230 COMPARISON OF SEMEN SAMPLES FROM NORMAL AND MUSCULAR DYSTROPHY AFFECTED ANIMAL MODELS BY THIOBARBITURIC ACID REACTIVE SUBSTANCES TEST AND FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS

Abstract

Duchenne’s muscular dystrophy (DMD) is the most common human muscular affliction with high frequency in male individuals. The condition is characterized by progressive muscle degeneration, weakness, and loss of motion capacity. These individuals exhibit longer lifespans resulting from improvements in technical patient care and now raise new questions ranging from ethical to physiological issues never observed before, such as the possibility of reproduction. Thus, this study used Golden Retriever muscular dystrophy (GRMD)-affected dogs, a natural experimental model of DMD, to evaluate semen quality and oxidative stress by thiobarbituric acid reactive substances (TBARS) test. Thirty-seven ejaculates from 4 non-affected Golden Retriever and 5 GRMD-affected dogs (from 1.5 to 4.5 years old) were collected monthly and evaluated as 5 different replicates. Semen samples were processed, and initial analysis comprised of sperm concentration, percent of straightforward motility and morphology. Samples were then diluted to a final concentration of 106 spermatozoa mL^–1 of prior incubation with each fluorescent probe. Acrosome integrity assessment was conducted with PSAFITC (100 g mL^–1), and mitochondrial activity was assessed by JC1 (50 g mL^–1). Fluorescence-activated cell sorting was performed in 103 spermatozoa, from a pre-selected gate that contained only these cells. Seminal plasma was submitted to TBARS quantification by spectrophotometer under normal and oxidative conditions (stress). Parametric data were compared by Student’s t-test. There were no significant differences in ejaculate volume, sperm concentration, acrosomal integrity, mithocondrial activity, and ejaculate TBARS tests between GRMD and normal dogs. Data are summarized in the table. GRMD does not affect the production and quality of semen of dogs. This animal model suggests that ejaculates from DMD men can be obtained by the proper stimulation and that conventional in vitro fertilization techniques combined with preimplantation genetic diagnosis for single gene disorders may be able to satisfy the desire of paternity of DMD patients with the birth of non-affected children.

Table 1.  Sperm analysis and TBARS tests of ejaculates from GRMD and normal dogs

FAPESP for the financial support (06/50272-3); Surgery Department and Reproduction Department of FMVZ-USP; Canil GRMD Brazil.