Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.     

Effect of dietary protein and lipid levels on gonadal development of female redclaw crayfish Cherax quadricarinatus

The effect of two crude dietary protein (26% and 32% P) and two lipid levels (4.7% and 12% L) on gonadal development of female redclaw crayfish Cherax quadricarinatus was determined. The gonadosomatic index (GSI), hepatosomatic index (HSI), biochemical composition of gonad and hepatopancreas, oocyte diameter and frequency of the developmental stage of oocytes were analysed. The GSI was significantly less using 26% P to 4.7% L. A significant interaction between P and L content was found for GSI. A significantly higher HSI value was obtained when L was 12%. P content in the hepatopancreas was significantly higher using 32% P and 4.7% L. The maximum L content in the hepatopancreas was obtained from the 32% P and 12% L diet. A significant interaction between P and L content was found for L content in the hepatopancreas. A higher frequency of post‐vitellogenic oocytes was obtained with diets containing 32% P. There was an interaction between dietary P and L level causing lipid accumulation in the hepatopancreas and changes in GSI. For an optimum 32% P, excess dietary lipid is bioaccumulated in the hepatopancreas, indicating that there is excess available energy. When the P and L levels are restricted, there is a limited gonad development, thus affecting overall reproduction in female C. quadricarinatus.     

Water quality,phytoplankton composition and growth of Litopenaeus vannamei (Boone) in an integrated biofloc system with Gracilaria birdiae (Greville) and Gracilaria domingensis (Kützing)

A 28-day indoor trial was conducted to evaluate the water quality, phytoplankton composition and growth of Litopenaeus vannamei in an integrated biofloc system with Gracilaria birdiae and Gracilaria domingensis. The experimental design was completely randomized with three treatments: control (shrimp monoculture); SB (shrimp and G. birdiae) and SD (shrimp and G. domingensis), all with three replicates. Random sampling was done (6 % of total population per experimental unit) to confirm white spot syndrome Virus (WSSV) infection using nested-PCR analysis due to suspicion of presence of the virus in the experiment (treatment and control groups). Shrimp L. vannamei (2.63 ± 0.10 g) were stocked in experimental tanks at a density of 425 shrimp m^?3, and the Gracilaria was stocked at a biomass of 2.0 kg m^?3. Shrimp mortality began in both the experimental and control groups at 10 days of culture. The integrated biofloc system (shrimp and seaweed) increased settleable solids (by 26–52 %); final weight (by 6–21 %); weekly growth (by 17–43 %); weight gain (by 17–43 %); specific growth rate (by 16–36 %); and yield (by 5–7 %) and decreased feed conversion ratio (by 21–28 %) and Cyanobacteria density about 16 % as compared to the control (shrimp monoculture). The use of red seaweed Gracilaria in an integrated biofloc system can enhance shrimp growth and reduce Cyanobacteria density in the presence of WSSV.     

Composite Likelihood Inference for Multivariate Gaussian Random Fields

In the recent years, there has been a growing interest in proposing covariance models for multivariate Gaussian random fields. Some of these covariance models are very flexible and can capture both the marginal and the cross-spatial dependence of the components of the associated multivariate Gaussian random field. However, effective estimation methods for these models are somehow unexplored. Maximum likelihood is certainly a useful tool, but it is impractical in all the circumstances where the number of observations is very large. In this work, we consider two possible approaches based on composite likelihood for multivariate covariance model estimation. We illustrate, through simulation experiments, that our methods offer a good balance between statistical efficiency and computational complexity. Asymptotic properties of the proposed estimators are assessed under increasing domain asymptotics. Finally, we apply the method for the analysis of a bivariate dataset on chlorophyll concentration and sea surface temperature in the Chilean coast.     

Fertility in Holstein × Gyr cows in a subtropical environment after insemination with Gyr sex-sorted semen

Breeding records, including 649 inseminations during fall and winter at a dairy farm in a subtropical area of Western Mexico (24o N; 24°C, mean annual temperature 24°C) were analyzed to document effects of sex-sorted semen from commercial Gyr bullls, estrus synchronization protocol, inseminator, sire and environmental conditions on fertility of crossbred cows (Holstein × Gyr). Percentage of services resulting in pregnancies decreased sharply when sex-sorted semen was used (22.7 vs. 37.7%; P < 0.01). Although statistically not significant (P = 0.31), cows whose first insemination was in November experienced a numerically greater reduction (21 percentage points) in pregnancy rate compared to cows whose first insemination occurred in December. Substantial increases in services per pregnancy (4.71 ± 1.35 vs. 2.13–2.43; P < 0.01) were associated with the warmer month of the study period, November, compared to other winter months. Pregnancy rates of cows regardless of semen category (33%) were not affected by sire, temperature–humidity index and estrus synchronization protocol. Cows inseminated by one inseminator had higher pregnancy rates (P < 0.01) than cows inseminated by other two technicians. The sorted sperm produced 91% (142/156) female offspring. It was concluded that, under the field conditions of the present study, pregnancy rate with sexed semen was 15 percentage points lower than pregnancy rates using conventional semen, with 91% of female calves derived from sexed sperm.     

Insecticide resistance in field populations of Frankliniella occidentalis (Pergande) in Murcia (south-east Spain)

Thirty-nine field populations of Frankliniella occidentalis (Pergande) were collected from different crops (sweet pepper, tomato, lettuce, artichoke, melon, cucumber, carnation, broad bean, peach and plum) in Murcia (south-east Spain). All populations were reared separately in the laboratory to obtain enough individuals for bioassays. Female thrips were bioassayed, using a standard topical application method, against methiocarb, methamidophos, acrinathrin, endosulfan, deltamethrin and formetanate. Methiocarb was the only insecticide that showed a high efficacy against F occidentalis at field dose rates. Acrinathrin and methamidophos were moderately effective, while endosulfan and deltamethrin were ineffective. Only moderate levels of resistance (Resistance Ratios at LC50 of 10-30) were detected for the selective insecticides methiocarb, formetanate and acrinathrin used against F occidentalis in crops where these insecticides are used intensively. This generalized and low level of resistance to these insecticides, coupled with a lack of efficacy for the three broad-spectrum insecticides, was observed even in intensively managed vegetable crops. Implementation of IPM strategies in Murcia has contributed to more successful insecticide anti-resistance management.     

Serological Survey of Babesia bovis and Anaplasma marginale in cattle in Tete Province,Mozambique

A serological survey of bovine babesiosis and anaplasmosis in communal cattle was conducted in the northwestern province of Tete, Mozambique. Blood was collected from cattle ranging from 4 to 15 months old from randomly selected farms from six districts. Thirty-nine per cent of all 478 calves tested in Tete Province were seropositive to the ELISA for Babesia bovis antibodies and 63% of all calves were seropositive in the card agglutination test for Anaplasma marginale. Seroprevalence of B. bovis ranged from 22.8% in Tete City District to 48.1% in Angonia District. For A. marginale, it ranged from 34.4% in Angonia District to 87.3% in Moatize District. The dominant factor affecting seroprevalence for both haemoparasites was district and there was a trend for higher intensity of tick control to be associated with a higher seroprevalence of B. bovis and a lower seroprevalence of A. marginale. The obvious differences were the low prevalence of B. bovis in Tete City Council District and the low prevalence of A. marginale in Angonia District. The levels of exposure to B. bovis seen in our study are well below any that could be considered to be consistent with endemic stability, yet they are sufficiently high to ensure that clinical disease would be a risk. The seroprevalence of A. marginale, however, suggests that endemic stability with respect to this disease could exist in districts other than Angonia. There was no strong and consistent relationship between the intensity of control and the likelihood of seropositivity to either of the diseases.     

Amplified 16S ribosomal DNA restriction analysis for identification of Avibacterium paragallinarum

A molecular technique based on the restriction fragment length polymorphism of the 16S ribosomal genes amplified by a polymerase chain reaction (PCR), referred to as amplified 16S ribosomal DNA restriction analysis (ARDRA), was designed to identify 19 Avibacterium paragallinarum strains isolated from infraorbital sinus and nasal turbinate bone samples of broiler chickens, breeders, and laying hens from different regions of Peru. The 16S rDNA was amplified by PCR using a pair of bacterial universal primers and restriction analysis of 16S rDNA sequences was done to select endonucleases with the highest number of cutting points inside the 16S rDNA. The DNA patterns with DdeI and RsaI endonucleases were identical for the 19 A. paragallinarum strains, but differed from those obtained for Ornithobacterium rhinotracheale, a bacterium with a high genetic and phenotypic resemblance to A. paragallinarum, as well as from Escherichia coli, a bacterium associated with infectious coryza. The ARDRA method could prove to be valuable for molecular identification of A. paragallinarum, a microorganism implicated in respiratory diseases in commercial birds.     

Effects of carotenoids and retinol in oocyte maturation of crayfish Cherax quadrucarinatus

Bioactive additives in food to improve growth, development and reproductive success of crustaceans have received little attention. In the present study, the effects of carotenoid and vitamin A injections were characterized and correlated to the ontogenic development of oocytes in female Cherax quadricarinatus reproducers. During an acclimation period, 250 C. quadricarinatus females were fed a basal diet devoid of carotenoids and retinoids. Four treatments were administered by injection: retinol palmitate (RP), β‐carotene, astaxanthin and vegetable oil as the control treatment. Before the experiment, total gonadic mass of organisms was obtained to determine the gonadosomatic index. Histological techniques were used to evaluate the effects of the different treatments during the primary vitellogenic phase. Retinol palmitate had the greatest inductive effect on the primary vitellogenic phase and on indicators of ontogenic oocyte development. The bioactive and molecular foundations of the action of carotenoids and retinoids that actively participate during the process of nuclear signalling and differentiation of the oocyte are discussed, considering these compounds as essential nutritional additives during gonadic maturation.