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.

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).

Stock assessment of the demersal offshore fishery resources off the Karnataka coast

The total biomass of the demersal stocks in the depth range of 30-200m between 12°401 and 15°N latitudes in the Indian Exclusive Economic Zone is estimated to be a:round 1,23,000 metric tons (t). There is scope for doubling the present catch of about 40,000 t from demersal resources by extending exploitation to 50-200m depth zone. About 78% of this increase is expected to come from four species, namely, Nemipterus japonicus, Priacanthus macracanthus, Saurida tumbil and Arius spp.

Mass production of Penaeus monodon Fabricius juveniles in earthen nursery ponds

Different culture techniques were tried for rearing larvae of Penaeus monodon, in order to obtain preliminary data on stocking density, water management, fertilization versus feeding and effect of different types of vertical substrate. The results of the experiments showed that: (1) older fry have greater chances of survival; (2) the traditional nursery pond designs and practices used for milkfish in the Philippines are applicable to prawn only at very low densities and give acceptable high survival rates only when used with the older postlarval stage.

Spawning behaviour and induced breeding of an estuarine catfish, Mystus gulio (Ham.)

Spawning behaviour of hormone induced estuarine catfish, Mystus gulio was observed in captive condition. Spawning activities that include pairing, chasing and resting, nudging, and twisting, started about 5 hours post injection and ended with release of eggs within 1-2 hours of courtship. Three different dosages of "ovaprim" (1 ml/kg, 1.5 ml/kg, and 2 ml/kg in a single dose) were used in induced breeding of M gulio. The latency period was less (6-7 hours) with the dose of 1.5 and 2 ml/kg, while it was more (7-8 hours) with that of 1 ml/kg. However, all females spawned successfully with each of three different dosages, without any significant differences in the rate of fertilization and hatching. Eggs under all hormone dosages hatched between 18-20 hours after spawning. The hatching rate with 1, 1.5, and 2 ml/kg varied from 71.3-72.7%, corresponding to the fertilization rate of 80.7-84.7%.

Preliminary success on hormone induced captive breeding of goldspot mullet, Liza parsia (Ham.)

An attempt was made to breed goldspot mullet, Liza parsia in captivity through hormone induction. The fish started spawning 35-36 hours after a single dose of 2ml ova prim per kg body weight. Hatching of fertilized eggs completed within 42-48 hours after spawning. The mean hatching rate (%) was 71.33±12 corresponding to the fertilization rate (%) of 64±12. The larvae started its first external feeding on the third day and attained a length 2.5±0.25 mm. The salinity of both breeding and rearing cisterns was 20‰ and temperature was maintained at 22-23°C.

Effects of dietary vitamin E on the growth and breeding performance of Ompok pabda

A 90 day feeding trail was conducted to investigate the effect of vitamin E on the growth and breeding performance of Ompok pabda. A total of 84 healthy female brood fish (41.10±0.44g) were divided into 4 treatments i.e. treatment T1 T2, T3 and T4 having three replications each. The fish were fed twice a day with a standard feed (40% protein) having 4 doses of vitamin E viz. 0 (served as control), 50, 100 and 150mg vitamin E/kg feed. At the end of the feeding trial, the brood fish were induced to breed with PG extract to observe the effect of vitamin Eon feed. After rearing for 90 days with the experimental feeds, it was found that weight gain and specific growth rate of brood fish fed with 100mg vitamin E/kg feed (treatment T3) was the highest (14.78±0.38g and 2.99±0.11) while 150mg vitamin E/kg feed (treatment T4) fed fish gave the poorest result (2.97±0.89g and 1.21±0.32). There was no significant difference in terms of length gain of brood fish among the different treatments. The brood fish were induced to breed with equal dose of PG extract (18 and 12mg PG/kg body weight for female and male respectively) to observe the dietary effect of vitamin E on breeding performance. The highest ovulation, fertilization and hatching rate of eggs were found to be 81.48±6.41%, 84.04±3.53% and 68.59±5.03% respectively in the brood fish of treatment T1 while the poorest (33.33±00%, 52.35±5.02% and 45.70±7.24% respectively) were found in the brood fish under treatment T4. The results suggest that inclusion of 100mg vitamin E/kg feed is best for enhancing the breeding performance of O. pabda brood fish indicating that vitamin E content has a positive impact on reproduction of fish. The present results also imply that inclusion of higher level of vitamin E exerts an antagonistic effect in terms of growth and breeding performance of this species.

Effects of fish and prawn culture on physico-chemical parameters of water and rice yield in rice fields

An experiment was conducted with five treatments i.e. rice combined with fish having regular urea fertilization (T1), rice combined with prawn having regular urea fertilization (T2), rice combined with fish with supplementary feeding (T3), rice combined with prawn with supplementary feeding (T4) and without fish and prawn (T5) was kept as control. The dissolved oxygen values obtained in treatments with fish both in morning and afternoon were lower than the values of prawn containing treatments and control. The values of nitrate-N, ammonia-N, phosphate-P and chlorophyll-a were higher in fish containing treatments than the prawn containing treatments and control. Between the two fish containing treatments the higher gross (539.44 kg/ha) and net (440.14 kg/ha) yield were obtained in T3 with supplementary feeding and the lower gross (424.88 kg/ha) and net (314.32 kg/ha) yield were recorded in T1 without supplementary feeding. Again, between two prawn containing treatments the higher gross (108.69 kg/ha) and net (81.92 kg/ha) yield were obtained in T4 with supplementary feeding and lower gross (64.32 kg/ha) and net (30.98 kg/ha) yield were recorded in T2 without supplementary feeding. The highest yield of rice grain (3.45 mt/ha) and straw (6.37 mt/ha) were obtained in T1 with fish having urea fertilization without feeding.

Embryonic and larval development of Mystus gulio (Ham.)

Mystus gulio eggs are strongly adhesive and contain relatively small yolk (0.75-1.0 mm). The egg envelop is thick and transparent. First cleavage (two cells), four cells, eight cells, sixteen cells and multi cells stages were found 20, 25, 35-40, 60 and 70 minutes after fertilization, respectively. The morula stage was visualized within 1.5 h after fertilization. The heart beat visible and the circulatory system commenced after 16 h of fertilization. Embryos hatched 18-20h after activation of egg. The newly hatched larva measured 2.82±0.03 mm in length and 0.32±0.06 mg in weight. The yolk sac was fully absorbed by the third day though larvae commenced exogenous feeding even before completion of yolk absorption. A 5-day old post larva began wandering in search of food. Ten-day old post larvae endowed with eight branched rays in dorsal fin and seven in caudal fin. Fifteen-day old post larvae had the pectm:al spine become stout though the embryonic fin folds had to be disappeared. The length of fingerlings ranged from 25-30 mm after 30 days, and their external features were just like those of an adult except that they were not sexually matured.

Histology and hormonal effect of Ghrelin on ovary of Benni (Barbus sharpeyi) in order to study sexual maturity, zygote and larval generation

Benni (Barbus sharpeyi) is valuable fish that Khuzastan fisheries office propagated it artificially in Susangerd Fish Propagation Center every year. Pituitary gland is used for this aim but female fish lost their fertilization power after 2-3 years, so in present research, new hormone, that is called Ghrelin. The aims of this research are histology, hormonal, zygote and larval generation studies and comparing the results with each other. Ghrelin is a multifunctional peptidyl hormone which increases GTH-II in fish, amphibian, and birds and mammalian so its effect on Benni sexual maturation was studied. Human Ghrelin (hGRL) was obtained from ANASPEC, Canada, with 28 amino acids. In the present study, three levels of ghrelin including 0 (sham treatments), 0.10 (treatment 1) and 0.15 μg/g (treatment 2) body wt and one level of pituitary gland 4000 μg/g (pituitary treatment) with two replications were used. 56 specimens were injected intraperitonealy and their ghrelin level was evaluated immediately after injection and after 24 h. Control fish(n=16) were just injected by physiological saline. For hormonal studies sham and experimental fish(n=40) were anesthetized with MS-222 at a concentration of 250 mg l-1, and blood samples were collected and kept at 4ْC, then spun to collect serum. Serum samples were stores at -20ْC until the RIA for CTH-II. For histology studies immediately after injection a piece of ovary was collected from control fish (Sham zero) after being anesthetized. The sampled ovaries were fixed in Buin solution and embedded in paraffin, and stained to Sections of 5–6 μm using haematoxylin and eosin. The ovarian samples were performed with a compound microscope. Histology and micrometry studies had done. The mature oocytes had given from mature fish, then weighted and the working fecundity were counted. The mature oocytes fertilized, the eggs were incubated and the percentage of fertilization was calculated. After 72h the eggs hatched and the percentage of hatch was counted. The percentage of hindrance was calculated after 6 days. Hormonal results indicate that ghrelin and pituitary increase significantly the GTH-II level in comparison to sham. Macroscopic observations (before taking ovary) showed that ovaries with green colored have couple oval structure located in the abdominal cavity. Microscopic studies of dissected ovaries indicated simultaneous growth of 127 oocytes with 6 stages. The type of the ovary is asynchronous. The results indicated that both of the ghrelin treatment increased the percentage of mature follicles followed by decrease of immature follicles. There were significant differences (P<0.05) between the number of mature and immature follicles. Average diameter of follicle in both of the ghrelin treatment was significantly (P<0.05) declined in the stages of the vitellogenesis when the result compared to the other treatment. Just treatment 1 and pituitary treatment can give mature oocytes. The fecundity of pituitary treatment significantly increase in comparision to ghrelin treatment (P<0.05). In food-restricted fish where endogenous ghrelin levels are known to be increased, a chronic administration of ghrelin induces overt negative effect in releasing mature oocytes. The percentage of fertilization was significantly increase (P<0.05) in ghrelin t. in comparison to pituitary t. and the percentage of hatch was significantly increase (P<0.05) in pituitary t. in comparison to ghrelin t. There was no significant difference (P>0.05) in terms of percentage of hindrance between treatments. In conclusion, the present study demonstrated that ghrelin has positive effect on the level of GTH-II, oocyte maturation, ovarian vitellogenesis and the number of mature follicles of Barbus sharpeyi ovary. Increasing of the mature follicles number reduces their average diameter, indicating stimulating effect of ghrelin in sexual maturation of Barbus sharpeyi.The ghrelin and pituitary treatment have equal chance in the post-stage of spawning.

Determination of some dominant genetic characteristics and morpho-meristic features for selection of rainbow trout (Oncorhynchus mykiss) broodstocks

In this study, Iranian and French male and female Oncorhynchus mykiss broodstocks were divided into two groups 50 and 24 respectively in Research center of genetic and breeding of coldwater fishes, Yasouj, Iran and the genetic structure of them was investigated using 6 microsatellite markers. Then 19 morphometric and 5 meristic of broodstock were measured and compared in two populations. Along with broodstock maturation, fertilization 1:1(female:male) were randomly assigned and occurred in 25 of 12 Iranian and French treatment respectively. Reproductive parameters were recorded for the whole family. Average number of observed alleles in Iranian and French stocks was 6.68 and 6.83, respectively. Average number of effective alleles in Iranian and French stocks was 3.13 and 3.45 respectively. Fixation index Fst was calculated based on allelic frequency between two stocks was 0.058 with significant difference between 2 stocks. Morphometric analysis showed significant difference between two stocks in 8 characteristics. Meristic characters was without significant difference in broodstock groups. Eyed percentage for french broodstock calculated zero and deleted. Fertilization rate (100-0), the eyed percentage (98- 0), The hatch rate (98-0), the average fecundity 4114.708, the average eggs size 4.88 mm, Survival in the first three months 19-73% calculated for Iranian broodstocks. Considering the quality of eggs and larvae at different stages and selection between the different family and the within family remained 10 treatments and are kept as future broodstocks. The relationship between fecundity - egg size, fecundity - weight , fecundity - length, egg size- weight was performed using regression. The results showed that Fecundity was influenced more by weight and productive length. The research is beginning to ID the broodstock in our country.

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

Cryopreservation of Kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P<0.01). The results of NF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P>0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants. (C) 1997 by Elsevier Science Inc.