sperm analyzer (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), plasma and acrosomal membrane integrity and mitochondrial membrane potential were evaluated by flow cytometry (FACSaria-Beckton-Dickeson, San Jose, USA). The data were analyzed by the SAS program, at 5% level of significance. Beat cross frequency (BCF) test was higher (p < 0.05) for spermatozoa treated with laser 34.8 ± 0.7% as compared to Control 1 33.4 ± 0.8%. At time 0, mitochondrial membrane potential was lower in spermatozoa treated with laser before freezing 40.7 ± 1.5% compared to Control 2 47.4 ± 2.4% (P < 0.05). Two hours after thawing, plasma and acrosomal membrane integrity was higher (P < 0.05) in spermatozoa treated with laser before freezing (8.3 ± 0.7%) than Control 2 (6.2 ± 0.6%). The spermatozoa treated with laser after thawing were no different (P > 0.05) from the other groups at time 2, but at time 0 the percentage of progressive motility was lower (2.0 ± 0.3%) and different (P < 0.05) from the other groups (6.5 ± 1.3% in spermatozoa treated with laser before freezing; 5.5 ± 1.1% in Control 2). These results indicated that the irradiation with 650-nm wave length diode laser improves beat cross frequency in fresh semen and provides a long-term (2 h) protection for plasma and acrosomal membranes of equine spermatozoa. New studies with different diode laser wave length should be driven in order to improve stallion semen cryopreservation.
ANIREP 3632 1–59
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
9
Acknowledgements This research was supported by FAPESP (Proc. 04/09028-6; Proc. 05/55368-6; Proc. 06/609662). To Biotech-Botucatu-Ltda/ME for the donation of extender Botu-CrioTM . doi:10.1016/j.anireprosci.2008.05.086 8 Effect of cooling prior to cryopreservation of stallion spermatozoa J.E. Bruemmer ∗ , Q.P. Hardy, D.D. Denniston, J.K. Schumacher, E.L. Squires Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523, USA E-mail address: [email protected] (J.E. Bruemmer). Cooling equine spermatozoa to 5 ◦ C and shipping is a routine breeding farm procedure because it requires few additional supplies, equipment, or expertise compared to processing semen for immediate use. Currently, the majority of stallion spermatozoa are cryopreserved at specialized facilities. The ability to cool and ship spermatozoa for 18 h prior to cryopreservation compared to conventionally cryopreserved spermatozoa would allow stallion owners an additional marketing tool. The first objective of this study was to determine the effects of cooling prior to freezing on motion characteristics, viability and acrosome status on equine spermatozoa. In this experiment, we compared these parameters in conventionally frozen–thawed (CONTROL) spermatozoa with those cooled for either 6 or 18 h prior to cryopreservation. The second objective was to compare results with two commercial freezing extenders: skim milk-egg yolk (SMEY) and lactose-EDTA (L-EDTA). Neither total (TM) nor progressive (PM) motility, nor average velocity (VAP) of spermatozoa were affected by cooling prior to freezing in either extender. The SMEY extender was superior in preserving motion characteristics in all treatments compared to L-EDTA (P < 0.05). The mean percentage of live spermatozoa as determined by flow cytometry was similar between control (25.8%) and 6 h (26.4%) or 18 h (28.2%) cooled–frozen treatments extended in SMEY (P > 0.05). Control spermatozoa extended in SMEY maintained a higher percentage of live acrosome-intact cells compared to 18 h cooled–frozen spermatozoa (P < 0.05). SMEY was superior in preserving live and live, acrosome-intact spermatozoa compared to L-EDTA in control and 18 h cooled–frozen treatments (P < 0.05). The third objective was to compare fertility of spermatozoa cooled prior to cryopreservation. In this experiment, we tested the effect on pregnancy rates in mares inseminated with control or 18 h cooled–frozen spermatozoa. Timed inseminations occurred at approximately 30 and 48 h post-deslorelin injection with 800 × 106 cooled–frozen (n = 19) or control (n = 17) spermatozoa. No difference in pregnancy rates was detected when using either 18 h cooled–frozen or control (30% and 38%, respectively, P = 0.673) spermatozoa. Based on these experiments, stallion spermatozoa can be effectively cooled and stored at 5 ◦ C for 18 h prior to cryopreservation without a decrease in TM, PM, VAP, or viability compared to the current cryopreservation protocol. Although 18 h cooled–frozen spermatozoa had a lower percentage of live, acrosome-intact spermatozoa compared to control, the 18 h cooled–frozen spermatozoa were as fertile as those frozen immediately following collection. doi:10.1016/j.anireprosci.2008.05.087
ANIREP 3632 1–59
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
unlikely as lactoferrin, an iron-binding protein present in stallion seminal plasma, catalyzes the production of the hydroxyl radical in vitro in a Fenton type Haber–Weiss reaction. It should also be considered that other antioxidative enzymes than catalase might be more important for the protection of the condensed sperm chromatin structure. doi:10.1016/j.anireprosci.2008.05.085 7 Effect of diode laser on motility, plasma and acrosomal membrane integrity, and mitochondrial membrane potential of cryopreserved stallion spermatozoa A.C. Brand˜ao ∗ , R.P. Arruda, A.F.C. Andrade, F.G. Zaffalon, O.F.B. Tarrag´o, J.A. Visintin, M.E.O.A. Assumpc¸a˜ o Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of S˜ao Paulo, S˜ao Paulo, Brazil E-mail address: [email protected] (A.C. Brand˜ao). Sperm motility depends on energy consumption. In some species sperm mitochondria play an important role in the production of energy for tail activity. Low-level laser irradiation increases this production as a modulation tool. The objective of this study was to analyze the effect of a continuous 650-nm wave length diode laser irradiation with 6 J/cm2 for 120 s, in the motility, plasma and acrosomal membrane integrity and mitochondrial membrane potential in fresh and frozen equine spermatozoa. Five ejaculates were obtained from each of five stallions (n = 25). Semen was packaged into 0.5-mL straws with 200 × 106 cells/mL in a Botu-CrioTM (BiotechBotucatu-Ltda/ME, Botucatu, Brazil) and frozen by automated technique using a programmed machine (TK3000® , TK Tecnologia em Congelac¸a˜ o-Ltda, Uberaba, Brazil). Fresh samples were divided in two groups: spermatozoa treated with laser and Control 1—non-treated spermatozoa. Frozen samples were divided in three groups: spermatozoa treated with laser before freezing; spermatozoa treated with laser after thawing and Control 2—cryopreserved spermatozoa. Cryopreserved samples were analyzed immediately after thawing (time 0) and 2 h after thawing (time 2). Motility was evaluated by computer assisted sperm analyzer (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), plasma and acrosomal membrane integrity and mitochondrial membrane potential were evaluated by flow cytometry (FACSaria-Beckton-Dickeson, San Jose, USA). The data were analyzed by the SAS program, at 5% level of significance. Beat cross frequency (BCF) test was higher (p < 0.05) for spermatozoa treated with laser 34.8 ± 0.7% as compared to Control 1 33.4 ± 0.8%. At time 0, mitochondrial membrane potential was lower in spermatozoa treated with laser before freezing 40.7 ± 1.5% compared to Control 2 47.4 ± 2.4% (P < 0.05). Two hours after thawing, plasma and acrosomal membrane integrity was higher (P < 0.05) in spermatozoa treated with laser before freezing (8.3 ± 0.7%) than Control 2 (6.2 ± 0.6%). The spermatozoa treated with laser after thawing were no different (P > 0.05) from the other groups at time 2, but at time 0 the percentage of progressive motility was lower (2.0 ± 0.3%) and different (P < 0.05) from the other groups (6.5 ± 1.3% in spermatozoa treated with laser before freezing; 5.5 ± 1.1% in Control 2). These results indicated that the irradiation with 650-nm wave length diode laser improves beat cross frequency in fresh semen and provides a long-term (2 h) protection for plasma and acrosomal membranes of equine spermatozoa. New studies with different diode laser wave length should be driven in order to improve stallion semen cryopreservation.
ANIREP 3632 1–59
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
9
Acknowledgements This research was supported by FAPESP (Proc. 04/09028-6; Proc. 05/55368-6; Proc. 06/609662). To Biotech-Botucatu-Ltda/ME for the donation of extender Botu-CrioTM . doi:10.1016/j.anireprosci.2008.05.086 8 Effect of cooling prior to cryopreservation of stallion spermatozoa J.E. Bruemmer ∗ , Q.P. Hardy, D.D. Denniston, J.K. Schumacher, E.L. Squires Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523, USA E-mail address: [email protected] (J.E. Bruemmer). Cooling equine spermatozoa to 5 ◦ C and shipping is a routine breeding farm procedure because it requires few additional supplies, equipment, or expertise compared to processing semen for immediate use. Currently, the majority of stallion spermatozoa are cryopreserved at specialized facilities. The ability to cool and ship spermatozoa for 18 h prior to cryopreservation compared to conventionally cryopreserved spermatozoa would allow stallion owners an additional marketing tool. The first objective of this study was to determine the effects of cooling prior to freezing on motion characteristics, viability and acrosome status on equine spermatozoa. In this experiment, we compared these parameters in conventionally frozen–thawed (CONTROL) spermatozoa with those cooled for either 6 or 18 h prior to cryopreservation. The second objective was to compare results with two commercial freezing extenders: skim milk-egg yolk (SMEY) and lactose-EDTA (L-EDTA). Neither total (TM) nor progressive (PM) motility, nor average velocity (VAP) of spermatozoa were affected by cooling prior to freezing in either extender. The SMEY extender was superior in preserving motion characteristics in all treatments compared to L-EDTA (P < 0.05). The mean percentage of live spermatozoa as determined by flow cytometry was similar between control (25.8%) and 6 h (26.4%) or 18 h (28.2%) cooled–frozen treatments extended in SMEY (P > 0.05). Control spermatozoa extended in SMEY maintained a higher percentage of live acrosome-intact cells compared to 18 h cooled–frozen spermatozoa (P < 0.05). SMEY was superior in preserving live and live, acrosome-intact spermatozoa compared to L-EDTA in control and 18 h cooled–frozen treatments (P < 0.05). The third objective was to compare fertility of spermatozoa cooled prior to cryopreservation. In this experiment, we tested the effect on pregnancy rates in mares inseminated with control or 18 h cooled–frozen spermatozoa. Timed inseminations occurred at approximately 30 and 48 h post-deslorelin injection with 800 × 106 cooled–frozen (n = 19) or control (n = 17) spermatozoa. No difference in pregnancy rates was detected when using either 18 h cooled–frozen or control (30% and 38%, respectively, P = 0.673) spermatozoa. Based on these experiments, stallion spermatozoa can be effectively cooled and stored at 5 ◦ C for 18 h prior to cryopreservation without a decrease in TM, PM, VAP, or viability compared to the current cryopreservation protocol. Although 18 h cooled–frozen spermatozoa had a lower percentage of live, acrosome-intact spermatozoa compared to control, the 18 h cooled–frozen spermatozoa were as fertile as those frozen immediately following collection. doi:10.1016/j.anireprosci.2008.05.087
ANIREP 3632 1–59