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A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.

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

Current data about the kinetics of exogenous DNA interaction with spermatozoa are controversial. The amount of DNA associated with the spermatozoa is proportional to the incubation period. However, a great amount of exogenous DNA, associated or internalized, can activate endonucleasis, which cleaves the exogenous and/or endogenous DNA similarly to apoptosis. The aim of this work was to evaluate the effect of the incubation period of bovine spermatozoa with exogenous DNA on transfection rate and induction of apoptosis, oncosis, or initial oncosis and/or late apoptosis of sperm cells. For that, 5 × 106 spermatozoa/mL were incubated with 500 ng of plasmid pEYFP-Nuc (Clontech, Mountain View, CA, USA) for 60, 90, and 120 min. Sperm cells were then subjected to electrophoresis to remove non-associated exogenous DNA. DNA (endogenous and exogenous) was extracted and real-time PCR was performed to quantify exogenous DNA/spermatozoa association. Each reaction was performed with 12.5 µL of Platinum SYBR Green; 0.5 µL of ROX; 5.6 µL of H2O; 5 µL of sample (1 µg); 0.7 µL of primer sense (5′-ATGGCCGACAAGCAGAAGAAC-3′) and 0.7 µL of primer anti-sense (5′-TGCCGTCCTCGATGTTGTG-3′); 500 nM each. Real-time PCR was conducted for 40 cycles of 95°C for 15 s and 64°C for 1 min. Apoptosis and oncosis analyses were evaluated by flow cytometry with 106 sperm/mL stained with 2 µL of Yo-pro (100 µM) for 20 min and with 10 µL of propidium iodide (6 µM) for 10 min at room temperature. Data were analyzed by MINITAB Realease 14 Statistical Software (Minitab, Inc., State College, PA, USA), subjected to ANOVA, and compared by Fisher's or Tukey's test (P ≤ 0.05) for transfection rate and apoptosis/oncosis, respectively. Linear regression obtained by the amplification of pEYFP-Nuc at different concentrations showed efficiency of R2 = 0.9987. The results summarized in Table 1 show that increase in length of the incubation period increases the amount of exogenous DNA associated with spermatozoa and does not affect the average of live spermatozoa in apoptosis, oncosis, and initial oncosis and/or late apoptosis groups. Table 1.Quantification of exogenous DNA associated with bovine sperm cells and percentages of live, apoptotic, and oncotic spermatozoa This work was supported financially by FAPESP 03/07456-8 and 03/10234-7.

Several studies have been performed over the years to promote the understanding and improvement of the sperm-mediated gene transfer technique. However, little is known about the effect of exogenous DNA in the sperm cells. The aim of this study was to evaluate the effect of the incubation period and exogenous DNA addition on mitochondrial activity and acrosomal status of bovine spermatozoa. Frozen-thawed semen was separated by Percoll gradient (45/90%) at 600g for 30 min, and the pellet was resuspended and washed by centrifugation in sperm-TALP at 200g for 5 min. The spermatozoa were resuspended in fertilization medium (without heparin) at a concentration of 5 × 106 spermatozoa/mL and incubated at 39°C, 5% CO2 in air with 500 ng pEYFP-Nuc/mL (Clontech, Mountain View, CA, USA) (DNA group) for 1 and 2 h or without DNA (control group) for 0, 1 and 2 h. JC-1 (Molecular Probes; Invitrogen Brasil, Ltd., Sao Paulo, Brazil) was used to determine mitochondrial potential and fluorsecein isothiogyanate (FITC-PSA; Sigma-Aldrich, Brazil) was used to assess acrosomal status. Two microliters of JC-1 (76.5 μM in DMSO) and 50 μL of FITC-PSA (100 μg/mL in DPBS) were added to 150 μL of IVF medium + 5 × 106 spermatozoa/mL; the mixture was incubated at 25°C for 10 min. DNA incorporation was evaluated by p-EYFP-Nuc PCR amplification. All treatments were repeated ten times and data were analyzed by ANOVA and Tukey test (P < 0.05). The results are described in Table 1. The sperm were classified as: Class 1 (intact acrosome and high mitochondria potential), Class 2 (intact acrosome and low mitochondria potential), Class 3 (reacted acrosome and high mitochondria potential) and Class 4 (reacted acrosome and low mitochondria potential). In both groups, the Class 1 sperm decreased over time, whereas an increase in number was verified for Class 2 sperm. There was no significant difference in numbers among the incubation periods in both groups for Class 3 sperm. However, there was an increase in Class 4 sperm number over time, indicating that the main effect of the incubation period was the loss of mitochondria potential. There was no effect of the exogenous DNA addition on sperm viability in relation to the control group, indicating that the exogenous DNA had no effect on mitochondrial activity and acrosomal status of bovine semen. Table 1. Effect of incubation period and exogenous DNA addition on sperm viability This work was supported by FAPESP 03/10234–7; 03/07456–8.