Effect of diode laser on motility, plasma and acrosomal membrane integrity, and mitochondrial membrane potential of cryopreserved stallion spermatozoa

Abstract

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/cm 2 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 × 10 6 cells/mL in a Botu-Crio TM (Biotech- Botucatu-Ltda/ME, Botucatu, Brazil) and frozen by automated technique using a programmed machine (TK3000 ® , TK Tecnologia em Congelac ¸ ˜ ao-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. Cryop- reserved 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) 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.