CB诱导熊本牡蛎三倍体及其存活率与倍化率的变化关系

为诱导熊本牡蛎三倍体,研究了细胞松弛素B (CB)浓度、诱导起始时间、诱导持续时间等因素对卵裂率、D幼率、三倍体率的影响,并分析了幼虫、稚贝及成贝的存活率和三倍体率的变化特征。结果显示,CB浓度为0.5~0.6 mg/L,诱导起始时间为40%受精卵释放第一极体,诱导持续时间为20 min时可获得87%的三倍体率。卵裂率、D幼率、三倍体率的最大影响因素分别为CB浓度、诱导持续时间、诱导起始时间与诱导持续时间。三倍体率与卵裂率无显著负相关性,而与D幼率呈显著正相关。因此,减小CB浓度或诱导持续时间,可同时获得较高的三倍体率与幼虫产量。3~15日龄三倍体组与对照组的存活率分别由71.27%与96.09%降低至34.14%与58.80%,成贝期450日龄(9月)三倍体组与对照组的存活率分别为53.62%与44.67%。3~9日龄三倍体率从87%降低至77%,而90~450日龄三倍体率平均值为59.21%±4.99%,表明幼贝与成贝期三倍体率变化较小,三倍体率的维持与存活率无显著相关性。     

Induction of triploidy in large yellow crocker Pseudosciaena crocea (Richardson, 1846): effects of pressure shocks and growth performance in the first rearing year

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow‐cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 °C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P<0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).     

静水压休克法诱导三倍体鲶鱼（silurus asotus L）的研究

采用静水压休克法诱导三倍体鲶鱼。通过对受精时间、静水压力及持续施压处理时间三方面进行筛选试验的结果表明，鲶鱼卵受精4－5min，用600－649kg/cm^2的静水压力处理3min，可以获得100％的三倍体鲶鱼，而且胚胎存活率也较高，孵化率达对照组的90％以上，是静水压休克法诱导三倍体鲶鱼的最佳条件。三倍体鲶鱼的倍性用细胞遗传学方法验证。     

Effect of High Pressure Processing on the Quality and Endogenous Enzyme Activities of Grass Carp (Ctenopharyngodon idellus) Fillets Stored at 4ºC

ABSTRACT

This study assessed effects of high pressure processing on physicochemical quality and endogenous enzyme activities of grass carp fillets stored at 4 ºC for 21-day storage. Fresh fillets were vacuum-packed and subjected to high pressure processing (0.1, 200, 300, 400, 500, and 600 MPa at room temperature for 15 min). Results showed that high pressure processing significantly delayed microbial growth, and reduced total volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substances (TBARS) levels of grass carp fillets, especially for the 600 MPa group. On the 21st day, the corresponding increases of 6%, 35%, 45%, 43%, and 53% in hardness were observed for 200 MPa, 300 MPa, 400 MPa, 500 MPa, and 600 MPa samples compared with control samples, respectively. In addition, enzyme activities in different groups decreased during storage. Among them, calpain, and myofibril-bound serine proteinases (MBSP) activities were immediately reduced after high pressure treatments, while the activities of collagenase, cathepsin B, and cathepsin D were significantly (P < 0.05) inhibited when the pressure exceeded 400 MPa. However, the pressure treatments activated cathepsin L initially, and then the activity gradually decreased in treated samples. Generally, high pressure processing reduced activities of most of the endogenous enzymes and improved the quality and extended the shelf life by at least 4 ~ 6 days of refrigerated grass carp fillets.     

Effect of triploidy on paternal and maternal variance components in brown trout, Salmo trutta L.

The covariation between diploid and triploid progenies from common breeders and the effect of triploidy on the parental variances were investigated in brown trout (Salmo trutta L.) using two progeny testing experiments, sampling, sires and dams respectively, from the same population. The traits studied were body weight, growth, condition factor and red spotting of the skin. Triploidization generated some interactions with the parental breeding value, but their effect was minor (less than 20% of the genetic variance, in most cases) as compared with the amount of variation common to both ploidy levels. These interactions were mainly caused by a scale effect, triploidy reducing the variance attributable to sires and increasing the variance attributable to dams. Actual lack of correlation (genetic correlation significantly less than 1) between diploid and triploid familial performances was observed in a single instance out of 18. The modification of respective parental variance components by triploidy, already observed in the rainbow trout Oncorhynchus mykiss (Walbaum), appears as a logical consequence of the genetic make‐up of triploids, and should be taken into account in selective breeding.     

Early survival rates and subsequent morphological abnormalities in landlocked,anadromous and hybrid (landlocked × anadromous) diploid and triploid Atlantic salmon

Upwards of 95% triploidy was induced in landlocked, anadromous and hybrid (landlocked female × anadromous male) Atlantic salmon using heat shocks (5 min at 32°C, 20 min after fertilization and incubation at 10°C). On average, 23% of the treated eggs died within 24 h of fertilization. Most of the subsequent mortality occurred prior to hatching. In all cases, triploid hybrids had better survival rates than triploids of either pure form; triploid hybrids had survival rates comparable to those of diploid hybrids, whereas the survival rates of triploid pure forms were substantially lower than their respective diploid pure forms.Two types of morphological abnormalities were apparent in underyearling parr derived from control and heat shocked groups. Short gill covers were present in both diploid and triploid groups, and were not associated with the heat shock. Protruding lower jaws, however, were found almost exclusively in triploid fish, but were not present in all triploid fish within any particular treated group. No diploid fish originating from previously heat shocked eggs had protruding jaws, so the predisposition to this abnormality seems to depend on the triploid condition and not the heat shock.     

Studies on triploid oysters in Australia: Evaluation of cytochalasin B and 6-dimethylaminopurine for triploidy induction in Sydney rock oysters Saccostrea commercialis (Iredale and Roughley)

Naturally spawned Sydney rock oysters Saccostrea commercialis (Iredale and Roughley),were used to determine the appropriate stage of development for inducing triploidy and to compare the effectiveness of cytochalasin B (CB) and 6-dimethylaminopurine (6-DMAP) in dose-optimization trials. Induction should commence at 50% first polar body (PB1) extrusion in eggs (approximately 17-19 min post-fertilization at 25^oC). By day 5 the highest triploidy percentage and yield (number of triploid larvae per 100 fertilized eggs) were achieved in the ranges of 0.75-1.5 mg CB 1^-1 (1.6-3.1 μm CB)or 200-400 μm 6-DMAP (32.6-65.3 mg 6-DMAP l^-1). However, CB treatment resulted in greater survival and triploidy percentage than 6-DMAP in Sydney rock oysters.     

Effect of three triploidy induction methods on the growth and survival of larvae and post‐larvae of the Caribbean scallop Argopecten nucleus

Argopecten nucleus is a small scallop from the Caribbean Sea and a relatively new species for aquaculture. One of the key challenges to develop the farming operations for this species from the current pilot scale to commercial level is to improve its harvest size. In this study, we tested three different methods for triploidy induction. Additionally, the effect of these protocols on survival, developmental rate and size of larvae and post‐larvae were assessed. Three different mechanisms to stimulate the inhibition of the release of the second polar body were tested; (1) cold shock (18°C); (2) 6‐dimethylaminopurine (6‐DMAP); (3) cytochalasin B (CB) and (4) dimethylsulphoxide (DMSO). The treatment with 6‐DMAP yielded the highest percentage of triploid larvae (39%). The survival and development rate, however, were higher in non‐treated larvae (control) than in the treatment groups. Interestingly, larvae from CB and the DMSO control groups exhibited lower growth rates in length than those from control and the other two treatments. No influence of the triploidy induction treatments was observed on post‐larvae survival, but the size of post‐larvae was larger for the cold shock treatment and DMSO control group. Our results indicate that the use of 6‐DMAP has the greatest potential to produce triploid larvae of A. nucleus without affecting negatively growth and survival of post‐larvae.     

Influence of dietary protein level on growth performance,digestibility and activity of immunity‐related enzymes of leopard coral grouper,Plectropomus leopardus (Lacépède, 1802)

An 8‐week feeding trial was conducted to assess the interaction between dietary protein levels and fish growth, digestibility and activity of immunity‐related enzymes of Plectropomus leopardus. Five diets with different protein levels (400 g/kg, 450 g/kg, 500 g/kg, 550 g/kg and 600 g/kg protein) were designed. P. leopardus fed with 500 g/kg, 550 g/kg and 600 g/kg dietary protein, showed higher weight gain rates than fish fed 400 g/kg and 450 g/kg dietary protein. Ingestion rate in fish fed with 500 g/kg dietary protein was significantly higher than those with other diets. P. leopardus fed with 500 g/kg, 550 g/kg and 600 g/kg dietary protein, showed that feed coefficients were significantly lower than those fed with 400 g/kg and 450 g/kg dietary protein. Net protein utilization was significantly lower in fish fed with 400 g/kg diet than those with other diets. Fish fed with 400 g/kg and 450 g/kg dietary protein had an apparent feed digestibility coefficient for dry matter that was significantly lower than that with other diets. Protease activity was highest in fish fed on 500 g/kg dietary protein. Fish fed with 500 g/kg dietary protein, had the highest superoxide dismutase activity. Fish fed with 600 g/kg dietary protein, had the highest alkaline phosphatase activity. Thus, a diet containing 500 g/kg protein is recommended for P. leopardus aquaculture.     

The breeding potential and biochemical composition of triploid Manila clams, Tapes philippinarum Adams and Reeve

In 1989 and 1990. triploid Manila clam, Tapes philippinarum Adams and Reeve, seed were reared to 15-20 mm at the Fisheries Laboratory, Conwy, and planted out in the Menai Strait, North Wales. In each of the summers of 1992 and 1993, three of these batches, at 2, 3 or 4 years old, were returned to the laboratory to assess ploidy, size, spawning potential and biochemical composition. Percentage triploidy at this time was similar to that in the seed. After 6 and 8 weeks of warmwater conditioning. Only 45 out of l21 triploids (37%) were induced to spawn by thermal shock, with only one spawning as a male. By comparison, 75% of diploid clams spawned with a 1:1 ratio of males to females. Mean fecundity of triploids was significantly lower than that for diploids, at 0.497 compared with 1.54 million eggs per female. Compared with eggs from diploid females, eggs from triploids were larger and significantly fewer of them developed into D-larvae when fertilized by sperm from diploid males. Triploid clams were heavier and had a higher condition index and carbohydrate content than diploids of the same age, but lipid levels were similar. Potential advantages of producing and cultivating 100% triploid batches of Manila clam seed are discussed.     

Triploidization of brown trout (Salmo trutta) by heat shocks

This study was made to optimize heat shock conditions for producing triploidy in the brown trout, Salmo trutta. Heat shock at 29°C for 10 min duration, initiated between 5 and 45 min after insemination, gave high frequencies of triploid embryos (77–91%) as assessed by chromosome observation. Shocks initiated between 90 and 260 min following insemination had no effect on polyploidization. Other groups heat shocked at 29°C for 5–15 min duration initiated 10 min after insemination resulted in moderate rates of triploidy (50–63%). A high temperature shock of 32°C for 6 min duration gave 100% triploidy but a lower temperature shock of 26°C, even for 30 min duration, had only a moderate effect (57%). Rates of hatching were generally decreased in the groups giving moderate to high frequencies of triploidy. In most treated lots haploid embryos were observed and were considered to be a cause of decreased survival.     

Oxygen utilization by triploid landlocked Atlantic salmon (Salmo salar L.)

There was no difference between diploid and triploid Atlantic salmon in either oxygen consumption rate or PO₂ at asphyxiation, in spite of the increase in erythrocyte volume and lower blood haemoglobin content associated with triploidy. It is suggested that triploids should adapt to reduced oxygen levels as well as diploids.     

Artificial Propagation and Induction of Triploidy in Largemouth Bass Micropterus salmoides and Ploidy Discrimination using Erythrocyte Length

We describe an artificial propagation procedure and simple ploidy discrimination techniques using erythrocyte major axis length for largemouth bass Micropterus salmoides. Hormonal treatments of 5 mg/kg of carp pituitary and 50 μg/kg of leutinizing hormone-releasing hormone (LHRH) produced viable gametes in 21-24 h, and triploidy was induced using a pressure treatment of 563 kg/cm² on embryos for a 1-min duration exactly 5 min following fertilization. We produced about 500 fingerling triploids and about 500 diploid controls, and verified genetic status of a subset of each group using flow cytometry. Erythrocyte length was measured for 10 known diploid and 10 known triploid individuals. Remaining fish were internally microtagged with group-specific tags and mixed to test the model. We developed ploidy discrimination intervals based on the 99% confidence limits of mean erythrocyte length (MEL, N = 25 erythrocytes) for individual fish, which were 14.43-16.66 μ.m for triploids, and 10.23-13.62 μm for diploids. Logistic regression provided the discrimination model: Ploidy status (±) = -196.16 + 13.97 x MEL, with positive (+) outcomes considered triploid. Both discrimination techniques were 100% effective at differentiating ploidy of the 22 microtagged largemouth bass recollected from the mixed population. We did not observe a significant change in erythrocyte length as fish size increased, indicating that erythrocyte length is an accurate predictor of ploidy for all sizes of largemouth bass.     

Experimental production of triploid brown trout, Salmo trutta L., using heat shock

Abstract. This study was designed to investigate the potential of heat shock to produce triploidy in brown trout, Salmo trutta L., and to develop a methodof routinely identifying triploids in this species. Triploids were produced in all heat-shocked batches and were identified by the size of their erythrocyte nucleus, which had a volume ratio of 1:1-57 relative to diploid controls. Cytogenetic and flow cytometric analyses confirmed that trout with the larger nuclei were triploid. Heat shock of 28°Cof 10 min duration initiated 5-15 min post-insemination produced high rates of triploidy in experimental batches (88-2-100%), later shocks at 20-25 min producing lower rates (down to 60%). Reproducibilicy of tripioid rates was generally good, a maximum difference between replicates of 21.9% being observed, the majority of differences being considerably less. The highest triploid yield was produced with a heat shock of 28°C for 10 min initiated at 10 or 20 min post-insemination, the difference between replicates being due to variability in survival to hatch. Survival to hatch was generally lower in groups having higher rates of tripioidy.     

Viability of diploid and triploid larvae of Siberian sturgeon and bester hybrids

The aim of the present study was to produce Acipenser baeri× (Huso huso×Acipenser ruthenus) hybrids in a diploid and triploid state and to study their viability in comparison with the A. baeri from the fish farm stock. A heat shock (37°C) in the 18th minute after fertilization was applied to induce triploidy. The survival rate and the ploidy level of the hybrids obtained were studied. The mortality of triploid hybrids was approximately twice as high as the mortality of diploid hybrids. No significant difference in the survival rate between Siberian sturgeon and their diploid hybrid with bester was noted. Cytogenetic analysis was performed by preparing chromosomes from the gill epithelium. The results showed that all studied fish from the heat‐shocked group were triploid.     

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus,reared in intensive culture

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17β-estradiol (E₂)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant. Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3′-triiodo-L-thyronine (T₃) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T₃ and 11kT.     

Induced triploidy in the Pacific oyster,Crassostrea gigas: Optimal treatments with cytochalasin B depend on temperature

Triploidy was induced in Crassostrea gigas using cytochalasin B (CB) (1 mg CB/l) at three temperatures: 18, 20 and 25°C. Between 3 and 5 million eggs/l were treated with CB at 15-min intervals following fertilization.Large differences in survival to straight hinge among mass spawns were observed. These were attributed to variable quality of strip-spawned eggs and treatment with CB. The negative effect of CB treatment was most apparent during critical periods of zygotic development (e.g., fertilization, polar body formation). After 48h, larvae from control and treatment groups had equivalent survival and growth rates.Replicates yielded similar percentages of triploids with standard errors of generally 10% or less. Induction curves were calculated for each temperature; triploid maxima at 18, 20 and 25°C were 52, 76 and 90%, respectively. The highest mean percentages obtained empirically at 18, 20 and 25°C were 62, 74 and 88%, respectively. No evidence for bimodal distributions to separate meiotic I and meiotic II triploids was found. Treatments at lower temperatures delayed triploid maxima which occurred approximately 30, 45 and 50 min after fertilization at 25, 20 and 18°C, respectively. Overall, the optimal treatment for inducing triploidy in the Pacific oyster (C. gigas) appears to be 30–45 min post-fertilization at 25°C, which yielded 88±9% (SE) triploidy over four replicates.     

Preliminary study on performance of triploid Thai silver barb, Puntius gonionotus

The triploid chromosome condition was induced in Thai silver barb (Puntius gonionotus) by application of cold shock (2°C) to eggs at time intervals after activation of 0.5 min with a duration of 10 min which resulted in mean triploidy yield of 72.5% at 9 months of age. Growth rate of the 2–9-month-old, cold shock group (0.1–6.2 g/month) did not differ from that of the control (0.1–5.7 g/month). Gonadal somatic indices (GSI) of presumed triploid males and females were lower than that of control (GSI values of the presumed triploids were 35.0–60.2% and 28.7–75.9% of control males and females, respectively). Spermatogenesis and oogenesis were retarded in triploids. However, all stages of spermatogenic cells were observed in triploid males, including few spermatozoa. Oocytes of triploid females did not undergo vitellogenesis while normal oogenesis was observed in diploids. Nuclear volume of red blood cells (RBCs) of triploid fish was 1.63 times larger than that of diploids.     

Induction of triploidy in the South African abalone using cytochalasin B

An investigation into triploidy induction in the South African abalone, Haliotis midae, was conducted. It was found that 0.5 mg l–1 of Cytochalasin B (CB) in seawater induced triploidy when administered to coincide with the normal timing of the release of either polar body one (PB1) or two (PB2). This concentration of CB produced 70.9% triploid induction in the PB2 treatment and 48.4% induction at PB1. Significant numbers of tetraploid larvae were found in the PB1 treatment. These resulted from the presence of excess sperm (polyspermy) but only when CB was present. Although larval survival after triploid induction was lower than the control animals, it was considered high enough for use in commercial hatcheries. © Rapid Science Ltd. 1998     

Induction of triploidy in the turbot (Scophthalmus maximus): II. Effects of cold shock timing and induction of triploidy in a large volume of eggs

Triploidy was induced in the turbot (Scophthalmus maximus, L.) by applying cold shocks shortly after fertilization. The combined effects of the timing of cold shock commencement after fertilization, cold shock duration and cold shock temperature were investigated. Ploidy was assessed by counting the number of nucleoli per nucleus (NOR) in larvae and also by measuring erythrocyte size in juveniles. A clear peak in triploidy induction was obtained when shocks were started between 6 and 7 min after fertilization at a pre-shock temperature of 13–14°C. With this timing, shocks of 20-min duration at 0°C gave >90% triploidy, with survival about 80% of the untreated controls. In order to ensure both high triploidy rates and high survival, it was necessary to carefully maintain the water temperature just below 0°C. Experiments with small and large volumes of eggs were performed in order to determine how changes in the relative volumes of eggs and chilled water could affect survival and triploidy induction. The best combination to induce triploidy in the turbot was as follows: shock commencement 6.5 min after fertilization, shock duration 25 min, and shock temperature between 0 and −1°C. With this combination, 100% triploidy could consistently be induced with survival 60% of the untreated control. This was successfully applied to a large volume of eggs (300 ml; 1 ml 800 eggs) in order to mass-produce triploid turbot. Triploids had lower survival rate than diploids at hatching but similar thereafter, with the ability to complete the different stages of larval rearing, indicating the viability to produce triploid turbot under farming conditions.