Cold-Shock Test Is a Practical Method for Selecting Boar Ejaculates Yielding Appropriate Seminal Plasma for Post-Thawing Supplementation

Artificial insemination (AI) with cryopreserved semen is still unreliable for extensive pig industry application. Adding seminal plasma (SP) could improve post-thawing quality, but its suitability could vary. We applied a simple cold-shock test (CST, 5 min at 0 °C) on neat semen for classifying ejaculates (n = 63) as resistant or sensitive, obtaining two SP pools (CST-resistant: SPr, sensitive: SPs). Subsequently, frozen/thawed spermatozoa from six boars were incubated (37 °C) in MR-A® extender (control), 20% SPr, or 20% SPs, and analyzed at 0, 2, and 4 h. SP improved total and progressive motility, with a higher effect for SPr and STR (p < 0.05), decreasing kinematic parameters VCL and VAP, ALH, and BCF. Sperm viability was unaffected. SP increased apoptotic and membrane disorder ratios, and acrosomal damage, not affecting the chromatin structure (DNA fragmentation and immaturity by SCSA), protamination (CMA3), or disulfide levels (mBBr). However, the proportion of spermatozoa with elevated free thiols (disulfide bridges reduction) significantly increased. Results support a stimulatory role of SP on thawed semen, with additional benefits from SPr. The effect of SP and especially SPr after AI should be tested since CST could be a practical test for selecting suitable ejaculates in AI centers.

Time Dependent Impact of Reactive Oxidants on Seminal Attributes of Murrah Bull during Cryopreservation and Storage

Background: Cryopreservation is an invaluable technique yet it is also known to be detrimental to sperm function and fertility due to cryo-injury and concomitant generation of reactive oxidants. During laboratory manipulation for the cryopreservation and freeze-thaw process, spermatozoa undergo osmotic stress, ionic imbalance, metabolic decoupling, membrane phase transition, destabilization of the cytoskeleton and antioxidant depletion which communally hampers the semen quality.Methods: With the aim of determining implications of cryopreservation and storage, semen samples were collected by artificial vagina technique from 12 Murrah bulls and subsequently examined at 0 hour (before cryopreservation) and at 24 hour, 1 month and 2 month of storage for various seminal attributes. Simultaneously seminal plasma was separated and preserved at -20oC till the analysis of biochemical indicators of semen quality viz., nitric oxide (NO), total antioxidant quantity (TAC) and lipid peroxidation status (TBARS). Result: A sharp reduction (p less than 0.01) in the semen quality was observed only at 24 h after cryopreservation except for viability. Significant reduction (p less than 0.05) in viable counts was observed up to 1 month interval. The capacitated sperm percentage was greater (p less than 0.01) in the cryopreserved semen as compared to fresh ejaculate. The mean ± SE levels of NO (μmol/L), TAC and TBARS (Units/ml) was 2.31±0.27, 0.73±0.04 and 1.11±0.16 respectively in seminal plasma of neat semen stored at -20oC, while the values in the extended seminal plasma after cryopreservation was 2.37±0.31, 0.44±0.03 and 0.65±0.03 respectively. So it can be inferred that most of the damage encountered by spermatozoa is during the initial period of freezing, but the damage associated by various stressors cannot be ignored.

Presence of Round Cells Proteins do not Interfere with Identification of Human Sperm Proteins from Frozen Semen Samples by LC-MS/MS

In sperm proteomic experiments round cells and leukocyte proteins are profiled along with sperm proteome. The influence of round cell and leukocyte proteins on the sperm proteome has not been investigated. The objective of this study was to identify if the proteins from round cells, including leukocytes, interfere with the proteomic analysis of spermatozoa in frozen semen samples. Proteomic profiling of sperm was performed using liquid chromatography-tandem mass spectrometry in four groups: Group 1 contained neat semen with round cells and leukocytes ≥ 1 × 106/mL, group 2 contained neat semen with round cells ≥ 1 × 106/mL that was processed by 65% density gradient to remove the round cells and leukocytes, group 3 contained neat semen with round cells < 1 × 106/mL, and group 4 contained neat semen with round cells < 1 × 106/mL that was processed by 65% density gradient to remove the round cells. Pure leukocyte culture was used as control group. A total of 1638, 1393, 1755, and 1404 proteins were identified in groups 1, 2, 3, and 4, respectively. Comparative analysis of group 1 vs. 3 revealed 26 (1.18%) differentially expressed proteins (DEPs). On the other hand, only 6 (0.31%) DEPs were observed with group 2 vs. 4. Expression of these DEPs were either absent or very low in the control group. The results of our proteomics analysis failed to show any influence of non-spermatogenic round cell proteins on sperm proteome identification. These results validate the use of neat semen samples for sperm proteomic studies.

Effects of human sperm cryopreservation on apoptotic markers in normozoospermic and non-normozoospermic patients

SummaryThe negative effects of cryopreservation on sperm parameters are well documented but little information is known about molecular basis of the process. The aim of the present study was to investigate the possible effects of sperm cryopreservation on main apoptotic signs including DNA fragmentation and caspase-3 activation and to determine if these effects vary according to sperm parameters. Sperm samples of 72 patients were cryopreserved. The patients were sub-grouped as normozoospermic or non-normozoospermic patients according to their semen parameters. DNA fragmentation rates and caspase-3 activation levels were analyzed before and after cryopreservation in both groups. Mean DNA fragmentation rate was increased significantly from 23.98% in neat semen samples to 27.34% after cryopreservation (P = 0.03). DNA fragmentation rates were slightly higher in non-normozoospermic patients compared with the normozoospermic patients in both the neat semen and after cryopreservation (23.25 and 24.71% vs. 26.32 and 28.36%, respectively) although the difference obtained were not statistically significant. An increasing trend for caspase-3 activations (0.093 vs. 0.116) was observed after cryopreservation but the differences were not statistically significant. Caspase-3 activation was found to be slightly higher in non-normozoospermic patients both in the neat semen and after cryopreservation compared with the normozoospermic patients but the differences were not statistically significant. Caspase-3 expression was also shown using immunocytochemistry in both fresh ejaculated sperm and thawed sperm after cryopreservation but at different localizations. The cryopreservation process had detrimental effects on sperm quality but the quality of the sperm samples was not adversely effective for the apoptotic markers including DNA fragmentation and caspase-3 activation patterns. In fact, it was the cryopreservation process itself that adversely effected the above apoptotic markers and apoptosis. It was concluded therefore that sperm cell cryopreservation triggers apoptosis after thawing and this process adversely affects semen parameters.

280 BOVINE SPERM DNA FRAGMENTATION RATES AND EXTENDER pH: A DYNAMIC EXPERIMENTAL APPROACH

Flow cytometry technology for the sorting of X- and Y-chromosome bearing sperm is currently utilised in research and for commercial applications. During sample preparation before the sex-sorting process, sperm go through different pH treatments that may affect their quality. The hypothesis to test in this study is that sperm DNA damage could occur due to differences in the pH of sperm extenders. Bull semen doses from 15 dairy and 15 beef bulls (2 ejaculates per individual), ranging in age between 13 and 96 months, were randomly selected from a bull stud in Texas (Sexing Technologies, Navasota, TX, USA). Each semen sample was divided into 4 separate aliquots. One neat semen sample was kept as a control and the other 3 aliquots were treated with 16 μL of 8.1 mM Hoechst 33342 (Molecular Probes, Eugene, OR, USA). A calculated amount of modified Tyrode’s albumin lactate pyruvate (Clear TALP), pH 7.4, was added based on neat ejaculate concentration. For the separation of live and dead sperm during the sex-sorting process, 2 mL of red TALP (Red Food Dye FD&C #40; Sensient Technologies Corp., Milwaukee, WI, USA) at 3 different pH treatment levels (5.5, 6.4, and 7.4) were added to the sperm samples. The dynamics of sperm DNA fragmentation were assessed immediately following the addition of red TALP treatment and after 24 h of incubation at 34°C. Sperm DNA fragmentation was measured using the commercial variant of the sperm chromatin dispersion test, the bull Sperm-Halomax® kit (Halotech DNA, Madrid, Spain), and counting 300 sperm cells under fluorescence microscopy. Analysis of variance was used to determine if there were statistical differences (α = 0.05) among mean values of the groups (SPSS v.17.0 for Windows, SPSS Inc., Chicago, IL, USA). Bonferroni post hoc tests were utilised to determine the pairwise directional differences between groups. Differences in DNA fragmentation were not observed at 0 h (P > 0.05); however, at 24 h this value was significantly higher (P < 0.05) in semen samples treated with red TALP, pH 6.4, v. samples treated with red TALP, pH 7.4, (44.0 ± 24.9% v. 34.8 ± 24.9%), and even higher when using red TALP pH 5.5 (50.8 ± 23.8%). Further experiments should be designed to demonstrate that the DNA molecule could be affected by pH fluctuations, which may be important in refining the sex-sorting process. Notably, the undiluted neat semen controls had greater levels of DNA fragmentation overall (57.5 ± 29.4%), suggesting a negative effect of high sperm concentrations and seminal plasma. The authors thank Mike Evans, Eddy Valenzuela, David Del Olmo, Jamie Jeffers, and the staff at Sexing Technologies for their technical assistance. This research was funded by Sexing Technologies and XY Inc.