Effect of ascorbic acid on metabolic status, lipid peroxidation, antioxidant activity and quality of frozen Indian red jungle fowl (Gallus gallus murghi) semen.

The Indian red jungle fowl population is decreasing in its natural habitat. Its conservation through semen cryopreservation with sufficient live sperm recovery rate is requisite where ascorbic acid could play significant role to mitigate cryo-incited injuries. The objective was to elucidate the effect of ascorbic acid on freezability of Indian red jungle fowl sperm. Pooled semen was aliquoted and diluted (1:5) with red fowl extender having ascorbic acid: 0.0 (control), 1.0, 2.0 and 4.0 mM. Diluted samples were cryopreserved and semen quality was assessed at post-dilution, cooling, equilibration and freeze-thawing stages. Sperm metabolic status, antioxidant potential and lipid peroxidation were studied at post-dilution and freeze-thawing. Sperm motility did not differ (p > .05) in experimental extenders and control at post-dilution and cooling; however, it was recorded higher (p < .05) with ascorbic acid at 2.0 mM compared with other levels at post-equilibration and post-thawing stage. Sperm viability, plasma membrane and acrosome intactness were recorded higher (p < .05) with 2.0 mM ascorbic acid compared with other concentrations of ascorbic acid at all stages of cryopreservation. Sperm metabolic status and antioxidant potential were recorded higher (p < .05), while lipid peroxidation was recorded lowest (p < .05) with 2.0 mM ascorbic acid compared with 1.0, 4.0 mM and control. In conclusion, ascorbic acid at 2.0 mM in red fowl extender improves quality, metabolic status and antioxidant potential of frozen Indian red jungle fowl semen through ameliorating lipid peroxidation.

Sperm binding to hyaluronan is an excellent predictor of Nili-Ravi buffalo bull fertility.

This study reports the first evaluation of sperm hyaluronan binding assay (HBA) for predicting the fertility of Nili-Ravi buffalo bulls in relation to standard parameters of sperm quality. Cryopreserved semen doses of low (n = 6), medium (n = 3) and high fertility (n = 8) bulls based on their respective return rates were used. Significantly, more spermatozoa bound to hyaluronan from the most fertile bulls (57.15% ± 1.44) compared with medium (42.46% ± 1.08) and low fertility bulls (29.70% ± 0.78). A strongly positive correlation (r = .824, p < .01) was found between HBA and fertility that predicts a 67.9% variability (r2  = .679, p < .01) in fertility. HBA was also strongly positively correlated with sperm viability (r = .679, p < .01) followed by their live/dead ratio (r = .637, p < .01), uncapacitated spermatozoa (r = .631, p < .01), normal apical ridge (r = .459, p < .01), motility (r = .434, p < .01), mature spermatozoa with low residual histones (r = .364, p < .01), high plasma membrane integrity (r = .316, p < .01) and nonfragmented DNA levels (r = .236, p < .05). It was negatively correlated with spermatozoa having reacted acrosome (r = -.654, p < .01). A fertility model built using a combination of sperm HBA and either sperm livability or viability predicts, respectively, 86.1% (r2  = .861, p < .01) and 85.9% (r2  = .859, p < .01) variability in buffalo bull fertility. In conclusion, sperm HBA may prove to be a single robust predictor of Nili-Ravi buffalo bull fertility.

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone.

The Indian red jungle fowl is a sub-species of the genus Gallus native to South Asia; facing high risk of extinction in its native habitat. During cryopreservation, permeable cryoprotectants like glycerol are usually employed and we previously showed encouraging results with 20% glycerol. Because bird spermatozoa contain very little intracellular water, the possibility of replacing an internal cryoprotectant by an external one is opened. In the present study, we tested the replacement of internal cryoprotectant glycerol by the external cryoprotectant Polyvinylpyrrolidone (PVP). PVP is a non-permeable cryoprotectant and keeps the sperm in glassy state both in cooling and warming stages without making ice crystallization within the sperm cell. We evaluated the effect of various levels of polyvinylpyrrolidone (PVP) on Indian red jungle fowl semen quality and fertility outcomes. The qualifying semen ejaculates collected from eight mature cocks were pooled, divided into five aliquots, diluted (37 °C) with red fowl semen extender having PVP [0% (control) 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v)]. Diluted semen was cryopreserved and stored in liquid nitrogen. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) with 6% PVP at post-dilution, cooling, equilibration and freeze-thawing. Higher (P < 0.05) no. of fertile eggs, fertility, no. of hatched chicks, percent hatch and hatchability was recorded with 6% PVP compared to control. It is concluded that 6% PVP maintained better post-taw quality and fertility of Indian red jungle fowl spermatozoa than glycerol and can be used in routine practice avoiding the contraceptive effects of glycerol.

Honey as an Alternative to Antibiotics for Cryopreservation of Nili-Ravi Buffalo Bull Spermatozoa.

The antimicrobial properties of honey have stimulated interest in evaluating it as an alternative to antibiotics for cryopreserved buffalo semen. Acacia nilotica, Brassica campestris and Ziziphus jujuba honey were analyzed and Z. jujuba honey was found suitable in terms of quality and purity. Buffalo semen (24 ejaculates) was studied for in vitro dose tolerability to Z. jujuba honey (0.1%-1%), and up to 0.2% (v/v) was not toxic to buffalo spermatozoa. Afterward, semen from three bulls (24 ejaculates) was cryopreserved (four replicates) in tris-citric egg yolk extender supplemented with 0.1% or 0.2% honey, with or without streptomycin-penicillin (SP); extender with SP used as a control. After dilution and cooling, extender without antibiotics but with 0.2% honey was better (p < 0.05) than control in terms of sperm motility and plasma membrane integrity. After thawing, the extenders containing 0.1% honey with antibiotics and extender having 0.2% honey without antibiotics consistently yielded good results in terms of all parameters studied compared to control and other extenders. The extender containing 0.2% honey without antibiotics was better (p < 0.05) in terms of total aerobic bacterial count. In conclusion, 0.2% honey improves the post-thaw quality of buffalo spermatozoa and can replace the use of antibiotics in extender through its antimicrobial activity.

Effect of Synergism Between Carboxylated Poly-l-Lysine and Glycerol on Freezability of Nili-Ravi Buffalo (Bubalus bubalis) Semen.

Carboxylated poly-l-lysine (CPLL), an ampholytic polymer, has remarkable cryoprotective properties. It was hypothesized that CPLL will reduce/replace glycerol in extender and improve the freezability of buffalo semen. The objective was to evaluate various combinations of CPLL and glycerol in extenders for any synergism toward cryosurvivability of Nili-Ravi buffalo sperm. Semen collected from four Nili-Ravi buffalo bulls (ejaculates = 40; replicates = 5) was diluted in tris-citric acid based extenders, Control: (0% CPLL +7% Glycerol); E1: (1% CPLL +6% Glycerol); E2: (2% CPLL +5% Glycerol); E3: (3% CPLL +4% Glycerol); E4: (4% CPLL +3% Glycerol); E5: (5% CPLL +2% Glycerol); E6: (6% CPLL +1% Glycerol), and E7: (7% CPLL +0% Glycerol), and cryopreserved using a programmable cell freezer. Percentages of post-thaw sperm motility, plasma membrane integrity, and acrosomal integrity were found to be higher (p < 0.05) in extenders E1, E2, E3, E4, and E5 compared to E6 and the control. Sperm livability (%; live/dead ratio) and viability (%; live sperm with intact acrosome) were higher (p < 0.05) in extender E4 compared to all the other extenders. Sperm DNA integrity was higher (p < 0.05) in extender E2, E3, E4, and E5 compared to control, E1, and E6 extenders. Sperm lipid peroxidation levels were lower (p < 0.05) in E3, E4, E5, and E6 compared to control, E1, E2, and E7 extenders. Total antioxidant capacity of seminal plasma was higher (p < 0.05) in extenders E5, E6, and E7 than control, E1, E2, E3, and E4 extenders. It is concluded that synergism between CPLL and glycerol (4% CPPL and 3% Glycerol) seems to improve the freezability of Nili-Ravi buffalo semen by reducing oxidative stress.

A Hybrid Approach for Modeling Type 2 Diabetes Mellitus Progression.

Type 2 Diabetes Mellitus (T2DM) is a chronic, progressive metabolic disorder characterized by hyperglycemia resulting from abnormalities in insulin secretion, insulin action, or both. It is associated with an increased risk of developing vascular complication of micro as well as macro nature. Because of its inconspicuous and heterogeneous character, the management of T2DM is very complex. Modeling physiological processes over time demonstrating the patient's evolving health condition is imperative to comprehending the patient's current status of health, projecting its likely dynamics and assessing the requisite care and treatment measures in future. Hidden Markov Model (HMM) is an effective approach for such prognostic modeling. However, the nature of the clinical setting, together with the format of the Electronic Medical Records (EMRs) data, in particular the sparse and irregularly sampled clinical data which is well understood to present significant challenges, has confounded standard HMM. In the present study, we proposed an approximation technique based on Newton's Divided Difference Method (NDDM) as a component with HMM to determine the risk of developing diabetes in an individual over different time horizons using irregular and sparsely sampled EMRs data. The proposed method is capable of exploiting available sequences of clinical measurements obtained from a longitudinal sample of patients for effective imputation and improved prediction performance. Furthermore, results demonstrated that the discrimination capability of our proposed method, in prognosticating diabetes risk, is superior to the standard HMM.

Sperm sexing in Nili-Ravi buffalo through modified swim up: Validation using SYBR® green real-time PCR.

Sperm sexing through flow-sorting technology is relatively expensive, requires considerable technical support and is actually not practicable in many developing countries. The aim of this study was to investigate the feasibility of producing enriched pools of X or Y chromosome-bearing sperm by a modified swim-up method. For this purpose semen was collected from five mature Nili-Ravi buffalo bulls for a period of six weeks. The qualifying ejaculates were divided into two aliquots for further processing through modified swim-up or control (untreated). After processing, semen was cryopreserved in tris citric acid extender using standard techniques. Semen quality was assessed at pre dilution, post dilution and post thawing. Validation of technique was done by using SYBR® green real time PCR using two sets of primers, PLP and SRY for X and Y chromosome of buffalo genes, respectively. Sperm recovery rates, pre freeze and post thaw sperm quality were found significantly higher in X chromosome bearing sperm fraction than Y chromosome bearing fraction and control. Mean fold relative expression of X bearing sperm was significantly higher (4-5 fold) in X chromosome bearing fraction of supernatant than Y chromosome bearing fraction (0.06 fold), similarly mean fold relative expression of Y chromosome bearing sperm was significantly higher in Y chromosome bearing fraction (4 fold) of supernatant than X chromosome bearing fraction (0.15 fold) compared to control (1.00). In conclusion, a modified swim up method proved to be an effective method for Nili-Ravi buffalo sperm sexing as validated by real time PCR.

A comparative analysis of sperm selection procedures prior to cryopreservation for Nili-Ravi buffalo bull (Bubalus bubalis) semen-: Assessment of its impact on post-thaw sperm functional quality.

Sperm selection techniques have been developed to get sperm suspensions enriched in motile and functional cells. Studies show that selection before cryopreservation improves post-thaw quality of cryopreserved sperm but information on buffalo bull sperm is scarce. The study was aimed to 1) perform a comparative analysis of sperm selection procedures; Swim-Up (SU), Sephadex™-G15 Filtration (S-G15) or Glass Wool Filtration (GWF) for total and motile cell recovery, 2) to assess the impact of sperm selection prior to cryopreservation on sperm quality (motility, morphology, cell membrane and normal apical ridge, viability and livability, chromatin integrity) and sperm functionality (Embryo Cleavage after IVF with selected sperm) in post-thawed suspensions of buffalo bull sperm. Semen was collected from 5 Nili Ravi buffalo bulls maintained at the Semen Production Unit Qadirabad, District Sahiwal, Pakistan. Ejaculates were divided into four aliquots for SU, S-G15 and GWF and control. After sperm selection, total and motile sperm recovery was highest in GWF samples (total sperm=84.08±8.39%; motile sperm=80.42±3.57%). An improvement (P<0.05) in all post-thaw parameters was observed in S-G15-selected sperm and, in some parameters in GWF-filtered sperm suspensions compared to control. The highest (P<0.05) embryo cleavage rate (%) was achieved with frozen-thawed sperm selected with S-G15 prior to cryopreservation (44.72±4.18) compared to control (21.98±3.00). In conclusion, post thaw sperm quality was improved after sperm selection from fresh buffalo bull semen through S-G15 and GWF procedures compared to SU and control while, the fertility rate (cleavage rate) was improved with sperm processed using the S-G15 procedure.