Effect of dietary selenium on boar sperm quality.

The primary objective of this research was to evaluate the effect of long-term dietary selenium supplementation of commercial swine diets on semen production and sperm quality. The dietary treatments were a non-supplemented basal diet or the basal diet supplemented with 0.3ppm selenium in either an organic or inorganic form. A secondary objective was to determine if there were any beneficial effects of dietary selenium supplementation on changes in sperm quality during storage of semen post collection. Boars were fed dietary treatments from weaning at 20.97±0.18 d of age until the study was terminated when they were 382.97±0.18 d of age. Boars (n=6 per treatment) were maintained on a 1 time per week collection frequency for 5 months. Immediately after this, boars were collected six times over a 4 day period. Ejaculates were extended in a commercially available, 5-day semen extender and evaluated on day 1 and 6 of storage post-collection. Boars fed the organic selenium had higher (P<0.01) plasma levels of selenium compared to control boars and similar levels to those supplemented with the inorganic form (P=0.18). Dietary treatment did not affect (P>0.2) volume, concentration, total sperm in the ejaculate, sperm motility, progressive motility, morphology, lipid peroxidation, or glutathione peroxidase activity. These results indicate that supplementing a basal diet with organic or inorganic selenium did not affect semen quantity or sperm quality in fresh ejaculates nor did it appear to have any beneficial latent effects in extended semen stored post collection.

Molecular markers of sperm quality.

Light microscopic semen evaluation provides useful information about a given sperm sample, but due to its subjective nature has limited prognostic value for the reproductive performance of males or the outcome of assisted fertilization. Cryptic sperm abnormalities (occurring at the molecular level) are not easily detectable by light microscopy, but can be revealed by an array of biomarkers. The latter include fluorescent markers of acrosomal status, fluorochromes detecting altered sperm chromatin or DNA integrity, vital dyes revealing sperm mitochondrial activity, probes detecting apoptotic events, and antibodies detecting proteins that are either up- or down-regulated in defective spermatozoa. Many of the above biomarkers are best tested by flow cytometry, permitting rapid, automated, high throughput, objective measurement of the relative abundance of these biomarkers in semen. This review summarizes a strategy for the identification of novel male fertility/sperm quality biomarkers based on proteomic, biochemical and immunocytochemical analyses of defective spermatozoa. This approach identifies proteins or ligands uniquely associated with defective spermatozoa, regardless of whether they carry gross morphological defects or subtle, but critical hidden defects (e.g. DNA strand breaks) not detected with conventional, light microscopic analysis. Such markers, including ubiquitin, sperm thioredoxin SPTRX3/TXNDC8, 15LOX, and Lewis(y)-terminated N-glycans, are associated with poor semen quality and reduced fertility, warranting a designation of "negative" markers of fertility. The significance of sperm cytoplasmic droplet, a structure that accumulates several of the discussed biomarker proteins, is also discussed with regard to sperm quality and fertility.

High resolution light microscopic evaluation of boar semen quality sperm cytoplasmic droplet retention in relationship with boar fertility parameters.

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.

Arachidonate 15-lipoxygenase and ubiquitin as fertility markers in boars.

Accurate semen analysis is an important issue in the swine industry. We evaluated two candidate fertility marker proteins associated with sperm cytoplasmic droplet (CD), including 15-lipoxygenase (15-LOX) and ubiquitin (UBI) in a controlled single-sire artificial insemination (AI) trial. Ejaculates (n=116) were collected from 18 fertile Large White boars monthly for 8 mo, and analyzed by semi-quantitative, densitometry-based Western blotting and flow cytometry with antibodies against 15-LOX and UBI. Data were correlated with farrowing rates (FR) and total numbers of piglets born (TNB) from 1754 AI services by 13 of 18 boars, and compared with a conventional microscopic semen analysis. In semi-quantitative Western blotting, both 15-LOX and UBI were correlated with seasonal changes in the percentage of normal (r=-0.38, P<0.01; r=-0.27, P<0.05, respectively) and CD-bearing spermatozoa (r=0.35, P<0.01; r=0.27, P<0.05, respectively). In flow cytometry, UBI and 15-LOX levels showed seasonal changes coinciding with seasonal changes of FR and TNB, representing 13 boars, 88 ejaculates and 1,232 AI services. There were correlations between flow cytometric values of UBI and FR (r=0.31; P<0.05), adjusted FR (r=0.30; P<0.05), TNB (r=-0.38; P<0.01) and adjusted TNB (r=-0.37; P<0.01). Flow cytometric measurements of 15-LOX correlated negatively with TNB (r=-0.33; P<0.05) and adjusted TNB (r=-0.34; P<0.05). These data suggested that boar fertility estimation could be achieved within a group of fertile boars by the use of objectively measurable fertility markers. Flow cytometry appeared more informative and more practical than semi-quantitative Western blotting. This technology could be further optimized for the selection of the most fertile sires in an artificial insemination program.

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet.

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.