Alpha-tocopheryl Succinate in Extender Improves the Post Thaw Quality of Water Buffalo Spermatozoa.

BACKGROUND: Alpha-tocopheryl succinate, a major chain-splitting antioxidant, is the most effective form of vitamin E and may be used in the semen extender for cryopreservation of buffalo spermatozoa. OBJECTIVE: To use different concentrations of alpha-tocopheryl succinate (T1, 0.3 mM, T2, 0.6 mM, and T3, 0.9 mM) and control (0.0 mM) in extender for dose optimization and hence improve the frozen-thawed quality of water buffalo spermatozoa. MATERIALS AND METHODS: Semen samples were collected from three mature buffalo bulls with artificial vagina (42°C) and this study was replicated for five times. Semen was cryopreserved by conventional method which included filling of semen per experimental treatment into 0.5 mL French straws, sealing with polyvinyl alcohol powder and keeping them 5 cm above the liquid nitrogen vapors for 12 min and storing in liquid nitrogen tank. Frozen-thawed semen was also processed for total antioxidant capacity content (TAC) and lipid peroxidation (LPO) level by thiobarbituric acid (TBA). Computer-assisted semen analysis (CASA) and other assays were also performed. RESULTS: TAC levels were higher (P<0.05) with T2 and T3 as compared to T1 and control. LPO levels were lower (P<0.05) with T2 and T3 as compared to T1 and control. Sperm progressive motility (%) and rapid velocity (%) were higher (P<0.05) with T2 and T3 as compared to control. The extender containing T3 had higher (P<0.05) sperm average path velocity (µm/s) and straight line velocity (µm/s) as compared to control. At 1 and 2 h incubation period (37 °C) T2 and T3 in extenders had higher (P<0.05) progressive motility and rapid velocity compared to control. Sperm supra vital plasma membrane integrity (%), mitochondrial transmembrane potential (%), viable and intact acrosome (%) and DNA integrity (%) were higher (P<0.05) with T2 and T3 as compared to T1 and control, respectively. CONCLUSION: The supplementation of alpha-tocopheryl succinate in extender, either at 0.6 (T2) or 0.9 (T3) mM concentrations improves the post thaw quality of water buffalo spermatozoa by sustaining the TAC levels and keeping the LPO levels lower as compared to the control. It is suggested that future study should be aimed to explore the influence of these optimal concentrations of alpha-tocopheryl succinate on in vivo fertility of buffalo bull spermatozoa.

Effect of Cryopreservation on Casa Characteristics, Mitochondrial Transmembrane Potential, Plasma and Acrosome Integrities, Morphology and in vivo Fertility of Buffalo Bull Spermatozoa.

BACKGROUND: Application of frozen-thawed semen is an important tool for improving the vivo fertility, but the process of freezing and thawing causes significant damage to spermatozoa. OBJECTIVE: The aim of this study was to evaluate the effect of cryopreservation on CASA characteristics, mitochondrial transmembrane potential, plasma, and acrosome integrities, morphology and in vivo fertility of buffalo bull spermatozoa. MATERIALS AND METHODS: Semen was collected from four mature buffalo bulls with artificial vagina at 42 °C. Ejaculates having > 1 mL volume, > 60 % sperm visual motility and > 0.5 x 109 sperm/mL concentration from each bull were diluted in Tris-citric acid egg yolk glycerol extender (TCA) making two aliquots per bull for analysis at post dilution and cryopreserved respectively. RESULTS: Analysis of variance (ANOVA) showed that the process of freezing and thawing significantly reduced (P < 0.05) CASA characteristics including total motility (TM, %), progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), beat cross frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %). Furthermore, the process of freezing and thawing significantly reduced (P < 0.05) subjective motility (SM, %), Supra-vital plasma membrane integrity (SVPMI, %), high mitochondrial membrane potential (HMMP, %), viable spermatozoa with intact acrosome (V/IACR, %). Moreover, it was observed that the freezing thawing process significantly decreased the in vivo fertility (%, 50.35 % vs. 61.39 %; P < 0.05) as compared to post diluted semen. CONCLUSION: It is concluded that the process of freezing and thawing significantly reduced semen quality and in vivo fertility of buffalo bull in terms of various functional parameters.

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender.

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (µM/L) and lipid peroxidation levels (µM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, µm/s; straight-line velocity, µm/s; curved-line velocity, µm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender.

Programmable fast-freezing method improves the post-thaw motion dynamics, integrities of plasmalemma, mitochondrial transmembrane, DNA and, acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN2 ] for 10 min, plunging in LN2 ; FR2, programmable medium, +4°C to -15°C at 3°C min-1 , from -15 to -80°C at 10°C min-1 and final holding for 1 min at -80°C, plunging in LN2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to -20°C at 10°C min-1 , from -20°C to -100°C at 30°C min-1 , final holding for 1 min at -100°C and plunging in LN2 ) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (µm s-1 ), straight line velocity (µm s-1 ), curved line velocity (µm s-1 ), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa.

Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa.

The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2 ) for 10 min, plunging in LN2 ; FR2, programmable ultra-fast, holding at +4 °C for 2 min, from 4 to -10 °C at -10 °C/min, from -10 to -20 °C at -15 °C/min, from -20 to -120 °C at -60 °C/min, holding at -120 °C for 30 sec, plunging in LN2 ), and thawing rates (T1, 37 °C for 30 sec; T2, 50 °C for 15 sec; T3, 70 °C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, µm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa.

Variation in genotypic responses and biochemical analysis of callus induction in cultivated wheat.

Wheat is notorious for callus induction, which is a major hindrance in direct gene transfer and consequently for genetic improvement programs. In order to provide a successful platform for gene transformation, good callus quantity and quality is important. We investigated the variation in callus induction capabilities of Pakistani wheat cultivars and measured the reducing sugar content in the induced calluses. Ten elite wheat varieties, developed and cultivated in Pakistan were selected on the basis of agronomic and stress tolerance parameters. Significant differences were found between and among wheat cultivars for callus induction response, shoot length and callus quality. The callus induction responses of Punjab-81, Punjab-96 and Zarghoon-79 were found to be the best among the 10 varieties. The induced calluses were of two types, embryogenic (hard) and non-embryogenic (soft). The seeds gave good germination. The highest reducing sugar concentration was found in cultivar Sutlaj-86, which needs to be tested for stress resistance, a measure of its utility for genetic engineering programs. The relative callus induction rate and reducing sugar content of the wheat cultivars were found to be genotype-dependent.

Decolorization potential of mixed microbial consortia for reactive and disperse textile dyestuffs.

Four different aerobic mixed consortia collected from basins of wastewater streams coming out of dying plants of Crescent Textile (CT), Sitara Textile (ST), Chenab Fabrics (CF) and Noor Fatima Textile (NF), Faisalabad, Pakistan were applied for decolorization of Drimarene Orange K-GL, Drimarene Brilliant Red K-4BL, Foron Yellow SE4G and Foron Blue RDGLN for 10 days using the shake flask technique. CT culture showed the best decolorization potential on all dyestuffs followed by ST, NF and CF, respectively. CT could completely decolorize all dyes within 3-5 days. ST cultures showed effective decolorization potential on Foron Yellow SE4G and Drimarene Brilliant Red K-4BL but complete color removal was achieved after 4 and 7 days, respectively. NF culture showed 100% decolorization efficiencies on Foron Yellow SE4G and Foron Blue RDGLN but it took comparatively longer time periods (5-7 days). Where as, the NF culture had decolorized only 40% and 50% of Drimarene orange and red, respectively, after 10 days. CF caused complete decolorization of Foron Blue RDGLN and Drimarene Brilliant Red K-4BL after 4 and 8 days, respectively but it showed poor performance on other two dyes.