Influence of Bacterial Contamination and Antibiotic Sensitivity on Cryopreserved Sperm Quality of Indian Red Jungle Fowl.

Aims: Bacterial contamination may occur in feces during collection and processing of semen. Bacteria not only compete for nutrients with spermatozoa but also produce toxic metabolites and endotoxins and affect sperm quality. The aim of the present study was to investigate the effect of antibiotic supplementation on the sperm quality of Indian red jungle fowl, estimation and isolation of bacterial species and their antibiotic sensitivity. Materials and Methods: Semen was collected and initially evaluated, diluted, and divided into six experimental extenders containing gentamicin (2.5 µg/mL), kanamycin (31.2 µg/mL), neomycin (62.5 mg/mL), penicillin (200 U/mL), and streptomycin (250 µg/mL), and a control having no antibiotics were cryopreserved and semen quality was evaluated at post-dilution, post-cooling, post-equilibration, and post-thawing stages (Experiment 1). A total aerobic bacterial count was carried out after culturing bacteria (Experiment 2) and subcultured for antibiotic sensitivity (Experiment 3). Results: It was shown that penicillin-containing extender improved semen quality (sperm motility, plasma membrane integrity, viability, and acrosomal integrity) compared with the control and other extenders having antibiotics. The bacteria isolated from semen were Escherichia coli, Staphylococcus spp., and Bacillus spp. Antibiotic sensitivity results revealed that E. coli shows high sensitivity toward neomycin, kanamycin, and penicillin. Staphylococcus spp. shows high sensitivity toward streptomycin, neomycin, and penicillin. Bacillus spp. shows high sensitivity toward kanamycin and penicillin. Conclusions: It was concluded that antibiotics added to semen extender did not cause any toxicity and maintained semen quality as that of untreated control samples, and penicillin was identified as most effective antibiotic. It is recommended that penicillin can be added to the semen extender for control of bacterial contamination without affecting the semen quality of Indian red jungle fowl.

The Effect of Adding Different Levels of Reduced Glutathione to Extender on the Quality of Cooled Ring-Necked Pheasant Semen.

Aim: Artificial propagation of ring-necked pheasant through semen preservation is of significance, as this species is facing enormous threats in its natural habitat. Semen preservation inevitably induces oxidative stress, and exogenous antioxidants need to be investigated for the preservation of ring-necked pheasant semen. Therefore, the current study was conducted to investigate the role of glutathione (GSH) in extender on the liquid storage of ring-necked pheasant semen. Materials and Methods: Semen was collected from 10 sexually mature males, evaluated for sperm motility, and pooled. Pooled semen was aliquoted for dilution with Beltsville poultry semen extender (1:5) at 37°C having GSH levels of 0.0 mM (Control), 0.2, 0.4, 0.6, and 0.8 mM. Extended semen was gradually cooled to 4°C and stored in a refrigerator (4°C) for 48 hours. Semen quality, that is, sperm motility, membrane integrity, viability, acrosomal integrity, and DNA integrity, was assessed at 0, 2, 6, 24, and 48 hours. Results: Sperm motility (%), plasma membrane integrity (%), viability (%), and acrosomal integrity (%) were recorded higher (p < 0.05), whereas DNA fragmentation (%) was recorded lower in extender supplemented with 0.4 mM GSH up to 48 hours of storage compared with 0.2, 0.6, and 0.8 mM GSH concentrations and control. Conclusion: It is concluded that 0.4 mM GSH in extender improves sperm quality parameters of ring-necked pheasant during liquid storage up to 48 hours at 4°C.

Effect of Quercetin on Oxidative Stress, Mitochondrial Activity, and Quality of Indian Red Jungle Fowl (Gallus gallus murghi) Sperm.

Aim: The study was designed to elucidate the effects of quercetin in an extender on oxidative stress, mitochondrial activity and quality of Indian red jungle fowl (Gallus gallus murghi) sperm during cryopreservation. Materials and Methods: Semen was collected from seven adult males through abdominal massage and evaluated for semen volume, concentration, and motility. The qualifying semen ejaculates having >80% motility were diluted in red fowl extenders with 0 (control), 5, 10, 15, and 20 mM quercetin. Diluted semen was frozen following a glycerol-based protocol. Semen quality (motility, plasma membrane integrity, viability, acrosome integrity, and chromatin condensation status) and biochemical parameters (mitochondrial activity, ferric reducing antioxidant power, and malondialdehyde [MDA]) were determined at various stages of cryopreservation. Results: Sperm motility, plasma membrane integrity, viability, acrosome integrity, and chromatin condensation were recorded highest (p < 0.05) with 15 mM quercetin compared with 5, 10, and 20 mM quercetin and control at post-dilution, cooling, equilibration, and freeze-thawing. Nevertheless, mitochondrial activity and antioxidant potential were recorded highest with 15 mM quercetin compared with all experimental extenders at post-equilibration and freeze-thawing. MDA concentration in sperm and seminal plasma were recorded lowest (p < 0.05) in the extender having 15 mM quercetin at post-equilibration and freeze-thawing. Cryopreservation stages showed negative effects (p < 0.05) on semen quality parameters, irrespective of experimental extenders. Conclusions: It is concluded that quercetin (15 mM) supplementation in red fowl extender improves sperm motility, plasma membrane integrity, viability, acrosome integrity, chromatin condensation, and mitochondrial activity by elevating the total antioxidant potential and ameliorating lipid peroxidation during cryopreservation.

Synthesis of Hollow Pt-Ni Nanoboxes for Highly Efficient Methanol Oxidation.

In direct methanol fuel cell technology, highly stable electrochemical catalysts are critically important for their practical utilization at the commercial scale. In this study, sub ~10 nm hollow Pt-Ni (1:1 at. ratio) nanoboxes supported on functionalized Vulcan carbon (Pt-Ni/C-R2) were synthesized through a facile method for the efficient electrooxidation of methanol. Two reaction procedures, namely, a simultaneous reduction and a modified sequential reduction method using a reverse microemulsion (RME) method, were adopted to synthesize solid Pt-Ni NPs and hollow nanoboxes, respectively. To correlate the alloy composition and surface structure with the enhanced catalytic activity, the results were compared with the nanocatalyst synthesized using a conventional NaBH4 reduction method. The calculated electroactive surface area for the Pt-Ni/C-R2 nanoboxes was 190.8 m2.g-1, which is significantly higher compared to that of the Pt-Ni nanocatalyst (96.4 m2.g-1) synthesized by a conventional reduction method. Hollow nanoboxes showed 34% and 44% increases in mass activity and rate of methanol oxidation reaction, respectively, compared to solid NPs. These results support the nanoreactor confinement effect of the hollow nanoboxes. The experimental results were supported by Density Functional Theory (DFT) studies, which revealed that the lowest CO poisoning of the Pt1Ni1 catalyst among all Ptm-Nin mixing ratios may account for the enhanced methanol oxidation. The synthesized hollow Pt-Ni/C (R2) nanoboxes may prove to be a valuable and highly efficient catalysts for the electrochemical oxidation of methanol due to their low cost, numerous catalytically active sites, low carbon monoxide poisoning, large electroactive surface area and long-term stability.

Evaluation of Enrofloxacin for Use in Cryopreservation of Zebu Bull (Bos indicus) Semen.

Aim: The present study was designed to evaluate enrofloxacin (EN), a fluoroquinolone broad-spectrum antibacterial drug for use in cryopreservation of Zebu bull semen. Materials and Methods: Semen was collected from three Zebu bulls of the Sahiwal breed (Bos indicus) for 3 weeks (replicates) for each experiment. Experiment I: semen samples were cultured for isolation of bacteria and in vitro sensitivity tests against streptomycin, penicillin, and EN. Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were isolated. P. aeruginosa was found to be resistant to streptomycin and penicillin (SP), while Proteus spp. was resistant to streptomycin. EN effectively inhibited the growth of all four bacteria in an in vitro sensitivity test. Experiment II: in vitro dose toxicity of EN (0-2000 µg/mL) for bull sperm was assessed in 2.9% sodium citrate buffer. EN up to a concentration of 1200 µg/mL was found to be safe for motility and viability of bull spermatozoa. Experiment III: semen was cryopreserved in a tris-citric acid extender containing either SP or EN (400 µg/mL) or without antibiotics as the control. Results: Post-thaw sperm motility was found to be higher in the extender containing EN and SP compared to control. Nevertheless, sperm viability was recorded higher (p < 0.05) with EN compared to SP and control. Sperm chromatin integrity did not differ (p > 0.05) in EN, SP, and controls. The total aerobic bacterial count was recorded as zero in the extender containing EN compared to SP and control. The fertility rate was higher (p < 0.05) with 400 µg/mL EN (59%) compared to SP (44%). Conclusions: It is concluded that EN is capable of controlling the bacterial load in frozen Zebu bull semen, resulting in a higher fertility rate under field conditions.

Effect of Cryopreservation on Lipid Peroxidation, Antioxidant Potential, Chromatin Integrity, and Mitochondrial Activity of Indian Red Jungle Fowl (Gallus gallus murghi) Semen.

Aim: The study elucidates the impact of cryopreservation on lipid peroxidation (LPO), antioxidant potential, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm. Materials and Methods: Semen from eight mature cocks was pooled and diluted at 37°C using a specific medium for red fowl species. The diluted semen samples were immediately refrigerated at 4°C for 2 hours (0.275°C min-1). Glycerol was then added to a final concentration of 20%, and the samples were equilibrated for 10 minutes at 5°C before loading into 0.25 mL French straws. These straws were then frozen by placing them in liquid nitrogen vapor for 10 minutes and plunged into liquid nitrogen. Motility, viability, DNA integrity, antioxidant activity, and LPO were assessed before dilution (fresh semen), after equilibration (processed semen), and post-thawing (frozen semen). Results: Sperm motility, viability, DNA integrity, and mitochondrial activity decreased (p < 0.05) in processed and frozen semen compared with fresh semen. Nevertheless, the concentration of malondialdehyde (MDA) in sperm and seminal plasma was greater (p < 0.05) in frozen-thawed and processed semen compared with fresh semen. Multivariate regression analysis showed a negative impact of MDA concentration in sperm (R2 = 0.90, Wilk's λ = 0.003, p < 0.001) and seminal plasma (R2 = 0.84, Wilk's λ = 0.02, p < 0.001) on motility, viability, DNA integrity, and mitochondrial activity. Nonetheless, total antioxidant capacity (TAC) had a positive impact on sperm variables (R2 = 0.82, Wilk's λ = 0.096, p < 0.001). Conclusions: The decrease in motility, viability, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm was associated with an increase in LPO during cryopreservation. Furthermore, TAC was reduced during the freeze-thaw process, which was insufficient in protecting the sperm against high reactive oxygen species levels.

Evaluation of Tris-citric acid, skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa.

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris-citric acid, skim milk and sodium citrate) at 37°C. Extended semen was cooled from 37°C to 5°C in 2h, and stored for three days at 5°C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p>0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p<0.05) in Tris-citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p<0.05) in Tris-citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris-citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n=18) were split into five aliquots for dilution (37°C; 50×10(6)spermatozoaml(-1)) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4°C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN(2) level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4h post-thawing (37°C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p<0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n=2) spermatozoa was higher (p<0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.

Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen.

This study was designed to compare the effect of straw size (0.25 vs. 0.5 ml) and thawing time (30 vs. 60 sec) on the quality of cryopreserved buffalo bull semen. Sperm motility, plasma membrane integrity and viability were higher (p ≤ 0.05) in 0.25 ml than 0.5 ml straw, thawed at 37°C either for 30 or 60 sec. In conclusion, cryopreservation of buffalo semen in 0.25 ml straw resulted in a higher post-thaw quality.

Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa.

This experiment was designed to compare powdered egg yolk with fresh egg yolk in an extender for cryopreservation of Zebu bull semen. Sperm motility, plasma membrane integrity and viability were assessed at different stages of cryopreservation (post-dilution, pre-freezing and post-thawing). Sperm plasma membrane integrity remained similar at all the stages of cryopreservation. Sperm motility and viability were significantly higher after thawing in the extender containing powdered egg yolk. In conclusion, powdered egg yolk may be used in an extender for the cryopreservation of Zebu bull spermatozoa.