Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm.

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0-5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0-5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (-0.0002 ± 0.07 vs. -0.266 ± 0.09), kurharm0 (-0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0-5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. -0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

Evaluation of carboxylated poly-l-lysine for cryopreservation of Labeo rohita sperm.

Carboxylated poly-l-lysine (CPLL) is an anti-freeze agent having pronounced non-permeating yet membrane stabilizing cryoprotective capabilities. The objective was to evaluate the CPLL supplementation in extender in terms of post-thaw quality (sperm), total anti-oxidant activity (milt) and fertilization potential of cryopreserved Labeo rohita sperm. For this purpose, male brood fish reared at a fish seed hatchery, Rawal Town Islamabad, Pakistan were captured from different rearing ponds and acclimatized in hatchery ponds for 6 h. The brooder was injected with Ovaprim (0.2 mL/kg), and milt was collected after 8 h in cooled sterilized falcon tubes, maintained at 4°C and evaluated for sperm motility. The milt collected from three brooders (n = 3) was diluted in extenders viz., modified Kurokura-2 extender having 10% methanol (control); experimental extenders with CPLL supplementation at the rate of 0.5%, 1% and 1.5%. Diluted milt was filled in 0.5 mL straws, exposed to liquid nitrogen vapours and cryopreserved. Cryopreserved milt was thawed at 25°C and assessed for post-thaw sperm quality. Sperm motility, motility duration, viability, total anti-oxidant capacity and DNA integrity was significantly higher (p < 0.05) in the extender having 1.5% CPLL than control. To evaluate the fertilization rates, male and female brooders were injected with Ovaprim at 0.2 mL/Kg and 0.5 mL/Kg body weight respectively. Fresh eggs and milt were collected through abdominal stripping. Batches of 10 g of eggs from each female (n = 2) were fertilized with one straw, each from frozen sperm with KE + methanol (control), KE + methanol + 1.5% CPLL and 50 µL fresh milt (negative control). After 1.5 h of fertilization, eggs were collected from all jars and a total of 200 eggs were counted. The fertilized eggs appeared clear and transparent while unfertilized eggs looked opaque with disintegrated nuclei. Sperm fertilization rate (%) was higher (p < 0.05) in extender KE + methanol + 1.5% CPLL (78.7 ± 0.5) compared to control (KE + methanol) (52.0 ± 0.4) however, it was lower compared to that of negative control, the fresh milt (85.2 ± 0.6). In conclusion, supplementation of carboxylated poly-l-lysine (1.5%) to modified Kurokura-2 extender having 10% methanol improves post-thaw motility, motility duration, viability, DNA integrity, anti-oxidant capacity (milt) and fertilizing ability of cryopreserved L. rohita sperm.

Antioxidant effect of Moringa olifera leaves extract in extender improves post-thaw quality, kinematics, lipid peroxidation, total antioxidant capacity and fertility of water buffalo bull semen.

Our objectives were to determine the antioxidant effect of Moringa olifera leave extract (MLE) in tris extender on post-thaw quality, kinematics, lipid peroxidation, total antioxidant capacity and field fertility of water buffalo bull semen. Semen was collected on a weekly basis for 5 weeks (n = 5 bulls & n = 25). After collection, every sample was diluted individually with extender (tris egg yolk) containing MLE concentrations (0%, 5%, 10%, 15% and 20%) at 37°C. After dilution, semen was filled in 0.54 mL straws and frozen in automatic freezer. After thawing, sperm motility (%) (progressive & total), path velocity (µm/s), straight velocity (µm/s), curvilinear velocity (%), beat cross frequency (Hz), linearity (%), straightness (%), length of average path (µm), length of straight-line path (µm), length of curvilinear path (µm), plasma membrane integrity (%), acrosome membrane integrity (%), DNA integrity (%) and morphology (%) were higher (p < 0.05) in spermatozoa preserved in extender containing 15% MLE as compared to control. The fertility rate (60%, vs. 45%) was higher (p < 0.05) with semen doses frozen with 15% MLE in extender than the control. In conclusion, 15% MLE improves semen quality, kinematics and field fertility in water buffaloes.

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.

Asphaltum Improves the Post-thaw Quality and Antioxidant Status of Nili Ravi Buffalo Bull Sperm.

Asphaltum, a mineral exudate from the mountains, is an ayurvedic medicine believed to be a panacea for male reproductive health issues. The objective of the study was to evaluate asphaltum in terms of phytochemical components, radical scavenging activity (RSA), in vitro dose tolerability, and cryosurvivability of buffalo sperm. Asphaltum was procured from an authentic source and confirmed for the presence of flavonoids, terpenoids, saponins, tannins, alkaloids, steroids, and glycosides. It showed good RSA as confirmed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. In vitro dose tolerability of buffalo sperm (n = 3, replicate = 4, ejaculates = 24) for asphaltum was assessed at 0.75%, 1.5%, 2.25%, 3.0%, 3.75%, 4.5%, 5.25%, and 6.0% (w/v). Buffalo sperm showed good tolerance up to 3% of asphaltum in terms of sperm progressive motility and plasma membrane integrity. Buffalo semen (n = 3, replicates = 4, ejaculates = 24) was cryopreserved in extender supplemented with 0.0%, 0.75%, 1.5%, 2.25%, and 3.0% (w/v) asphaltum and sperm quality was assessed at post-dilution, post-cooling, and post-thaw. After dilution motility, viability and livability; post-cooling motility and plasma membrane integrity; and post-thaw motility, plasma membrane integrity, viability, livability, DNA integrity, sperm RSA, sperm total lipids, sperm mitochondrial activity, and total antioxidant activity of semen were improved by 3%. In conclusion, asphaltum supplementation in an extender at 3% improves the post-thaw quality and antioxidant activity of buffalo semen.

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.

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.

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.