Liofilização de espermatozóides bovinos: viabilidade estrutural e funcional

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Lyophilization is a procedure developed for cell preservation, as a result of restriction of active water from biological systems by ice sublimation. Recently, freeze-drying process has been used to preserve mammalian spermatozoa, becoming an alternative tool for genetic material preservation. In the present thesis the effect of lyophilization with different protecting medium on the bovine sperm cells structure and function was evaluated. The following medium were tested: (T 1) - TCM Hanks with 10% FCS; (T 2) - TCM Hanks with 10% de SFB and trehalose 0,2 M; (T 3) EGTA solution, composed of 50mM/L de EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid] and 10mM/L of Tris-HCl. Effects of sperm lyophilization were evaluated using optical microscopy analysis to assess motility, maintenance of the tail, and acrosome integrity by trypan-bleu/giensa stain; electron microscopy analysis was used to evaluated ultrastructure of important components such as plasma membrane, acrosome and microtubules; fluorescence microscopy was used to analyzed nuclear integrity by Acridine Orange Test (AOT) and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL); and finally, intracytoplasmic sperm injection (ICSI) to verify the fecundity and embryo development potential of lyophilized spermatozoa. The results of the first experiment indicated that nuclear viability preservation depends on the protector media utilized. In addition, it showed that TUNEL technique is more sensitive than AOT to detect nuclear damage, being the most indicated for nucleus evaluation. In the second experiment, data demonstrated that lyophilization is an aggressive procedure for the sperm cells, causing damage in the plasma membrane and lost of motility. However, other cell components such as acrosome and mitochondria were similarly preserved in all treatments. Nuclei were better conserved in medium containing EGTA and trehalose. Those treatments, which had lower of fragmented DNA, also presented higher rates of decondensed sperm head, pronucleus formation and embryo development, confirming TUNEL evaluation. Theses results suggested that freeze-drying spermatozoa participate in the process of fertilization when used for ICSI. However, additional studies are needed, being the improvements in this technology reliant on the adequate protector media and in the higher efficiency of ICSI technique

ASSUNTO(S)

egta tunel biologia molecular egta bovinos trehalose tunel icsi sperm lyophilization liofilização espermática trehalose bovine icsi

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