Fraction Of Ejaculate Research Articles

The Seminal Plasma of the Boar is Rich in Cytokines, with Significant Individual and Intra-Ejaculate Variation.

The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-γ, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-β3. Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.

Individual and seasonal variations in the quality of fractionated boar ejaculates

Reproductive seasonality has been shown to affect the quality of boar semen. In this study, effects of seasonal variations in the characteristics of spermatozoa and seminal plasma (SP) of fractioned ejaculates from individual boars have been investigated. Fractionated ejaculates, designated as fraction 1 (F1), fraction 2 (F2), and fraction 3 (F3), were collected from five mature boars during the autumn-winter (October through March) and spring-summer periods (April through September). A total of 10 fractionated ejaculates (F1, F2, and F3) were collected from each boar within each seasonal period. Assessments of the sperm quality characteristics included computer-assisted sperm analysis motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity, normal apical ridge acrosomes, and DNA fragmentation. Besides SDS-PAGE and densitometric analyses of the SP proteins, the antiperoxidant activity was monitored. There were marked differences in the sperm quality characteristics among the boars, except for sperm MMP. Distinct seasonal differences (P < 0.05) were observed in the ejaculate volume of F3 during the autumn-winter and spring-summer periods (107.78 ± 5.45 and 87.80 ± 4.75 mL, respectively). Significantly higher (P < 0.05) sperm concentration and the total number of spermatozoa in the fraction were observed during the autumn-winter period. Seasonal effects in MMP and plasma membrane integrity were manifested in significantly higher (P < 0.05) percentages of spermatozoa with functional mitochondria and intact plasma membrane during the autumn-winter period. However, the seasonal effects were less marked in either sperm normal apical ridge acrosomes or sperm DNA fragmentation. Sodium dodecyl sulfate–PAGE and densitometric analyses revealed marked variations in the protein composition of the SP profiles among the boars, regardless of the ejaculate fraction and seasonal period. Distinct seasonal variations, observed in the SDS-PAGE profiles, were associated with an abundance of protein fractions of low-molecular and high-molecular weight components, particularly during the autumn-winter period. There were wide variations in antiperoxidant activity in the SP among the boars, being significantly higher in the autumn-winter period, irrespective of the ejaculate fraction. It can be suggested that marked deterioration of the quality of fractionated ejaculates during the spring-summer period was probably caused by impaired reproductive function in the boar.

The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility.

Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.

Seminal quality in the first fraction of ejaculate

Semen samples from 40 patients were collected in consecutive fractions. The variability in semen quality of each fraction was then determined. The first ejaculated fraction (FEF) primarily contained prostatic secretions, while the second ejaculate fraction (SEF) held the majority of the spermatozoa suspended in the secretions from the seminal vesicle. Differences in sperm quality were observed when the FEF was compared to the SEF and the total ejaculate fraction (TEF). These included the seminal parameters (volume, sperm concentration, motility) and sperm DNA fragmentation (SDF). When compared to TEF and SEF, the FEF presented a lower volume, higher sperm concentration, higher motility rates, and lower SDF. The data suggest that the first fraction renders an improved subpopulation of spermatozoa, with lower SDF. Spermatozoa from this fraction and hence their use for ART may have a positive effect on fertilization and embryo development.

Effect of the holding time at 15 °C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15°C prior to cryopreservation (2, 4 and 8h), thawing rate (37°C for 20s or 70°C for 8s) and post-thaw incubation temperature (15°C or 37°C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240min of incubation. The results show that with the holding time at 15°C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70°C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37°C for 20s. In addition after thawing the sperm samples incubated at 15°C had a sustained sperm quality for longer, up to 4h post-thawing.

Improvement in overall seminal quality by separate collection of first sample of ejaculate

We aim to assess the improvement of sperm quality observed in the first ejaculate fraction compared to the second ejaculate fraction and total ejaculate. Usually, the ejaculate to be used for ART is collected in just one pot, producing a sustained contact between sperm and seminal liquid that can compromise chromatine stability. After a detailed explanations to the patients, semen samples were collected in two separate fractions in order to compare overall sperm quality in FEF. 30 patients were included in the study. Their semen sample was collected in two separate fractions, after 2 to 5 days of sexual abstinence, and analyzed within an hour. In both fractions and the TEF, classic seminal parameters and Zinc, Citric Acid and Fructose content were assessed. DNA fragmentation test was performed using the Sperm Chromatin Decondensation assay with Halosperm kit. Compared to TEF and SEF, we observed in FEF a disminution of volume, higher sperm concentration, higher motility rates, better morphology according to Kruger criteria and lower DNA fragmentation (DIF), all statistically significant.Tabled 1FEFSEFTEFVolume (ml)0,631,53p=0,02Motility (a+b%)48,531,940,3p=0,01Sperm count89,54065p=0,01Morphology4,93,44,5p=0,01DFI1732,324,7p=0,01Chart 1: Parameters assessed in FEF, SEF and TEF. Open table in a new tab Chart 1: Parameters assessed in FEF, SEF and TEF. Collection of semen sample in two separate fractions offers the possibility to achieve a improved sperm population, which may act synergically for the selection of high quality spermatozoa with further techniques to be applied in later stages, such as DL-DGc and MACS prior to ICSI.

Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa

The goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.

Testicular Steroids, Prolactin, Relaxin and Prostate Gland Markers in Peripheral Blood and Seminal Plasma of Normal Dogs and Dogs with Prostatic Hyperplasia

Concentrations of 17β-oestradiol (E(2) ), testosterone (T), 5α-dihydrotestosterone, prolactin (PRL) and relaxin (RLN) were determined in peripheral blood serum or plasma and prostatic secretion of 77 physically healthy intact male dogs (19 Rhodesian Ridgebacks/RR, 58 dogs of other breeds, 1-9 years of age). Furthermore, the concentrations of acid phosphatase in prostatic secretion and canine prostate-specific esterase (CPSE) were measured in blood plasma. All dogs were submitted to a complete breeding soundness examination, including B-mode sonography. Prostatic volume was larger, and blood plasma levels of CPSE were higher in ageing dogs and in dogs with benign prostatic hyperplasia (BPH) compared with young dogs and dogs with normal prostate. Furthermore, a higher E(2) /T ratio was found in dogs with BPH. Despite missing significant differences in PRL concentrations, the slight increases in PRL concentrations in the prostatic secretion observed both with increasing age and in dogs with BPH and the observed correlations between concentrations of PRL and testicular steroids may possibly indicate a role of PRL in the pathogenesis of canine BPH. Serum RLN concentrations were at similar level in all dogs. Regarding breed differences, an appreciably larger prostatic volume and higher concentration of CPSE were verified in RR than in other pure-bred dogs, confirming our suspicion of a premature enlargement of the prostate gland, which may result from a genetic disposition for BPH in this breed.

Sperm quality following 4 days of cooled storage: impact on fertility

degrade protein components of extracellular matrix and basement membranes, and are thought to be involved in pre-fertilization events. The purpose of this study was to compare the levels of MMP-2 and MMP-9 in different fractions of SP, and to evaluate the correlation of the MMP levels with fertility. During the breeding season, semenwas collected from 43 stallions (20 Standardbreds, 17 Finnhorses, 4 warmblood riding horses, 2 ponies; ages 4-23 years) residing at nine different stud farms in Finland. The breeding history and current use of the stallions was variable, with the stallions serving 2 to 150 (median 14) mares during the season. Semen jets were collected as 1-4 fractions using an open-ended artificial vagina (AV) or a modified closed AV. After semen collection, the gel was removed and the volume of each fraction was measured. A sample representing the whole ejaculate was formed by combining 10% of the volume of each fraction. The samples were centrifuged at 4000 x g (15 min), and the supernatant was filtered using 0.45 mm filters. The SP samples were stored frozen in 1-mL aliquots in -75 C until analyzed. The levels of the pro-form of MMP-2, active MMP-2 and total MMP-9 were evaluated using gelatin zymography and densitometry. Fertility data were provided by the stud farms and the Finnish trotting and breeding association (Suomen Hippos). The first cycle pregnancy rate was calculated from all of the reported matings in the year in question for the stallions included in the study, and all mares that foaled or aborted were considered pregnant. Pro-MMP-2, active MMP-2 and total MMP-9 were detected in stallion seminal plasma, with the highest levels found in the sperm-rich fraction. In all fractions, the levels of proMMP-2 were significantly higher than the levels of active MMP-2 and total MMP-9, which did not differ. The levels of the different types of MMP were not correlated with the first cycle pregnancy rate. Pro-MMP-2, active MMP-2 and MMP-9 are present in all fractions of the stallion's ejaculate, with the highest levels in the sperm-rich fraction. Despite a possible role in the fertilization process, the levels of MMP-2 and MMP-9 were not correlated with fertility in this study on a limited number of animals.

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H2DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.

Seminal Plasma Proteins as Modulators of the Sperm Function and Their Application in Sperm Biotechnologies

Seminal plasma (SP) is known to play an important role in mammalian fertilization. However, the variability found in its composition among species, males and even fractions of the same ejaculate has made difficult to completely understand its effect in sperm function. Proteins are one of the major SP components that modulate sperm functionality. During the last years, intensive work has been performed to characterize the role of these proteins. They have been found to influence sperm capacitation, formation of the oviductal sperm reservoir and sperm-oocyte interaction. Sperm biotechnologies, such as sperm cryopreservation and flow cytometric sex-sorting, that involve a substantial dilution of the SP are detrimental to sperm quality. Attempts to improve the outcome of these biotechnologies include the restoration of SP, which has produced contradictory results. To overcome this variability, different research groups have proposed the application of isolated SP proteins. Herein, we will review the current knowledge in the role of the major SP proteins as modulators of sperm functionality. Furthermore, we will discuss the possible applications of the SP proteins in sperm cryopreservation and flow cytometric sex-sorting.

Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate.

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.

Measurement of Alkaline Phosphatase in Canine Seminal Plasma – An Update

In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut-off value of 5000 IU/l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre-analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre-analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at -18 °C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107,328 IU/l. After 24 h of frozen storage, activities did not differ significantly (96,844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1:100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU/l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24h before analysis.

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.

Effects of Seminal Fluid Fractions on Plasma and Acrosome Membrane Integrity and Mitochondrial Membrane Potential Determined by Flow Cytometry in Chilled Canine Spermatozoa

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third[prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential(Δψm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm-rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5C for 72 h in egg yolk-TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT-SF) or third (EYT-PF) ejaculated fractions. After cold storage,groups EYT-SF and EYT-PF showed significantly higher percentages of sperm cells with an intact acrosome[68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1%respectively]. Only in EYT-SF was PS translocation significantly reduced compared to EYT-PF and EYT [3.9 ± 0.4%,10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT-SF and EYT-PF compared to EYT[36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in Δψm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6).Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation.In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 – 766.5pg/mL) and IL-10, (73.7 – 837.3pg/mL), was found. For IL-6, the concentration was low (range 11.5 – 30.9pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (−20°C or −80°C) did not affect the seminal plasma TGF-β1 level after one year of storage.To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca2+]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca2+]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

Bacteriological findings in different fractions of canine ejaculates showing normospermia, teratozoospermia or azoospermia

There is little available information on the distinction between physiological and pathological bacterial flora in dog semen, or in fractions of the canine ejaculate. In order to improve the diagnostic basis, the present study describes the bacteriological findings in canine semen samples. The ejaculates were assigned to three groups: Group A, normospermia (n = 30); Group B, teratozoospermia (n = 33); Group C, azoospermia (n = 29). Our data show that irrespective of semen quality, bacterial contamination and stronger bacterial growth is more common in the pre-sperm fraction than in the other two fractions. The number of bacteria and bacterial species did not vary between the three fractions or the three groups of dogs. Bacteria regarded as potentially pathogenic (β-haemolytic Streptococci, Escherichia coli variation haemolytica) could be isolated in every group, with β-haemolytic Streptococci tending to occur more frequently in all ejaculate fractions from dogs with teratozoospermia. These data emphasise that the first fraction is more contaminated with bacteria than the second and third fractions which may be due to bacteria ascending from the urethra. The use of a fractionated collection of canine ejaculates seems to be beneficial to reduce or prevent bacterial contaminations.

Ejaculate fractions of asthenozoospermic and teratozoospermic patients have differences in the sperm DNA integrity

The initial fraction of the human ejaculate mainly contains prostatic secretions and the subsequent fraction holds majority of the spermatozoa suspended in the secretions from the seminal vesicle. Apart from large series of proteins, human ejaculate also contains antioxidants and reactive oxygen species that are specific to certain accessory sexual glands; however, the influence of these components on the sperm DNA integrity has not been elucidated till date. The present investigation was conducted using split (first and second) ejaculate fractions of forty-one subjects having various semen abnormalities. Sperm DNA integrity was assessed in the individual fractions by comet assay and quantified. The amount of sperm DNA damage between the split fractions is not significantly different in normozoospermic semen samples. In contrast, split fraction-2 had significantly elevated level of DNA-damaged spermatozoa in asthenozoospermic (P < 0.01) and teratozoospermic groups (P < 0.001) when compared to whole ejaculate. The split fraction analysis using various types of ejaculates demonstrated the difference in sperm DNA integrity, which has not been reported till date. Hence, in a clinical point of view, the use of initial ejaculate fraction may be considered superior to whole ejaculate in assisted conception if the DNA integrity is a concern especially in asthenozoospermic and teratozoospermic samples.

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF–P1, usually 30–40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF–P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4–5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF–P1, 1.25 ± 0.14 × 109 spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF–P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.