Tolerance of Stored Boar Spermatozoa to Autologous Seminal Plasma: A Proteomic and Lipidomic Approach

Abstract

Long-term exposure of liquid preserved boar spermatozoa to seminal plasma (SP) can cause dramatic sperm injury. This study examined whether boar specificity exists in the sensitivity of spermatozoa to SP and whether correspondent biomarkers can be identified. Consecutive ejaculates (n = 4-5) collected from 19 boars were centrifuged, diluted with a pH-stablising extender with 10% (v/v) autologous SP and evaluated by computer-assisted semen analysis and flow cytometry. Up until 144 h storage, four boars showed consistently high sperm motility, viability and mitochondria activity, and one boar showed consistently low values. Intra-boar variability was high in the other boars. Screening of SP (n = 12 samples) for protein markers using mass spectrometry identified three protein candidates of which the granulin precursor, legumain and AWN were 0.5 to 0.9 log2-fold less abundant (p < 0.05) in SP-resistant compared to SP-sensitive samples. Lipidome analysis by mass spectrometry revealed 568 lipids showing no difference between the SP-groups. The most abundant lipids were cholesterol (42,442 pmol), followed by phosphatidylserine (20,956 pmol) and ether-linked phosphatidylethanolamine (13,039 pmol). In conclusion, three candidate proteins were identified which might be indicative of SP-tolerance of sperm during long-term storage. Noteworthy, a first lipidomic profile of boar SP is presented.

Keywords: biomarker; boar seminal plasma; lipidomics; proteomics; semen preservation; sperm quality.

Figure 1

Sperm motility in relation to the concentration of seminal plasma in the semen dose. Pearson’s correlation (r = −0.52, p < 0.0001) between sperm motility at 144 h of storage and the concentration of seminal plasma in uncentrifuged semen portions extended to 18 × 10⁶ sperm/mL in 100 mL volume; n = 87 semen samples from 19 different boars.

Figure 2

Sperm quality in stored semen doses containing different concentrations of seminal plasma (SP). Sperm pellets of centrifuged ejaculates (n = 4–5 from each of 19 boars) were extended with modified Beltsville thawing solution (mBTS) to 18 × 10⁶ sperm/mL containing three different concentrations of SP: 1%, 5% and 10% (v/v; final concentration) and semen portions were stored at 17 °C. Graphs present in A) total motility (%), B) membrane intact (%; propidium iodide negative and peanut agglutinin (PNA) negative) sperm (%); and C) viable sperm (%) with high mitochondria membrane potential (hMMP) (propidium iodide-negative sperm with J-aggregates). Values are presented as means and standard deviation (± SD). *: 10% SP differs from 5% and 1% SP (p < 0.05); **: 10% SP differs from 1% SP (p < 0.01).

Figure 3

Sperm quality after 144 h of storage in semen doses containing different concentrations of seminal plasma (SP). Box-whisker plots showing variation in the total motility (%), membrane intact (% propidium iodide negative and peanut agglutinin (PNA) negative) sperm (%), and viable sperm (%) with high mitochondrial membrane potential (hMMP) (propidium iodide negative sperm with J-aggregates). Semen samples contain three different final concentrations of autologous SP: 1%, 5% and 10% (v/v). Different letters indicate significant difference (p < 0.01).

Figure 4

Sperm motility after 144 h of storage in single semen doses containing 10% (v/v) seminal plasma. Scatterplot showing the total motility (%) of semen samples with 10% autologous seminal plasma collected from 19 boars (n = 4–5 ejaculates per boar). Dots show the values measured in each sample and the bars show the mean for each boar.

Figure 5

Exploratory data analysis of proteins identified in seminal plasma of boars showing high sperm quality (resistant) and low sperm quality (sensitive) in stored semen doses containing 10% (v/v) autologous seminal plasma. Overview of the results from the LC-MS/MS analysis of boar seminal plasma proteins. (A) Principal component analysis (PCA) of normalised protein intensity values shows no distinct separation of the two groups, whereas (B) volcano plot analysis reveals three differently abundant protein candidates (p < 0.05) in seminal plasma samples collected from ejaculates with resistant (blue boxes) and sensitive (orange boxes) sperm.

Figure 6

Proteins being significantly altered in abundance in seminal plasma of boars showing high sperm quality (resistant) and low sperm quality (sensitive) in stored semen doses containing 10% (v/v) autologous seminal plasma. Box plots of granulin (GRN), legumain (LGMN) and AWN intensity values in seminal plasma samples collected from ejaculates with resistant (blue boxes) and sensitive (orange boxes) sperm. * p < 0.05.

Figure 7

Exploratory data analysis of lipids identified in seminal plasma. Principal component analysis (PCA) showing how seminal plasma samples map onto principal components 1 and 2, which together contribute to explain 62% of the variance contained in the dataset. There is no distinct separation between samples collected from ejaculates with resistant (blue dots) and sensitive (orange dots) sperm to 10% (v/v) autologous seminal plasma in extended semen at 144 h of storage.

Figure 8

Lipid classes detected in seminal plasma, and profiles of total acyl chain length and unsaturation of nonstorage lipids. The mol% amounts of the different lipid features are presented as boxplots with dots. Each dot indicates a sample (n = 6 in each group). Values did not differ between sensitive and resistant samples (p > 0.05). (A) Lipid class composition. Mol% abundance of lipid classes in seminal plasma samples collected from ejaculates with resistant (blue bars) and sensitive (orange bars) sperm to 10% (v/v) autologous seminal plasma in extended semen at 144 h of storage. Prevalent lipid classes are cholesterol (Chol) and phosphatydilserine (PS). Classes appear in alphabetical order. The mole percent was calculated for all lipids. (B) Total acyl chain length profile. Lipids were regrouped according to the number of carbon atoms present in their acyl chains. The mole percent was calculated for sphingolipids, glycerophospholipids and diacylglycerides. Only length groups with a mean >1 mol l% are shown. Shorter chain lipids (lipids with 32 carbon atoms) are lowly abundant, as well as very long lipids (42 carbon atoms). The most represented group consists of lipids with 36 carbon atoms. (C) Unsaturation profile. Lipids were regrouped according to the number of double bonds present in their acyl chains. All species with more than six double bonds are indicated with “6+”. The mole percent was calculated for sphingolipids, glycerophospholipids and diacylglycerides. 0 = saturated lipids; 1 = mono-unsaturated lipids; 2 = di-unsaturated lipids; 3–6+ = poly-unsaturated lipids. Boar seminal plasma contains many mono-unsaturated phospho- and sphingolipids.