In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0 –native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H2DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.

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The aim of this experimental study was to evaluate the dynamics of nuclear DNA fragmentation in frozen-thawed (FT) boar spermatozoa incubated over time. Using the Sperm Chromatin Dispersion test (Sperm-Sus-Halomax), this study focused special attention on resolving the hypothesis that the original halo shapes around the sperm head could show dynamic changes over the postthawing incubation time. Twenty FT sperm samples from five boars (four per boar) were incubated at 37 °C during 168 hours and sperm motility (assessed using computer-assisted sperm analysis), viability (evaluated using the LIVE/DEAD Sperm Viability Kit), and nuclear DNA fragmentation were analyzed at 0, 0.5, 2, 4, 6, 24, 48, 72, and 168 hours. The percentages of motile and viable spermatozoa progressively decreased during incubation, with no motile and viable spermatozoa less than 10% in all boars at 24 hours of incubation. Four different halo shapes around the sperm head were considered in the Sperm Chromatin Dispersion test: normal, small, large scattered (typical fragmented nuclear DNA), and absent halo, all of them coexisting at the same time in the boar FT semen samples. Sperm with a large scattered halo did not change during postthaw, consistently showing percentages less than 5% over time in all boars. In contrast, the other three sperm populations showed a dynamic evolution over incubation time, characterized by a gradual reduction of sperm with normal halo, proportional to the increment in the sperm showing a small halo, followed by a switch between the sperm with a small halo and sperm with no halo. These results suggest that three of these four sperm populations, those showing small, large scattered, and absent halo, represent spermatozoa with different degrees of nuclear DNA damage, which should be taken into consideration to indicate the percentage of sperm with fragmented nuclear DNA in boar FT semen samples.

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