Poster Presentation IXth International Conference on Boar Semen Preservation 2019

Favorable seminal plasma environment to sperm fertility after L-arginine addition to boar diet (#2.1)

Seong B Park 1 , Tasha R Gruhot 2 , Brian J Rude 1 , Shengfa F Liao 1 , Benny E Mote 2 , Jean M Feugang 1
  1. Department of Animal & Dairy Sciences, Mississippi State University, Mississippi State, Mississippi , USA
  2. Department of Animal Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA

Several studies have reported the beneficial effects of L-arginine on sperm production and fertility. Recently, a study described the effectiveness of L-arginine-supplemented diets for alleviating heat-stress induced infertility of boars. Improvement of semen antioxidant capacity and other biochemical changes were observed, but the underlying molecular mechanisms related to spermatogenesis and sperm quality remain largely unknown. Here we performed a proteomic analysis to assess the changes of seminal plasma proteins due to nutritional L-arginine supplementation, followed by the prediction of effects on sperm fertilization potential using bioinformatic tools.

Mature (20 months of age) Nebraska Index Line boars (n=9) were individually housed and randomly assigned to a Control group (n=4), fed a soybean meal-based diet containing 0.77% arginine and an L-arginine group (n=5) fed the same basal diet but supplemented with 1% L-arginine. Semen was collected weekly before and during the experiment of 6 wks. Semen production outputs were recorded and sperm motion and morphology characteristics were subsequently analyzed using CASA. On wk 6, the semen from each boar was centrifuged (at 4 oC), and seminal plasma (SP) was collected for proteome analysis. Two-dimensional gel electrophoresis was used (300 µg of clarified/precipitated SP, resuspended in an appropriate buffer per IPG strip - pI 3-10, followed by the second-dimension gel electrophoresis). All electrophoresis gels were stained with Coomassie R250 dye for visualization, and differential protein spot intensities between Control and L-arginine groups were detected and analyzed (PDQUEST software). These spots were subsequently excised and digested for protein identification (liquid chromatography electrospray ionization tandem mass spectrometry), followed by functional bioinformatics analyses. Data were assessed by repeated measurements of ANOVA or Student’s t-test wherever appropriate. Significant data were called for at P<0.05.

Dietary L-arginine supplementation did not affect semen outputs and sperm characteristics throughout the experiment (P>0.05). After six weeks of the diet, semen volumes were comparable between boars fed with control (389±40mL) and L-arginine (393±35mL, P>0.05) diets. Gel electrophoreses of derived seminal plasma displayed over 200 protein spots, while the comparative analysis of gels (4x4) revealed 12 up- and two down-regulated spots in the L-arginine group. Following protein identification, annotations, and functional analyses, upregulated proteins mainly belonged to gene ontologies associated with extracellular localization and single fertilization process.

These results indicate that the increased dietary L-arginine for mature boars maintained in a thermo-comfort environment does not affect semen outputs nor sperm quality characteristics. However, the induced changes of seminal plasma proteins in boars fed with L-arginine may favor the fertility potential of spermatozoa. This work was supported by the USDA-ARS Biophotonics Initiative #58-6402-3-018.