Effect of glutathione on pre and post-freezing sperm quality of Indian red jungle fowl (Gallus gallus murghi).

During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules and insufficient natural antioxidant defence system. Therefore, present study was designed to identify the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi) sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen was divided in four aliquots, diluted with red fowl extender (1:5) at 37 °C having GSH 0 mM (control), 0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (-196 °C) in liquid nitrogen. Semen quality was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at 37 °C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling, equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scavenging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integrity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle fowl through enriching antioxidant potential and ameliorating the oxidative stress.

Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi).

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

Evaluation of α-linolenic acid for freezability and in vivo fertility of Nili Ravi (Bubalus bubalis) buffalo semen.

Alpha linolenic acid (ALA) is integral component of cell membrane that protects the cell in stressful events and involves in many metabolic pathways. It was hypothesized that ALA have the ability to protect the structural and functional integrity of buffalo spermatozoa during freeze-thawing. Therefore, study was designed to evaluate ALA supplementation (0, 5, 10 and 20 ng/mL) in extender on freezability and in vivo fertility of buffalo bull spermatozoa. Semen from three adult Nili-Ravi buffalo bulls of similar age was collected with artificial vagina (42 °C) for five weeks (replicates; N = 30). Qualified semen ejaculates (>1 mL volume, >60% motility; >0.5 billion/mL concentration) were diluted with tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/mL ALA at 37 °C and cryopreserved following established protocol. Sperm motility and plasma membrane integrity were recorded higher (P < 0.05) in extender containing 5.0 ng/mL of ALA compared to control. Nevertheless, sperm viability, live dead ratio and chromatin integrity were observed higher (P < 0.05) in all experimental extenders with ALA compared to control. The number of abnormal sperm reduced significantly in all experimental extenders having ALA. A total of 539 artificial inseminations were performed with the best evolved extender having ALA (5.0 ng/mL; 272 inseminations) and control (267 inseminations). In vivo fertility rates of buffalo semen were recorded higher (P < 0.05) with extender containing ALA (5.0 ng/mL) (58%) compared to control (46%). In conclusion, supplementing 5.0 ng/mL ALA in extender improved the post-thaw quality and in vivo fertility of cryopreserved Nili-Ravi buffalo bull semen.

Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study.

The reproductive potential of the adult males is expected to vary with age/season and largely differ not only in closely related avian species but even in subspecies, breeds and/or strains of the same species. Thus, it is pre-requisite to have knowledge of seminal parameters to achieve maximum production potential of at-risk species for ex situ in vitro conservation programs. A 4-year study was designed to evaluate the effect of age and season (spring, summer, autumn and winter) on semen characteristics of Indian red jungle fowl (Gallus gallus murghi) in a retrospective manner. Semen ejaculates (n = 1148) were regularly collected from eight adult cocks 6 to 54 months of age. Quantitative and qualitative semen parameters viz; volume (µL), concentration (1 × 109 mL-1), total sperm number per ejaculate (1 × 109 mL-1), motility (%), viability (%), plasma membrane integrity (%), acrosome integrity (%) and semen quality factor were recorded. A chronological increasing trend with age of most sperm quantitative and qualitative traits (semen volume, sperm concentration, total sperm number per ejaculate, plasma membrane integrity, viability, acrosomal integrity and semen quality factor) was observed. The highest values were observed at four years of age (P < 0.05) with the exception of sperm motility that was not affected by the age. Spring was the best season for sperm parameters viz; volume, motility, plasma membrane integrity, viability and acrosomal integrity (P < 0.05), however a remarkable sperm production was noticed all over the year. It is concluded that Indian red jungle fowl exhibits an evolution of sperm production that greatly differs in many points from other fowl sub-species. It is suggested that semen ejaculates of highest quality achieved for semen banking at the age of four year in the spring season.

Cryopreservation of Nili-Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation.

The study was designed to evaluate AndroMed® for the freezability and fertility of Nili-Ravi buffalo semen. Semen was collected from four adult Nili-Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 106 spermatozoa/ml) in tris-citric egg yolk or AndroMed® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post-thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris-citric egg yolk and AndroMed® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed® compared to tris-citric egg yolk post-thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen-thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris-citric egg yolk or AndroMed® extender (45.5% vs. 49%). It is concluded that AndroMed® is capable in protecting the buffalo bull sperm during freeze-thawing process and can be adopted safely for routine use replacing the tris-citric egg yolk extender in artificial insemination programme.