Proteomic Analysis of Proteins Involved in Spermiogenesis in Mouse

Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

Feed utilization and wastage in semi-intensive pond culture of mahseer, Tor putitora. (Ham.)

Mahseer, Tor putitora with 12.75, 12.11, and 12.02g of initial weight were fou · J to attain a net weight gain of 12.0 kg, ll.5 kg, and 11.4 kg respectively in pond-1 (commercial feed), pond-2 (farm-made feed), and pond-3 (farm-made feed), respectively against 78.2 kg, 70.3 kg, and 68.1 kg feed fed. Gross energy contents in fish were 1359.3 Kcal/kg, 1281.5 Kcal/kg and 1266.6 Kcal/kg, respectively in pond-1, pond-2, and pond-3 against 3630.4, 3876.9 and 3570.5 Kcal/kg energy in the feed fed. Only 9.4%, 10.5% and 13.7% of the protein, and 8.9%, 3.4% and 3.3% of the lipid fed to fish were converted into muscle respectively in pond-1, pond-2 and pond-3. It was observed that the higher the protein content in feed, the lower the rate of conversion in muscle; the same was also true for lipid. It is supposed that feed derived wastes contribute potentially to water quality deterioration and eutrophication. Lower feed conversion, higher nitrogenous and phosphatic concentrations and higher plankton biomass in the ponds are all supportive to this observation.

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.

Comparison of two hormone preparations on the reproductive performance of air breathing catfish Clarias batrachus (Lin.)

Two hormone preparations viz. Human Chorionic Gonadotropin (HCG) and pituitary gland (PG) suspension were compared for their comparative efficacy on the breeding performance of a air breathing catfish Clarias batrachus. It was found that HCG induced fish gave better ovulation response than PG. Both fertilization and hatching of eggs were significantly (p

Early developmental stages of Nandus nandus (Ham.)

In order to study the early developmental stages of Nandus nandus an experiment was conducted, where eggs and milt were obtained from the laboratory reared N nandus by stripping after 15 hours of 150 mg/kg body weight of carp PG extract injection. Then the eggs were fertilized in the laboratory and subsequent developmental stages were studied. First cleavage (two cell), four cell, eight cell, sixteen cell and multi cell stages were found 30, 50, 70, 105 and 160 minutes after fertilization respectively. Morula, early gastrula, middle gastrula, late gastrula and yolk plug stages were found 5, 8, 9, 11 and 13 hours after fertilization respectively. Hatching occurred within 20±2 hours after fertilization, and larvae were measured 1.60 mm in diameter. After one hour of hatching two melanophore bands were found at the caudal region of the body of the larvae. Eyes were first observed in l 0 hours, pectoral and pelvic fin buds appeared in 22 hours and well developed in 38 hours old larvae. Mouth cleft and brain lobes were visible when the larvae were 34 and 38 hours old respectively. Myomeres partially appeared in 16 hours, which were clearly visible in 74 hours old larvae. Larvae started wandering and searching for food after 56 hours of hatching. The yolk sac was completely absorbed when larvae became 62 hours old.

Effects of different dietary levels of vitamin E on the breeding performance of Heteropneustes fossilis (Bloch)

Experiments on the study of different dietary levels of vitamin E on the growth and breeding performance of Heteropneustes fossilis brood fish were carried out in two phases. The first phase consisted of studying its ovarian development and the second phase on breeding performance. Sixty female fishes were stocked in twelve experimental chambers of a raceway. The effects of four dietary vitamin E levels viz. 0 (served as control), 50, 100 and 200 mg/kg feed, on the somatic growth, ovarian development of brood fish and on their breeding performance were studied. Each treatment had three replications. It was observed that body growth in terms of length and weight was best with 0 mg vitamin E/kg feed and 200 mg vitamin E/kg of feed gave poorest result. The gonado-somatic index and fecundity, however, was highest in the fish fed with 100 mg vitamin E/kg of feed. In case of breeding performance such as ovulation rate, fertilization rate, hatching rate and survival rate, the best result was obtained with 200 mg vitamin E/kg of feed. The overall result of this experiment indicates that 200 mg vitamin E/kg of feed is the best vitamin E dose for H fossilis brood and vitamin E content has a positive impact on ovarian development.

Production of triploid African catfish, Clarias gariepinus (Burchell), using chromosome manipulation techniques

An experiment was conducted to induce triploidy in African catfish, Clarias gariepinus, using heat shock and cold shock techniques. Cold shock at a temperature of 0± 1°C and 5±1°C for a duration of 15, 30, 45 and 60 min and heat shock at a temperature of 40±0.5°C and 41 ±OS C for a duration of 1, 2 and 3 min was given to induce triploidy 5 min after fertilization. Maximum percentage of triploids (91.4%) were obtained in the heat shock at a temperature of 40±0SC for a duration of 1 min whereas cold shock at 0± 1 C for a duration of 60 min yielded 90% of triploids. Chromosome analysis revealed that diploids have 54 chromosomes and triploids have 81 chromosomes. The erythrocyte measurements of the minor axis and major axis were 1.17 times larger in treated fish than in controls. The growth studies showed that the growth rate was not significantly affected in triploids.

Comparison of effectiveness of heat and cold shocks applied in the induction of gynogenesis in Clarias gariepinus (Burchell)

An experiment was conducted to optimize the procedure of gynogenesis in African catfish, Clarias gariepinus by suppressing meiotic and mitotic cell divisions in fertilized eggs. Gynogensis was conducted by fertilizing normal eggs with UV-irradiated sperm followed by either heat or cold shocking Irradiation of spermatozoa was given for a duration of 1 min and the eggs were fertilized in vitro. Cold shock at a temperature of 3± 1°C for a duration of 30 and 60 min and heat shock at a temperature of 39± 1°C for a duration of 1 and 2 min was applied to induce diploidy. Higher percentage of hatching (68.66) was observed for meiotic gynogens at a shock temperature of 39± 1°C for a duration of 1 min, 5 min after fertilization (af). Higher percentage of mitotic gynogenetic induction (15.33) was observed at a temperature shock of 39± 1°C for a duration of 1 min, 30 min af.

Breeding biology and monosex male seed production of Genetically Improved Farmed Tilapia (GIFT) strain of Oreochromis niloticus L. in Bangladesh

A study on the breeding biology of the GIFT strain of Nile tilapia, Oreochromis niloticus, was conducted for a period of five months. The sex ratio of the parent individuals was optimized for performance in spawn production, where the best results were obtained with a sex ratio of female to male of 4:1 compared to those of 3:1 and 2:1. The diameter of eggs obtained from the GIFT stock had major and minor axes of 2.19±0.09 and 1.72±0.07 mm, respectively, with no significant differences between the treatments. The average number of eggs produced was 392±22 per female, with fertilization and hatching rates ranging between 94-96% and 85-88%, respectively. No significant variation was observed between the treatments. Breeding frequencies per female in the three treatment groups ranged between 10-40 days and the highest value was obtained at a female to male sex ratio of 4: l. In an other experiment, l7 aMethyltestosterone (MT) was applied orally to the fry at their first feeding stage with treatments- 1, 2, 3 and 4 at the dosage of 100, 80, 60 and 40 mg/kg feed respectively, for the period of 28 days. The mean percentage of males obtained in treatments, 1, 2, 3 and 4 were 98, 97, 95 and 68, respectively. Treatments-1, 2 and 3 did not differ significantly (P>0.05) from each other but treatment 4 showed significant variation (P<0.05) from other treatments. The results showed that MT-100, 80 and 60 mg/kg feed administered for 28 days produced close to cent percent male population of the GIFT strains in aquaria.

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation. (C) 2004 Wiley-Liss, Inc.

Effect of carp PG doses on induced breeding of Shing, Heteropneustes fossilis (Bloch)

Five hormone doses viz. 25, 50, 75, 100, and 125 mg of carp PG/kg of body weight of the recipient fish were tested and they were designated as T1 T2, T3, T4, and T5 respectively. Significantly higher fertilization (98%) and hatching rates (38%) were obtained from T3 (75 mg of carp PG extract/kg body weight). While T4 (100 mg of carp PG extract/kg body weight) and T5 (125 mg of carp PG extract/kg body weight) gave the highest (90%) ovulation rate. In June and July the highest fertilization rate of 96 and 96.4% respectively and hatching rate 42.5 and 48.7% respectively were obtained. In over all consideration carp PG extract at a dose of 75 mg/kg body weight appears to be the suitable dose for induced breeding of H. fossilis and June and July are the suitable time for its induced breeding.

Partial replacement of fishmeal protein by soybean meal protein in the diet of mrigal, Cirrhinus cirrhosus (Ham.) fry

To find out a suitable balance diet for Cirrhinus. cirrhosus fry substituting fish meal protein by full-fat soybean meal protein as dietary protein source for C. cirrhosus fry (1.29g) was studied in 12 aquaria for 60 days. The fishes were fed with four iso-nitrogenous (30% protein) and iso-caloric experimental diets viz. diet 1 (100% fish meal protein), diet 2 (75% fishmeal protein and 25% soybean meal protein), diet 3 (50% fishmeal protein and 50% soybean meal protein) and diet 4 (25% fish meal protein and 75% soybean meal protein). Among the diets the best growth, FCR and PER were obtained with diet 3 fed fishes and no significant difference (p<0.05) was observed between diet 1 and 2 and diet 4 offered the worst performance. Therefore, considering fish growth, FCR and PER, it can be suggested that fish meal protein might be replaced 50% by soybean meal protein in the diet of C. cirrhosus fry for better growth.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor,;encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization. (C) 2004 Elsevier Inc. All rights reserved.

Seed production and culture techniques of climbing perch, Anabas testudineus

Six treatments each with 12 replications designed to optimize the dose of inducing agent PG to achieve fertilization and hatching success of climbing perch, Anabas testudineus were tested. The females were given single injection of 7-12 mg PG/kg body weight and the males were given 4 mg PG/kg body weight. Fertilization and hatching rate varied from 67±4.55% to 66±3.0% and 59±4.88% to 57±6.21% for the doses of 10, 11 and 12 mg PG/kg of body weight, respectively. The hormone dose had significant (P<0.05) effect on fertilization and hatching. Six mini shallow cisterns (570 cm x 105 cm) were used to investigate the efficacy of zooplankton and Artemia nauplii as feed for spawn rearing. Three-day old spawns were stocked in six mini shallow cisterns at a stocking density of 100 individuals/L of water. Two treatments each with three replications were used to develop culture technique of the climbing perch. In case of treatment-1, the spawns were fed with Artemia nauplii three times daily, while in treatment-2, zooplankton were used as feed in the same manner as in treatment-1. After 14 days of rearing, mean final weight of the fry of treatments-1 and 2 were 95.55±6.71 and 57.69±5.40 mg, respectively. In treatment-1, spawn fed with Artemia nauplii showed significantly (P<0.05) higher mean weight than the spawn fed with zooplankton (treatment 2).

Ice storage characteristics of perch with special reference to its suitability for canning

Perch (Pagrus spinifer), one of the most abundantly available fishes of Gujarat coast, was subjected to a detailed study for assessing its storage life in ice and amenability of the iced fish for canning. Changes in the salt soluble nitrogenous material and myosin content of the iced fish showed good correlation with the changes in the organoleptic and physical qualities. The fish was found to have a storage life of 9 days in ice and samples stored up to 7 days were suitable for canning.