Measurable Cytokine Concentrations in Pig Seminal Plasma Are Modified by Semen Handling and Storage

Abstract

Sample handling and storing are critical steps for the reliable measurement of circulating biomolecules in biological fluids. This study evaluates how cytokine measurements in pig seminal plasma (SP) vary depending on semen handling and SP storage. Thirteen cytokines (GM-CSF, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNFα) were measured using Luminex xMAP^® technology in individual seminal plasma (SP) samples (n = 62) from healthy breeding boars. Three separate experiments explored the delay (2 h and 24 h) in SP collection after ejaculation (Experiment 1) and SP storage, either short-term (5 °C, -20 °C and -80 °C for 72 h, Experiment 2) or long-term (at -20 °C and -80 °C for two months, Experiment 3), before analysis. Levels in fresh SP-samples were used as baseline control values. Delays in SP harvesting of up to 24 h did not substantially impact SP cytokine measurements. Some cytokines showed instability in stored SP samples, mainly in long-term storage. Ideally, cytokines in pig SP should be measured in fresh samples harvested within 24 h after ejaculation. If storage of SP is imperative, storage conditions should be adjusted for each cytokine.

Keywords: cytokines; pig; seminal plasma; storage.

Figure 1

Box-and-whisker plot (horizontal line: median; box: 25/75 percentile; whisker: 10/90 percentile) showing the differences in cytokine concentrations measured in pig seminal plasma samples (SP) harvested 2 h and 24 h after ejaculate collection with respect to those harvested immediately after ejaculation (baseline samples, point 0 on Y axis). Below the X-axis is the concordance correlation coefficient (ρc) and 95% confidence interval (CI) showing agreement between the cytokine concentrations of the baseline and the experimental SP samples. Cytokines: Granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFNγ), interleukin (IL)-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and tumor necrosis factor-α (TNFα). Asterisks indicate statistical differences with baseline data. ** p < 0.01; * p < 0.05.

Figure 2

Box-and-whisker plot (horizontal line: median; box: 25/75 percentile; whisker: 10/90 percentile) showing the differences in cytokine concentrations measured in pig seminal plasma samples (SP) stored at 5 °C, −20 °C and −80 °C for 72 h with respect to those measured in fresh samples (baseline samples, point 0 in Y axis). Below the X-axis is the concordance correlation coefficient (ρc) and 95% confidence interval (CI), showing agreement between cytokine concentrations of baseline and experimental SP samples. Cytokines: Granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFNγ), interleukin (IL)-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and tumor necrosis factor-α (TNFα). Asterisks indicate statistical differences with baseline data. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 3

Box-and-whisker plot (horizontal line: median; box: 25/75 percentile; whisker: 10/90 percentile) showing differences in cytokine concentrations measured in pig seminal plasma samples (SP) stored at −20 °C and −80 °C for two months with respect to those measured in fresh samples (baseline samples, point 0 on the Y axis). Below the X-axis is the concordance correlation coefficient (ρc) and 95% confidence interval (CI), showing agreement between cytokine concentrations of the baseline and experimental SP samples. Cytokines: Granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFNγ), interleukin (IL)-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and tumor necrosis factor-α (TNFα). Asterisks indicate statistical differences with baseline data. *** p < 0.001; ** p < 0.01; * p < 0.05.