Reproductive ability of second generation ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate the reproductive ability of second generation (F₂) koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. Only diploid F₂ males and females were fertile and used in crosses. A significant increase was recorded in male fertility in F₂ versus F₁. In contrast with an earlier study in which only one fertile F₁ male was found, about 20% of F₂ males produced sperm. The observed reproductive ability of F₂ hybrids was similar to that demonstrated by the only fertile F₁ male and F₁ females. F₂ males produced diploid spermatozoa and generated triploids when crossed with koi females. All triploid fish in these progenies were males indicating that F₂ males had a sex chromosome constitution of XY. F₂ females produced diploid eggs and generated mostly triploids when crossed with koi males. In progenies obtained by crosses of F₂ males with F₁ and F₂ females, most of the surviving juveniles (63%–100%) were diploid; a minority of juveniles were aneuploid (ploidy ranged from 2.1n to 3.6n). Diploid fish in these progenies were presumably the result of spontaneous androgenesis and gynogenesis, by the same mechanisms observed earlier in progenies obtained by crossing the F₁ male with F₁ females.     

Variability in microsatellite DNA markers in gynogenetic and backcross progenies obtained from ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrid females

Inheritance and segregation at five microsatellite loci were studied in diploid gynogenetic and triploid backcross progenies obtained from koi × goldfish hybrid females, which produce diploid eggs. Gynogenetic and backcross progenies were obtained from three individual hybrid females by inseminating eggs with genetically inactivated and intact sperm of parental species respectively; no shock treatments were applied to the early embryos. Complete absence of paternally specific alleles at all investigated microsatellite loci has proven successful genetic inactivation of spermatozoa by irradiation and confirmed gynogenetic origin of progenies. Genotypic segregations at microsatellite loci showed almost complete homogeneity of gynogenetic progenies and their identity to female parents. These results correspond with previous cytogenetic data on the occurrence of premeiotic endomitosis in hybrid females producing diploid eggs. Fish from triploid backcross progenies had genotypes resulting from combination of entire diploid female genome and haploid genome from male.     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Comparison of food selection and growth performance of koi carp, Cyprinus carpio L., and goldfish, Carassius auratus (L.) in mono- and polyculture rearing in tropical ponds

To compare the effect of polyculture against conventional monoculture on ornamental carp production, investigations on food selection and growth performance of koi carp (K), Cyprinus carpio L. and goldfish (G), Carassius auratus (L.) were conducted in a 11‐week rearing experiment in two monoculture (100% K and 100% G) and five polyculture (90% K–10% G, 70% K–30% G, 50% K–50% G, 30% K–70% G and 10% K–90% G) conditions in tropical ponds. There were three replicates for each treatment. Environmental conditions and food availability were similar in all the treatments. Ivlev's electivity index showed that both fish species avoided phytoplankton and preferred cladocerans to other zooplankton groups (copepods and rotifers) in monotypic conditions. However, in the polyculture treatments, the positive electivity of goldfish towards cladocerans reduced significantly (P<0.05), while the percentage of copepods, rotifers and phytoplankton in the gut content increased. No significant differences in weight gain, specific growth rate and deformities were recorded at harvest for koi carp between the different treatments (P>0.05). Even the survival rate of koi carp recorded above 90% in all the treatments. However, the goldfish recorded significantly better weight gain, specific growth rate and survival in monoculture (100% G), compared with the polyculture treatments (P<0.05). Goldfish deformities were lowest (P<0.05) in the monoculture treatment (2.42%). The number of marketable fish above a set size limit of 4 g total weight was significantly higher in the two monoculture treatments, compared with the five polyculture treatments (P<0.05). Keeping in view of the dietary similarities of koi carp and goldfish, and the aggressive nature of koi carp in polyculture, it is suggested to refrain from polyculture of goldfish and koi carp until further documentations relating to optimum stocking density and management of polyculture of ornamental carps are available.     

Susceptibility of koi × crucian carp and koi × goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD)

Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV‐3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp.     

彭泽鲫(♀)与黑龙江野鲤(♂)的杂交利用研究

以彭泽鲫为母本、黑龙江野鲤为父本进行杂交，将杂交一代(F1)的生长速度、抗病力、群体产量、经济效益、同工酶、生物学性状等与双亲进行对比试验。结果表明，杂交一代(F1)具有明显的优势，其生物学性状及同工酶谱与母本彭泽鲫相似。     

Establishment and characterization of a new cell line from koi brain (Cyprinus carpio L.)

A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L‐15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The cell line was cryopreserved in liquid nitrogen at ?196°C and was recovered from storage after 1 year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 10^5.73TCID₅₀/0.1 ml within 7 days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.     

Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

雌核发育橙黄色锦鲤的遗传、性腺发育及外形等特征研究

用经紫外线灭活的团头鲂精子激活橙黄色锦鲤的卵子,在4℃的水温下冷休克处理卵子30 min使其染色体加倍,获得了孵化率为30.6％、存活率为24.2％的雌核发育锦鲤,表明该雌核发育锦鲤研制方法具有较高的效率.利用外周血细胞培养方法制备了雌核发育锦鲤染色体并利用核型分析方法对其核型进行了分析,发现雌核发育锦鲤染色体数目为100,核型公式为22 m+34 sm+22 st+22 t;表明雌核发育锦鲤为二倍体,其核型与锦鲤母本一致.利用石蜡切片方法,随机选取10尾1龄雌核发育锦鲤性腺进行检测,发现其性腺全部为卵巢;并且在繁殖季节随机检测100尾2龄雌核发育锦鲤,没有l尾能挤出精液,表明雌核发育锦鲤全部为雌性,为证明雌性锦鲤的性别决定类型为XX提供了重要证据.此外,还对雌核发育锦鲤与母本的的SOX基因遗传关系、外形、体色等进行了比较,结果表明雌核发育锦鲤与母本有相同的SOX基因扩增片段,为证明其为雌核发育锦鲤而非杂交后代提供了重要证据;同时,雌核发育锦鲤保留了母本体态优美的特点,且具有色泽更鲜艳等优点,表明该雌核发育方法产生了遗传改良效果.雌核发育锦鲤的获得为该观赏鱼的提纯复壮、遗传改良以及性别决定和性别控制研究奠定了基础.     

Impact of long‐term dietary exposure to lead on some reproductive parameters of a female Common carp (Cyprinus carpio L.)

During a long‐term 3‐year dietary exposure of mature carp females to lead, its bioaccumulation in the brain, changes in the neurohormonal activity at the level of the hypothalamus and the pituitary gland as well as the growth and maturation of ovaries were analysed. Moreover, an analysis of the effectiveness of hormone‐stimulated spawning during two subsequent spawning seasons was carried out. The results of the analyses show that chronic exposure of maturing and mature carp females to lead in feed results in its accumulation in the brain (1.365 μg g^?1). This impairs the endocrine activity of the hypothalamus, which is manifested by, among others, an increased secretion of dopamine and impaired spontaneous secretion of luteinizing hormone (LH). The observed decrease in the concentration of gonadotropin in blood inhibits vitellogenesis, which is manifested by a lower degree of the maturity of oocytes, lower fecundity (as measured by egg number, egg weight, egg maturity and egg survival) and lower gonadosomatic index values. The final negative impact of lead is the impairment of reproductive functions, as manifested by a smaller number of spawning females and in their lower fecundity during spawning.     

Carp edema virus in Polish aquaculture – evidence of significant sequence divergence and a new lineage in common carp Cyprinus carpio (L.)

Fish samples initially collected by local veterinarians on the common and koi carp farms in Poland between 2013 and 2015 as part of a KHV surveillance programme, when the water temperature was between 16 and 26 °C, and were also tested for CEV by qPCR. A partial 478 nucleotide fragment of the 4a gene was subsequently generated from 17 qPCR‐positive common carp Cyprinus carpio samples from 36 farm sites tested during the period. Sequence alignments and analysis revealed the presence of CEV in Poland both in common carp as well as in koi carp farms, and phylogenetic analysis assigned the Polish CEV sequences into three distinct genogroups. A lineage which includes the original sequences obtained from koi carp in Japan (genogroup II) included sequences from both koi carp and common carp, and the second lineage (genogroup I) contained sequences from common carp only. A third lineage (genogroup III) which was more closely related to the genogroup II also consisted of sequences from common carp only. The latter represents a lineage of CEV not previously described in the literature.     

二、三倍体乌克兰鳞鲤染色体核型分析

以二、三倍体乌克兰鳞鲤为材料,采用植物血球凝集素(PHA)体内注射,肾组织细胞短期培养,常规空气干燥法制备染色体.其核型分析结果:二倍体乌克兰鳞鲤染色体2n=100核型公式为:26m+30sm+30st+14t,染色体臂数NF=156;三倍体乌克兰鳞鲤染色体的核型公式为3n=150=39m+45sm+45st+21t,染色体臂数NF=234,二、三倍体染色体数之比为1:1.5.二倍体核型与已报道的鲤鱼染色体核型相似.未发现性染色体.     

镉对鲤血清促性腺激素和生长激素的影响

采用ＣｄＣｌ２浸没法处理鲤，研究镉对鲤血清促性腺激素（ＧｔＨ）和生长激素（ＧＨ）水平的影响，结果表明，较镐浓度镉（ＣｄＣｌ２９ｍｇ／Ｌ）处理引起血清ＧｔＨ水平降低，ＧＨ水平升高，提高水中钙浓度，能使由镉引起的ＧｔＨ水平的降低恢得取正常水平，并对镉引起的ＧＨ水平升高起延迟作用，镉处理使鲤对ＬＨＲＨ－Ａ刺激ＧｔＨ分泌的反应性下降，提高水中钙浓度也不能恢复正常，实验结果提示，镉引起的ＧｔＨ和ＧＨ的变化可     

Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio)

Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV‐3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV‐3‐specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV‐3‐infected carp. French CyHV‐3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV‐3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post‐infection. The results suggest that this non‐lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV‐3 disease.     

Induction of ovulation in ornamental common carp (Koi, Cyprinus carpio L.) using LHRHa ([d-Ser(tBu)6, Pro9-NEt]-LHRH) combined with haloperidol and carp pituitary extract

The effects of a single injection of mammalian superactive analogue [d ‐Ser(tBu)⁶,Pro⁹‐NEt]‐LHRHa (20 μg/ kg^?1) combined with the dopamine antagonist, haloperidol (HAL, 0.5 mg kg^?1), for induction of ovulation in the koi carp broodstocks were determined under routine hatchery conditions. The results were compared with classic carp pituitary extract (CPE, double injection) application (water temperature 22 °C). Physiological saline (0.7% NaCl)‐injected fish were used as a control group and no ovulation occurred in this group. The spawning ratio was high in the LHRHa+HAL treatment group and in the CPE treatment group (6/7 and 5/7 respectively). The latency period was 14–16 h in the LHRHa+HAL treatment group and 12–14 h in the CPE treatment group (after the second injection). There was no difference between the two ovulating groups with respect to the spawning index (the weight of eggs as a percentage of female body weight) and fertilization rate of eggs (P>0.05). As a result, ovulation can be induced successfully in koi carp broodstocks with 20 μg kg^?1 [d ‐Ser(tBu)⁶,Pro⁹‐NEt]‐LHRHa+0.5 mg kg^?1 HAL treatment in a single injection without decreasing the egg quality. Application of this combination can be useful for hatchery and broodstock management in koi carp culture.