Oestrogen induction of riboflavin-binding protein in immature chicks. Nature of the secretory protein.

The riboflavin-binding protein isolated from sera of oestrogen-treated male chicks as well as that synthesized and secreted in vitro by the chicken liver have the same molecular size as that of the egg-yolk protein. Functionally the serum and yolk proteins are similar. This is in contrast with the hormone-induced synthesis, secretion and deposition of phosvitin and lipovitellin in the ovary.

Rearing of Clarias batrachus (Lin.) larvae with formulated diets

An experiment was carried out for a period of 20 days using 7-day old Clarias batrachus larvae of initial total length of 7.4 ± 0.49 mm and weight of 2.9 ± 0.83 mg. Three artificial diets were used for the study having three replication of each. Among these, diet-I was formulated using 20% fish meal (FM), 30% powdered milk and 30% boiled egg yolk (BEY), diet-II using 27% FM, 20% Baker’s yeast (BY), 30% BEY and 3% agar and diet-III using 20% FM, 20% BY and 45% whole egg. The larvae fed on diets-II and III showed significantly (P<0.05) better length and weight gain than those of the larvae fed on diet-I. The larvae fed on diet- III showed the best survival rate (70%). However, the condition factor of the larvae fed on diet I was significantly better than those of the larvae fed on other two diets. The results of the study showed that C. batrachus larvae could be successfully reared with diet containing 45% whole egg, 20% yeast and 20% fish meal.

Cryopreservation of sperm of common carp, Cyprinus carpio and silver barb, Barbonymus gonionotus for genetically improved seed production

Experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of common carp, Cyprinus carpio and also for using the cryopreserved sperm for fertilization of eggs. Nine extender solutions as Alsever's solution, kurokura-1, kurokura-2, urea egg-yolk, egg-yolk citrate, 0.6% glucose, 0.9% NaCl, Ma and Mb, and five cryoprotectants namely ethanol, methanol, dimethylsulfoxide (DMSO), dimethylamine (DMA) and glycerol were tested. The cryoprotectants were mixed at 10% concentration of the extenders (v/v) to make the cryodiluents. Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever's solution, kurokura-1, kurokura-2, 0.6% glucose and 0.9% NaCl, 1:4 for urea egg-yolk, egg-yolk citrate, Ma and Mb. Among the cryodiluents Alsever's solution mixed with either ethanol or methanol was found to be suitable and it produced more than 90% and 80% spermatozoan motility at equilibrium and post-thaw periods, respectively. Kurokura-1 and kurokura-2 when mixed with the same cryoprotectants showed good spermatozoan motility at equilibrium period (80-90%) but the motility was reduced (30-55%) at post-thaw state. Other extenders did not produce acceptable sperm-motility and in some cases the frozen milt became clotted. Different dilution ratios (1:1, 1:2, 1:4, 1:5, 1:7, 1:9, 1:12, 1:15, 1:20) were formulated for obtaining a suitable milt dilution, the dilution ratio of 1: 9 (milt : cryodiluent) demonstrated the highest post-thaw spermatozoan motility (80%) in Alserver's solution. The optimum concentration of cryoprotectants in the cryodiluents was determined, 10% concentration level was found to be effective to produce the highest number of spermatozoan motility in comparison to the other concentrations (5%, 15%, 20% 30%). Sperm preserved with the cryodiluent Alsever's solution along with either methanol or ethanol was found to be effective to fertilize eggs and produce hatchlings. The hatching rates ranged between 1.48% and 14.76%, compare to control. The fish produced through use of cryopreserved sperm and normal sperm were found to grow well and no significant (P<0.05) growth difference was observed between them. In case of silver barb, Barbonymus gonionotus, sperm tested against six extenders such as egg-yolk citrate, urea-egg-yolk, kurokura-1, kurokura-2, 0.9% NaCl and modified fish ringer (MFR) solution. Cryoprotectants used were the same as those of C. carpio. Milt was diluted with the cryodiluent at a ratio of 1:4 for egg-yolk citrate and urea-egg-yolk, 1:5 for kurokura-1 and 1:9 for 0.9% NaCl, MFR and kurokura-2. The cryoprotectant concentration was maintained at 10% of the extender (v/v) in all the cases. Among the extenders, egg-yolk citrate and urea-egg-yolk mixed with 10% DMSO, methanol and ethanol produced 50% post-thaw spermatozoan motility, whereas DMA and glycerol provided only 10% motility. Trials on milt dilution ratio and cryoprotectant concentration are being conducted. Fertilization trials are also underway.

Sperm cryopreservation of Indian major carp, Labeo rohita: cryodiluents, sperm: cryodiluent dilution ratio and cryoprotectan t concentration

Cryogenic preservation trials of spermatozoa of Labeo rohita were carried out. Twenty four cryodiluents (extender + cryoprotectant), with the combination of six extenders such as egg-yolk citrate, urea-egg-yolk, 0.9% NaCl, Kurokura-2, Ma and Mb and four cryoprotectants viz. DMSO, glycerol, methanol and ethanol, were used to screen out the suitable cryodiluents. Sperm was preserved in 0.25ml plastic straw in programmable freezer. Two step freezing method was followed. Sperm preserved with egg-yolk citrate and urea-egg-yolk containing 10% DMSO showed best post-thaw motility (80%) followed by 0.9% NaCl (60%) and Kurokura-2(30%) solutions. Sperm with the extenders M" and Mb clotted at the time of equilibration and also after few days of preservation. Egg-yolk citrate mixed with ethanol and methanol also showed good percentage of motility (80%) but egg-yolk citrate with glycerol showed less sperm motility (>60%). To determine suitable dilution ratio of milt and cryodiluent two best extender eggyolk citrate and urea-egg-yolk with four cryoprotectants such as DMSO, glycerol, methanol and ethanol at different ratio viz 1:2,1:4,1:7,1:10,1:15 and 1:20 were used. Highest post-thaw motility (>80%) was observed when milt was preserved with egg-yolk citrate containing 10% DMSO at 1:2, 1:4, 1:7 and 1:10 dilutions. Meanwhile using glycerol as cryoprotectants provided less post thaw motility at lower dilution ratio but with the increase of its dilution showed good sperm motility compared with other cryoprotectants. Finally, evaluation on the effect of cryoprotectant concentration on post-thaw sperm motility was conducted. Egg-yolk citrate and four cryoprotectant i.e. DMSO, glycerol, methanol and ethanol with six different concentrations namely 5%,7%, 10%, 15%, 20% and 30%.were evaluated. Among the cryoprotectants DMSO, methanol and ethanol showed highest post-thaw motility (about 80%) at 7% and 10% concentrations. Although glycerol was not suitable at low concentration but its 20% and 30% concentration levels provided best post-thaw motility. No post-thaw motility was obtained with DMSO at 30% concentration. The overall analysis on cryoprotectant concentration indicated that below 5% and above 20% cryoprotectant concentrations could not be suitable for effective cryopreservation of spermatozoa.

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)) called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 mumol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 mug/ml enzyme was incubated for 90 min in the presence of PC and Ca2+. No detectable hemolysis was noticed when PC was not added. Ca2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC50 of 1.0 muM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D49 PLA(2)s. Also, the residues concerned to Ca2+ binding were conserved. This is the first report of cDNA sequence of T jerdonii venom PLA(2). (C) 2002 Elsevier Science Ltd. All rights reserved.

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender

Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. Methods: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris (mTTE) synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1%,3%, 5%, 10% and 15% [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. Results: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P < 0.05) for 5% glycerol (42.95 +/- 2.55 and 50.39 +/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%: 15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration (42.95 2.55 and 50.39 2.42) were better (P < 0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38. 98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Conclusion: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct Simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 mu g g(-1) DW. MCs reached 235 mu g g(-1) DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 mu g g(-1) DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g(-1) DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g(-1) DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g(-1) DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g(-1) DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife. (C) 2009 Elsevier B.V. All rights reserved.

Effects of adrenergic agonists on the extrahepatic expression of vitellogenin Ao1 in heart and brain of the Chinese rare minnow (Gobiocypris rarus)

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins and traditionally considered as the estrogen responsive precursors of the major egg yolk proteins. We identified five clones encoding VTGs, about 16% of the random EST clones from our constructed cDNA library from Chinese rare minnow liver tissue treated with 17 beta-estradiol (E2). Full-length vtgAo1 has been obtained based on the sequence information of four partial cDNA inserts by RACE. The inducibility of the vtgAo1 expression in liver by E2 was confirmed by RT-PCR. The presence of vtgAo1 transcripts have been observed primarily in liver. However. a significant level of the vtgAo1 was found in an unexpected location, heart, particularly in atrial cells by RT-PCR and whole mount in situ hybridization analyses. The vtgAo1 mRNA expression in heart and liver tissue could be suppressed by both alpha-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). The expression of VTG in the heart observed in the present studies suggested it may provide protection from surplus intracellular lipids in fish cardiomyocytes as triglyceride transport proteins do in mammals. The results also indicated that the production of teleost vtg in vivo can be regulated by riot only estrogenic agents, but adrenergic signals as well. (c) 2008 Elsevier B.V. All rights reserved.

Appropriateness of Largemouth Bass as a Model Species for Detection of Endocrine Dissruption

Intersex in largemouth bass (Micropterus salmoides) has been correlated with regional anthropogenic activity, but has not been causally linked to environmental factors. Four groups of hatchery-reared largemouth bass (LMB) and fathead minnows (FHM) of varying ages and sex were exposed to aqueous poultry litter mixtures, 17β- estradiol (E2), and controls. Water samples were analyzed for estrogens through liquid chromatography tandem mass spectrometry and estrogenicity through the bioluminescent yeast estrogen screen assay. Fish plasma was analyzed for the egg yolk protein vitellogenin (Vtg) using enzyme–linked immunosorbent assay and gonad tissue was examined histologically for enumeration of testicular oocytes (TO). Water chemistry revealed typical E2 conversion to Estrone with subsequent decay over the exposure periods. A modest prevalence of TO (9.4%) was detected with no apparent treatment effect. While significant Vtg induction was found in E2 exposed FHM, minimal Vtg induction was found in male LMB. Despite field findings of intersex in male LMB, this species may be poorly suited for laboratory investigations into endocrine disruption.

Inactivation of Mycobacterium avium subsp. paratuberculosis in milk during commercial pasteurisation

Four studies have been published relating to the inactivation of Mycobacterium avium subsp. paratuberculosis (Map) by commercial HTST pasteurization. Three of these were large surveys of commercially pasteurized milk at processing/retail level in the UK and Ontario, Canada, and the fourth a pasteurization study involving naturally infected milk and commercial-scale pasteurizing plant. Evidence that Map is capable of surviving commercial pasteurization was obtained in two of the studies: viable Map was cultured from 50 ml aliquots of commercially pasteurized milk after decontamination with 0.75% cetylpyridinium chloride for 5 h and then culture on Herrold's egg-yolk medium without antibiotics. In both studies culture did not commence until 24-72 h post-pasteurization and samples were stored at 4 degrees C in the interim period. In the other two milk surveys smaller volumes of milk were tested (1-5 ml and 15 ml) and no firm evidence of surviving Map was obtained. The three milk surveys differed in other respects - chemical decontamination, culture media used and use of antibiotics. Recent findings suggest that sub-lethally heat-injured Map in pasteurized milk have the potential to recover viability if stored at 4 degrees C for 48 h between heating and testing.

Deposition and depletion of five anticoccidials in eggs

Anticoccidials are compounds that are widely used as feed additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a certain time interval for broilers and pullets but not for laying hens. It was shown in the past that carry-over at the feeding mill is found to be the main reason for the presence of residues in eggs. An animal experiment was set up to investigate the effect of carry-over at the feeding mill on the presence of residues of anticoccidials in eggs. For the compounds diclazuril, robenidine, halofuginone and nicarbazin in combination with narasin, two concentration levels were tested: the maximum allowed concentration for broilers (100%) and a concentration corresponding to 5% carry-over during feed preparation. Also dimetridazole was included in the experiment but only at one concentration level. Eggs were sampled during treatment (14 days) and for a period of 30 days after withdrawal of the anticoccidial-containing feed. Residues were determined, and deposition and depletion curves were generated. Analyses were performed by ELISA and LC-MS/MS. For all compounds, substantial residues could be found in the 5% groups, which points out the risk of carry-over at the feeding mill. The distribution of the residues between egg yolk and white was determined by analyzing both fractions.