Development of Poly-NIsopropylacrylamide Surfaces for the Selection of Swine Sperm

The reproductive efficiency of pig farms is directly correlated with the fertility of the boars. The aim of this work was to develop polymeric materials that can be used as a platform to select a subpopulation of sperm with better cell physiological parameters. Polymeric hydrogels composed of Poly-N-isopropylacrylamide with different positive charges given by copolymerization with (3-acrylamidopropyl) trimethylammonium chloride (APTA, 5-10-15%), were synthesized. Subsequently, the interaction between the sperm cells and the polymeric surfaces was analyzed in TALP medium. Release of the spermatozoa from the polymeric surfaces was induced by changing to Ca2+ free media. Sperm motility, cell viability, plasma membrane and acrosome integrity were evaluated. The results indicated that a higher percentage of swine sperm attached to PNIPAM co-15% APTA hydrogels (62.86±3.33%). Ninety seven percent (97.19±1.45 %) of the sperm released from the PNIPAM co-15%APTA surfaces were viable (p<0.05 vs unbound population and raw semen), with acceptable motility (58.89±1.28%) and with intact plasma and acrosomal membranes (69±1.2% and 98.5±0.65% respectively). These results indicate that hydrogels can be used to select boar sperm with high viability and mobility for use in assisted reproductive techniques.

Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Gallic acid improves the viability and mitochondrial membrane potential of post-thawed goat buck semen

The aim of this study was to determine the effects of gallic acid (GA) on frozen-thawed goat spermatozoa. Four Honamli goat bucks were used at their breeding season, and ejaculates were collected by an electroejaculator. Mixed semen was divided into the following four groups: control (0 mM), low (L; 1 mM), medium (M; 2 mM), and high (H; 4 mM) concentration of GA. All the groups were frozen and thawed in a water bath for spermatological evaluation. The lowest motility was observed in the control group (47.60 ± 5.70%) (P < 0.05), while the highest viability (62.45 ± 1.68%), plasma membrane and acrosome integrity (44.81 ± 4.57%), and high mitochondrial membrane potential (35.96 ± 2.50%) were observed in the low GA group (P < 0.05). Also, the lowest hypo-osmotic swelling test (HOS +) value was found in the high GA group (47.60 ± 4.82%) (P < 0.05). In conclusion, supplementing a low concentration (1 mM) of GA to the Tris-based semen extender had a positive effect on spermatological parameters after freeze-thawing of Honamli goat semen. Further studies should be continued in other species with different doses and combinations using commercial and/or homemade semen extenders.

Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

Antioxidants Significantly Improve Qinling Giant Panda (Ailuropoda Melanoleuca Qinlingensis) Sperm Quality During Cryopreservation

Background: Semen cryopreservation has become an essential tool for the reproduction and long-term preservation of giant pandas (Ailuropoda melanoleuca). However, cryopreservation is severely detrimental to sperm quality, including motility, plasma membrane, acrosome integrity, and mitochondrial activity. It is necessary to screen a effective antioxidants for improvement of sperm quality. Eight groups of antioxidants were added to the freezing medium.Results:The results showed that lycium barbarum polysaccharide (LBP), Laminaria japonica polysaccharides (LJP), or goujresveratrol (RSV) added to the freezing medium significantly improved sperm motility, acrosome integrity, and mitochondrial activity during the cryopreservation process. Furthermore, the activities of glutathione and superoxide dismutase were also improved. However, the levels of reactive oxygen species and malondialdehyde in semen were notably reduced. Regarding fertility, acrosin activity was significantly increased in LBP-treated sperm. Unfortunately, sperm viability, DNA fragmentation, and hyaluronidase activity were not significantly different between the above groups. Conclusions: LBP (2.0 mg/mL) or RSV (50 μM) are the best candidate antioxidants for inclusion in the freezing medium for improving the quality of panda spermatozoa, during semen cryopreservation.

Bayesian Analysis of the Effects of Olive Oil-Derived Antioxidants on Cryopreserved Buck Sperm Parameters

The present study evaluates the effect of olive oil-derived antioxidants, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), on cryopreserved caprine sperm using Bayesian inference of ANOVA. For this proposal, sperm was collected, pooled and diluted in freezing media supplemented with different concentrations of HT, DHPG and the mixture (MIX) of both antioxidants. Sperm motility, viability, acrosome integrity, mitochondrial status, and lipid peroxidation (LPO) were assessed in fresh and frozen-thawed sperm samples. The results provided evidence that HT at low concentrations improves sperm motility and viability, and reduces the LPO. Contrastingly, DHPG and MIX exert a positive effect by reducing LPO values as concentration increases. Additionally, mitochondrial potential was reduced when samples were supplemented with HT at low concentrations and mixture of both antioxidants. Conclusively, the addition of olive oil-derived antioxidants (HT at 10 µg/mL and DHPG at 30 µg/mL) implements a protective effect in cryopreserved buck sperm. Bayesian analysis alternatives offer new possibilities to determine the repercussion of antioxidants on sperm, both quantitatively and qualitatively.

Glutathione supplementation in semen extender improves rabbit sperm attributes during refrigeration

In the present study, we evaluated the sustaining effect of various glutathione (GSH) concentrations in extender on rabbit sperm attributes during storage at 5°C for 24 h. Semen was collected from regular donor rabbit bucks using an artificial vagina and initially evaluated for sperm quality. The qualifying ejaculates were diluted with one of the extenders having 0, 1, 2, 4 or 8 mM GSH, to achieve a final concentration of 1×108 sperm/mL. The extended samples were stored at 5°C for 24 h. Sperm motility, motion kinetics, acrosome integrity and viability were assessed after 3, 6, 12 and 24 h of storage. The results showed that total sperm motility and sperm motion kinetics (oscillation index of the sperm, straightness index and beat cross frequency) were influenced (<em>P</em>&lt;0.05) by glutathione dose and refrigeration time. An interaction of (<em>P</em>&lt;0.05) GSH concentrations and refrigeration time was observed for sperm viability and acrosome reaction rate. In conclusion, the 4 mM GSH supplemented extender’s protective influence was remarkable to maintainrabbit sperm quality for 24 h 5°C.

Identification of Bacterial Profiles and Their Interactions with Selected Quality, Oxidative, and Immunological Parameters of Turkey Semen

This study focused on the identification of naturally occurring bacteria in the reproductive fluid and impact on the quality of ejaculates obtained from the turkey breed British United Turkeys (BUT) Big 6 (n = 60). We determined possible relationships between the bacterial load and advanced sperm quality parameters that are important for effective artificial insemination and high fertility, as well as the concentration of selected antimicrobial proteins and pro-inflammatory markers of turkey semen. Sperm motility was assessed with computer-assisted sperm analysis (CASA), while the membrane and acrosome integrity were examined with smearing and staining methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL assay, and the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cell lysates were prepared to investigate the extent of lipid and protein oxidation. Furthermore, levels of interleukins 1 and 6 (IL-1, IL-6), C-reactive protein, cathelicidin, and β-defensin were quantified in the seminal plasma using the ELISA method. The most dominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was Escherichia coli, Proteus mirabilis, Staphylococcus lentus, and Citrobacter braakii. The bacterial load had a negative effect on the sperm motility (p < 0.001), as well as membrane (p < 0.05) and acrosome integrity (p < 0.01). A strong positive relationship between the bacterial load and DNA fragmentation (p < 0.001) was detected as well. Positive associations were recorded between the increasing presence of bacteria, ROS overgeneration (p < 0.001), and a subsequent oxidative damage to the proteins (p < 0.001) and lipids (p < 0.01). It was revealed that the antimicrobial peptides β-defensin (p < 0.001) and cathelicidin (p < 0.001) had a positive relationship with the motility. In contrast, pro-inflammatory markers, such as IL-1 (p < 0.001) and IL-6 (p < 0.001), had a negative impact on the motion behavior of turkey spermatozoa. Our results suggest that the semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia is associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for a failed artificial insemination in turkey breeding.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.