Development of Pedigree Classification Using Microsatellite and Mitochondrial Markers for Giant Grouper Broodstock (Epinephelus lanceolatus) Management in Taiwan

Most giant groupers in the market are derived from inbred stock. Inbreeding can cause trait depression, compromising the animals’ fitness and disease resistance, obligating farmers to apply increased amounts of drugs. In order to solve this problem, a pedigree classification method is needed. Here, microsatellite and mitochondrial DNA were used as genetic markers to analyze the genetic relationships among giant grouper broodstocks. The 776-bp fragment of high polymorphic mitochondrial D-loop sequence was selected for measuring sibling relatedness. In a sample of 118 giant groupers, 42 haplotypes were categorized, with nucleotide diversity (π) of 0.00773 and haplotype diversity (HD) of 0.983. Furthermore, microsatellites were used for investigation of parentage. Six out of 33 microsatellite loci were selected as markers based on having a high number of alleles and compliance with Hardy-Weinberg equilibrium. Microsatellite profiles based on these loci provide high variability with low combined non-exclusion probability, permitting practical use in aquaculture. The method described here could be used to improve grouper broodstock management and lower the chances of inbreeding. This approach is expected to lead to production of higher quality groupers with higher disease resistance, thereby reducing the need for drug application.     

Unusual ovarian activity in a mare preceding the development of an ovarian granulosa cell tumour

An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a granulosa cell tumour; the left ovary was smaller than normal and non-functional. Surgical removal of the right ovary before increasing photoperiod resulted in a return to function of the left ovary and a pregnancy to frozen semen on the second cycle following removal. Figures showing concentrations of inhibin, progesterone, androstenedione, oestradiol and testosterone are presented for this entire period. Unusual ovarian activity in the mare might be a prelude to the development of a granulosa cell tumour.     

Pyaemia in pigs

Pyaemia is by far the most important cause of condemnation in pigs slaughtered in Singapore abattoirs. Between 1983 and 1987, 1757 from a total of 4,899,731 pigs were condemned by meat inspectors for pyaemia, accounting for 0.036% of the total condemnations. The common post-mortem findings of affected pigs during the 5-year period are presented. Abscesses were most commonly seen in the liver (22%), spleen (21%), gastrohepatic lymph node (20%) and bronchial lymph node (17%). Pseudomonas pseudomallei was the most predominant organism isolated, accounting for 39% of the pyaemic cases. The public health significance of abscesses in pigs is discussed with particular reference to melioidosis.     

Economic production of estuary grouper, Epinephelus salmoides Maxwell,reared in floating net cages

The factors affecting production of estuary grouper, Epinephelus salmoides, in floating net cages are discussed. The concept of economic production in cage culture is outlined. Economic production of estuary grouper could be achieved by providing a suitable culture site with good water quality; ensuring proper farm management and regular maintenance of cages; preventing disease and ensuring efficient treatment; providing optimum stocking density, a balanced diet, optimal feeding frequency; manipulating fish behaviour by providing artificial hides; and manipulating physiological characteristics, e.g. by the use of growth promoters.The paper futher describes environmental parameters, such as dissolved oxygen content, water temperature, salinity, water current, bacteria count, pH, and bottom sediments, as important factors in site selection. Their effects on the growth and production of fish are discussed and their optimal levels determined. The problems involving management and maintenance of the farm are also outlined. The main problem in cage culture is fouling which demands considerable labour and money for cage maintenance. Other problems such as predators, poaching, and cannibalism are discussed at length. It is suggested that by manipulation of stocking density, production could be increased. The optimal stocking rate was found to be 60 fish/m³ which gives a high yield of marketable fish. Stocking could be further intensified by providing artificial hides using used car tyres. The net production could be increased by 230% if hiding space of 251 cm³ per fish was provided and the stocking density increased to 156 fish/m³. Although formulated feeds enhanced growth by 27.1% over that of groupers fed on trash fish, the production cost was much higher, rendering the use of formulated feeds alone uneconomical for commercial fish culture. Growth promoters such as 17α-methyltestosterone and nitrovin have been found to promote growth of young estuary grouper by 43.4 and 62.8%, respectively.Using various combinations of culture techniques, it was shown that net production could be increased from 23.76 kg/m³ using conventional culture methods to 136.75 kg/m³ with a combination of hiding space, heavy stocking, appropriate feeding frequency and formulated feeds incorporating growth promoters. The production time was also shortened considerably from 6 months to 2.7 months.Cost-benefit analysis of the eight culture techniques tested indicated that the production cost could be reduced from US$2.00/kg with conventional methods to as low as US$1.28/kg using a sophisticated method. However, based on current market proces, the conventional culture technique was still economically feasible, as the net income over total capital costs was 33.3%. Improvements of the conventional culture technique will increase the ratio of net income over total capital costs by 1–3 times.     

Detection of betanodavirus in juvenile barramundi, Lates calcarifer (Bloch), by antigen capture ELISA

Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.     

Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks

Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry. An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R. anatipestifer infection in ducks. The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R. anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein. The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R. anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain. Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms. Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01). The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E. coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica. In addition, the DNA sequence encoding P45 was detected in R. anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R. anatipestifer serotypes. Thus, the ELISA described is applicable to the detection of R. anatipestifer infection in ducks.     

Effects of feeding frequency on the growth of young estuary grouper, Epinephelus tauvina (Forskål), cultured in floating net-cages

Studies on the effects of feeding frequency on the growth of young estuary groupers (Epinephelus tauvina) with initial size ranging from 16.2 to 16.9 cm in total length were conducted in floating net-cages. Seven feeding frequencies in the order of one feeding in 5 days, 4 days, 3 days, 2 days, 1 day, two feedings daily and three feedings daily were studied. Optimal growth and good food conversion ratio as well as higher survival rate were obtained in groups fed to satiation with one feeding in 2 days. Weight gains were substantially reduced in groups fed to satiation with one feeding in 5, 4 or 3 days and were not enhanced when the feeding frequencies were increased to two or three feedings daily. The fact that food conversion ratios were similar in fish fed to satiation with one feeding in 5, 4, 3 and 2 days suggests food intake to be important as a growth limiting factor. Total food intake per feeding was appreciably higher in fish fed once in 2 days. The intake of food was found to be closely related to the amount of food remaining in the stomach, intake being maximal when the stomach was empty. The food deprivation time in estuary groupers was found to be about 36 hours at which over 95% of the food was digested and less than 0.5% body weight of food remained in the stomach. Hence, feeding the fish at 48-hour intervals, i.e. once in 2 days, greatly enhanced maximum intake and efficient utilization of the food.