New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

摘要

The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.