328 FLUORESCENT PROBE ASSESSMENT OF POST-THAW QUALITY OF RAM SPERM TREATED WITH SEMINAL PLASMA

Abstract

A recently suggested alternative to improve post-thaw ovine semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryo-capacitation and helping sperm survival in the female tract. The aim of this study was to evaluate the effect of thawed ram semen incubation with seminal plasma for 15 or 30 min as assessed by the fluorescent probes, Hoechst 33342 (40 µg mL⁻¹), propidium iodide (0.5 mg mL⁻¹), JC-1 (76.5 µM in DMSO), and FITC-PSA (100 µg mL⁻¹ in Dulbecco's PBS (DPBS)), for cellular viability, plasmatic damage, mitochondrial activity, and acrosomal damage, respectively. Five ejaculates were collected from 4 different animals via artificial vagina and were pooled to eliminate individual differences. After thawing, semen was divided into 2 groups, one diluted with seminal plasma (1 : 1, 30% in DPBS) and the other in DPBS (1 : 1). After 15 and 30 min, fluorescent probes were added to 25 µL of each group and 100 cells were counted under a epifluorescence microscope (Olympus IX81, motorized inverted research microscope). Spermatozoa were classified under 8 categories: alive, damaged acrosome, and high mitochondrial activity (ADH); dead, damaged acrosome, and high mitochondrial activity (DDH); alive, damaged acrosome, and poor mitochondrial activity (ADP); dead, damaged acrosome, and poor mitochondrial activity (DDP); alive, intact acrosome, and high mitochondrial activity (AIH); dead, intact acrosome, and high mitochondrial activity (DIH); alive, intact acrosome, and poor mitochondrial activity (AIP); or dead, intact acrosome, and poor mitochondrial activity (DIP). Statistical analysis was performed by Fisher test at a 5% level. The results are presented in Table 1. There was no significant difference between groups except for the AIH category which was reduced during incubation in either DPBS or SP for 15 or 30 min. These last data allow the conclusion that ram sperm is highly susceptible to cryogenic damage. Nevertheless, the high percentage of the AIH category suggests that the spermatozoa that can resist the freezing protocol remain alive and intact. Hence, we can infer that ram frozen semen has approximately half the fertilizing potential of fresh semen. In addition, we observed a negative effect of the incubation period as the decreased percentage of the AIH category was accompained by an increase of the DDP and DIP ones. We conclude that seminal plasma incubation after thawing does not benefit sperm quality. It is necessary to study the seminal plasma constitution to conclude why this experiment differed from other published data.

Table 1.  Spermatozoa percentages in the categories during incubation periods with or without seminal plasma

This work was supported financially by FAPESP 05/55256-3