Sodium dodecyl sulphate, N-octyl ß-D glucopyranoside and 4-methoxy phenyl ß-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls.

Sodium Dodecyl Sulphate (SDS), N-Octyl ß-D Glucopyranoside (NOG), 4-Methoxy Phenyl ß-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -40 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); ii) Moderate P25 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -50 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C); and iii) Rapid P26 (freezing rate of -30 °C min-1 from 4 °C to -15 °C; -60 °C min-1 from -15 °C to -60 °C; and -50 °C min-1 from -60 °C to -140 °C; and then plunged in liquid Nitrogen at -196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls.

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml-1. Freezing of semen samples was conducted at 30 °C min-1 from temperature drop from 4 °C to -15 °C and -15 °C to - 60 °C followed by 50 °C min-1 from - 60 °C to -140 °C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml-1) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 °C min-1[Protocol (PRT) I], 40 °C min-1 (PRT II) and 50 °C min-1 (PRT III) in the critical temperature fall zone of -15 °C to -60 °C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml-1 in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml-1 as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml-1 as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml-1, resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 °C min-1 in the critical temperature fall zone of -15 to -60 °C perform better than the freezing rate of 30 °C min-1. It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml-1 improved post-thaw semen quality of cattle and buffalo.

Unraveling the molecular dependence of femtosecond laser-induced thermal lens spectroscopy in fluids.

Fluid systems exhibit thermal lens effects due to laser irradiation accompanied by convection and in contrast primarily conductive heat dissipation is observed in solids. The presence of a significant convective mode modifies the temperature gradient in fluids resulting in a deviation of the experimental results from theories that are based on pure conduction. Herein, we present a carefully designed femtosecond laser experiment that keeps the heat generation process constant in order to account for the effect of molecular properties on thermal dissipation. We derive a theoretical model that introduces and characterizes the additional convective heat transfer in thermal lens (TL) spectroscopy which explains our observed experimental results. We measured the TL signal for a series of liquid aliphatic alkanes, ranging from hexane to decane, and their comparison has proven the validity of our model. The influence of convective heat transfer on the TL signal is predicted in terms of the dimensionless Peclet number (PE). The lower values of PE for alkanes with longer carbon chains indicate that the convective flow of heat slows down substantially for larger molecules.

Expression pattern of GLUT 1, 5, 8 and citrate synthase transcripts in buffalo (Bubalus bubalis) preimplantation embryos produced in vitro and derived in vivo.

In vitro-produced (IVP) embryos are reported to be developmentally lesser competent than in vivo-derived (IVD) embryos and supposed to differ in the expression of genes related with glucose metabolism. So, the present study was conducted to analyse the expression pattern of GLUT 1, 5, 8 and citrate synthase (CS) in oocytes and embryos produced in vivo or in vitro in buffalo. IVD embryos were obtained from 18 superovulated buffaloes. IVP embryos were obtained from slaughterhouse-derived oocytes subsequently subjected to in vitro fertilization and culture. Total RNA was isolated from different stages of oocytes (immature and in vitro matured) and embryos (8-16 cell to blastocysts of IVP embryos and morula to blastocysts of IVD embryos). Results demonstrated that the expression of GLUT1, GLUT 8 increased from 8 to 16 cells to blastocyst and was significantly (p < .05) higher in IVP embryos. Expression of both genes was (p < .05) higher in IVD than in IVP blastocysts; though GLUT5 transcripts were not detected at 8- to 16-cell stage IVP embryos, significantly (p < .05) higher transcripts were found at morula and blastocyst stages irrespective of embryo source with significantly (p < .05) higher expression in IVD embryos compared to IVP embryos. No significant difference was observed in citrate synthase expression in embryos at morula stage irrespective of the embryo source while significantly (p < .05) higher transcript level was observed at blastocyst stage with no difference between in vivo and in vitro embryos. It can be concluded that expression of GLUTs and CS is upregulated with progression of embryonic stage and expression is higher in in vivo embryos than in vitro counter parts; thus, it can be said that in vivo-produced embryos are metabolically superior to in vitro embryos.

Measurement of pure optical nonlinearity in carbon disulfide with a high-repetition-rate femtosecond laser.

Using the close-aperture Z-scan technique, the pure nonlinear refractive index (n2) of carbon disulfide is measured with a 76 MHz repetition rate femtosecond laser. Strong interference of thermal effects exists with high-repetition-rate lasers that result in negative values of n2. We remove the thermal effect completely by continuously increasing the sample flow rate (F) in a sample cell as indicated by the change in sign of n2 from negative to positive. The positive value of n2 is due to Kerr-type nonlinearity. At sufficiently high values of F of >25 ml/min, all thermal effects are removed, resulting in an n2 value that matches low-repetition-rate experiments.

Thermal Lens Study of NIR Femtosecond Laser-Induced Convection in Alcohols.

We use time-resolved thermal lens (TL) experiments to examine the convective heat transfer at microscale in the first eight members of the homologous series of primary alcohols. TL measurements enable a direct study of these primary alcohols without adding any chromophore as a function of varying heat loads created via femtosecond laser pulses at 1560 nm. Convective heat transfer leads to the asymmetrical and reduced thermal gradient, which substantially weakens the TL signal. The inflection in the time profile of the TL signal of methanol at higher powers is attributed to the greater molecular convection in methanol compared to other samples. This inflection dies out with a decrease in laser power. Our results demonstrate that the convection is more prominent at higher laser powers in all samples, and it modifies the trend in the steady-state TL signal of different alcohols with pump laser power. Methanol also has the highest steady-state TL among the primary alcohol series at low laser powers. The maxima in the TL signal are shifted systematically from methanol to ethanol and then to propanol as the laser power increases. Semiempirical analysis of time-resolved TL signal by using the latest theoretical TL model enabled us to extract the coefficient of convective heat transfer in methanol at different laser powers. In addition to that, analysis of other members of alcohol series at the highest (7.3 mW) laser power shows that convection is more facile in short-chain alcohols compared to the long-chain alcohols.

Isolation and characterization of oviduct-specific glycoproteins from ampulla and isthmus parts of cyclic and acyclic buffalo for studying differential microenvironment.

The present study characterized the glycoproteins synthesized by buffalo oviduct. Scanning electron microscopy analyses of the ampullary and isthmic segments of cyclic and acyclic buffaloes showed ultrastructural variations in ciliated and nonciliated cells. Mucosal proteins were extracted by scrapping of different segments of oviduct and, after centrifugation, the remainder tissues were subjected to establish primary cell culture system of oviduct epithelial cells and conditioned media were prepared. Time- and concentration-dependent effects of trypsinization on the establishment of primary monolayer culture showed that 0.25% trypsin for 1-2 min at 37 °C were the optimal conditions. Total protein content in oviductal tissues and conditioned media was quantified and analyzed by SDS-PAGE which showed marked variation in different segments of the oviduct. Western blot analysis revealed five major oviduct-specific glycoproteins (OGPs) in cyclic oviduct (ampulla and isthmus) with Mw 180, 95, 75, 66 and 35 kDa in the oviduct extract and two glycoproteins with Mw 95 and 66 kDa in conditioned media. However, in acyclic oviduct (ampulla and isthmus), three glycoproteins were immunostained with Mw 180, 95 and 66 kDa in the oviduct extract and one glycoprotein with Mw 66 kDa in conditioned media. Indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) results showed significant differences of OGPs in different segments of cyclic and acyclic buffaloes and, thus, indicative of segmental variation in the synthesis and secretion of glycoproteins. Oviductal extract secretes more amounts of OGPs as compared to the conditioned medium. The role of these OGPs may be defined and exploited for influencing the fertilization process and/or subsequent embryonic development.

Effect of including growth factors and antioxidants in maturation medium used for in vitro culture of buffalo oocytes recovered in vivo.

This study examined the effect of including one of two growth factors (100 ng/ml IGF-1 or 20 ng/ml EGF) in combination with one of two antioxidants (50 microM cysteamine or 50 microM beta-mercaptoethanol) in maturation, fertilization and subsequent development of buffalo oocytes. The oocytes were recovered by in vivo ovum pick-up technique from six Murrah buffalo heifers twice a week over a period of 16 weeks. Immediately after ovum pick-up oocytes recovered from six donors were allocated randomly to five different maturation treatments. The control treatment was the basic maturation medium (MM; TCM-199 supplemented with 10% FBS, 10 IU/ml LH, 0.5 microg/ml FSH, 1 microg/ml estradiol-17beta and 50 microg/ml gentamicin). The other four treatments consisted of the control maturation medium (MM) plus one combination of a growth factor and an antioxidant viz. IGF-1+cysteamine; IGF-1+beta-ME; EGF+cysteamine or EGF+beta-ME. The total number of oocytes assigned to each maturation treatment ranged from 31 to 66. After maturation in different maturation medium, media used for in vitro fertilization and subsequent development of embryo was same for all groups. Data were analysed using Chi-square test. The maturation rate observed for the growth factor plus antioxidant treatments was similar to that for the control (90.4%). The highest cleavage rate recorded in the IGF-1+cysteamine treatment (71.9%) which was significantly higher (P<0.05) than the IGF-1+beta-ME (45.2%) and EGF+beta-ME (46.4%) treatments, but not significantly differ from the control (63.8%) and EGF+cysteamine treatment (60.7%). The proportion of cleaved oocytes those developed to blastocyst stage was significantly higher in the IGF-1+cysteamine treatment (52.2%; P<0.05) than in the control (23.3%), the EGF+cysteamine (13.5%) or the EGF+beta-ME (7.7%) treatments, but did not differ significantly from the IGF-1+beta-ME (28.6%) treatment. Following non-surgical transfer of 15 embryos to 14 synchronized recipients, four became pregnant and only one recipient sustained the pregnancy as long as 4.5 months when spontaneous abortion occurred. It was concluded that supplementing the maturation medium with IGF-1+cysteamine improved the production of buffalo embryos significantly in vitro culture.