Is There a Relationship Between the PT-Substructure Status and Acrosome Loss of Boar Spermatozoa Following Freezing-Thawing or Acrosomal Reaction?

4 Abstract: The aim of this study was to determine whether in vitro boar sperm spontaneous acrosomal reaction is accompanied by changes on the Perinuclear Theca (PT) and to test the hypothesis that early changes in sperm PT-substructure integrity on frozen-thawed spermatozoa affects this process. Semen was collected thrice from six boars, making a total of 18 ejaculates; each ejaculate was divided into 2 samples, one for use in fresh and the other to be processed by freezing. Both samples were incubated to induce spontaneous acrosome reaction. At the end of the incubation period, capacitating and acrosome-reacted spermatozoa were isolated from one another by a density gradient. An evaluation of acrosome viability and integrity was conducted using the technique of triple stain. In order to expose the PT, both samples were treated with non-ionic detergent Brij 36-T and processed for electron microscopy observation after negative stain. The substructure was absent in capacitated cells with spontaneous acrosome reaction, while in capacitated sperm in which the acrosome still remained, the substructure persisted. Frozen-thawed sperm had a greater proportion (p<0.05) of acrosome-reacted sperm, as compared to fresh sperm; the integrity of the substructure was lower after freezing-thawing (p<0.05). In fresh semen, prior to the induction of spontaneous acrosome reaction, 6.6% of the sperm showed damages in the PT-substructure and after such process, the percentage increased to 51.6% (p<0.001), while in frozen-thawed sperm, such values were 70 and 82.6%, respectively (p<0.005). The correlation analysis showed that there was a relationship between ultrastructural changes in the PT-substructure and the loss of sperm acrosome, both in fresh (r = 0.93; p<0.05) and in frozen-thawed (r = 0.71; p<0.05) sperm. However, in thawed sperm incubated for spontaneous acrosome reaction, the percentage showing a damaged PT-substructure was higher than (p<0.05) sperm without acrosome. In conclusion, the acrosome reaction produces spontaneous loss of the substructure of PT and this damage on cryopreserved sperm affects the process.