Amilcar Arenal

Mendelian transmission, transgene dosage and growth phenotype in transgenic tilapia (Oreochromis hornorum) showing ectopic expression of homologous growth hormone

Abstract Gene transfer has offered a new tool for the development of improved fish strains for aquaculture. However, characterization is required before these strains can be introduced into national aquaculture programs. Transgenic tilapia ( O. hornorum urolepis ) were produced by the microinjection into early embryos of a transgene containing the tilapia growth hormone (tiGH) cDNA under the regulatory sequences derived from the human cytomegolovirus (CMV). A male containing 1 copy/cell of the transgene was selected to establish a transgenic tilapia line. The transgene was transmitted to F 1 –F 4 generations in a Mendelian fashion. Previous studies showed ectopic, low level expression of tiGH in brain, heart, gonad, liver and muscle cells of transgenic tilapia. Biochemical analyses indicated lower levels of cholesterol, free alanine and aspartic acid in the muscle of transgenic animals. Four month old transgenic homozygous (F 2 +/+ ) and heterozygous (F 2 −/+ ) tilapia and non-transgenic siblings ( N TRANSGENICS =14; N CONTROLS =11; N F2 −/+ =8; N F2 +/+ =6) were studied for 3 months grown communally in the same pond. Transgenic (F 2 −/+ +F 2 +/+ ), F 2 −/+ , and F 2 +/+ progeny were larger than non-transgenic siblings at P =0.009, P =0.005 and P =0.07 (Student t -test), respectively, suggesting a transgene-dosage effect. These results indicate stable germ line transformation in this fast-growing transgenic tilapia line.

High fry production rates using post-thaw silver carp (Hypophthalmichthys molitrix) spermatozoa under farming conditions

Abstract Silver carp ( Hypophthalmichthys molitrix ) is not endemic to Cuba, and egg fertilization is totally artificial; males produce spermatozoa only between March and October. Cryopreservation of silver carp spermatozoa would reduce the number of males needed, minimize handling stress through less frequent stripping, and facilitate artificial propagation when eggs are available. The effects on motility and fry production from eggs fertilized with thawed sperm under farm-conditions were examined in this study. Five, seven and ten percent of dimethyl sulfoxide (DMSO), glycerol and methanol were tested as cryoprotectants. DMSO was a more suitable cryoprotectant than methanol or glycerol. The effect of equilibration time on the motility rate at 10% DMSO was evaluated. Hatching rates equal to the control ( P >0.01) were obtained under farm conditions with frozen spermatozoa, stored even for a year in liquid nitrogen, with a final DMSO concentration of 10%. Cryopreservation offers a useful routine method for sperm storage and silver carp handling. To our knowledge, this is the first report on the production of fry from post-thaw silver carp spermatozoa under farming conditions.

Helminth egg excretion in horses kept under tropical conditions—Prevalence, distribution and risk factors

Increasing levels of anthelmintic resistance observed in equine cyathostomin parasites have led to recommendations of selective anthelmintic treatment strategies to lower the selection pressure favoring resistant populations. This principle is based on determining strongyle fecal egg counts from all herd members, and treating those exceeding a predetermined treatment cutoff. However, epidemiological information is lacking from horses kept under tropical conditions, where parasite burdens may be of a different composition and magnitude compared to those of horses kept under temperate climate conditions. The aim of the present work was to characterize the strongylid fauna in horses kept in tropical Camagüey, Cuba and identify risk factors associated with strongylid and ascarid egg counts. A total of 396 horses from eight different establishments were included in the study. Coprocultures revealed that Strongylus vulgaris and cyathostomins, sensu lato, were detected in all of those establishments. Prevalence and mean value of strongylid eggs per gram of feces were 97% and 1436, respectively. Eggs of Parascaris spp. were observed in 10% of horses. A multivariate mixed linear model identified sex (p=0.022), month (p=0.044), operation type (p=0.037) and time since last deworming (p<0.001) to be significantly associated in with the magnitude of strongylid fecal egg counts. A multivariate logistic regression identified horses less than two years of age (p=0.010) and horses not receiving anthelmintic treatment (p<0.001) to be significantly more likely to harbor Parascaris spp. parasites. The high magnitude and prevalence of strongylid fecal egg counts observed and the common occurrence of S. vulgaris suggest that strongylid parasite burdens are substantially different from those typically observed in managed equines kept under more temperate conditions.

Diagnosis of resistance alleles in codon 167 of the beta-tubulin (Cya-tbb-1) gene from third-stage larvae of horse cyathostomins.

Anthelmintic resistance is a serious problem for the control of equine gastrointestinal nematodes. In the present survey, 173 third stage larvae of cyathostomins were investigated from three different locations for the presence of the resistant genotype at codon 167 of the beta-tubulin gene, as this is the most prevalent mutation. The larvae from the state of Parana (n=67), Sao Paulo (n=54) and Santa Catarina (n=52), showed 61.2; 31.5 and 38.5% of the heterozygous resistant genotype - TTC/TAC, respectively. An unpublished mutation at codon 172 that results in a serine (S) to threonine (T) substitution was found in 17.9% (12/67) of samples from Parana; and 13.0% (7/54) of samples from Sao Paulo. We have compared the molecular diagnostic with the fecal egg count data (R2=-0.79) from the same farms, and consider that the use of routine molecular diagnostic in individual larva may help to determine the population genetic distribution that is associated with drug failure.

PSYCHROBACTER SP 17-1 ENHANCES GROWTH AND SURVIVAL IN EARLY POSTLARVAE OF WHITE SHRIMP, PENAEUS VANNAMEI BOONE, 1931 (DECAPODA, PENAEIDAE)

The effects of Psychrobacter sp., isolated from the gut of shrimp Penaeus vannamei Boone, 1931, on growth, digestive enzyme activities, total protein content, osmotic stress resistance, immunity and disease resistance of postlarvae were investigated. For enzymatic activities assays whole postlarvae were homogenized. Shrimp subjected to Psychrobacter sp. treatment showed higher weight (40%) and length (5.6%) than that observed in untreated animals. Compared to the untreated control group, levels of protease and amylase activities in postlarvae, were higher in the probiotic group. Postlarvae treated with Psychrobacter sp. showed significantly increase in the survival compared to the untreated ones after osmotic stress. Nitric oxide metabolite levels and the enzymatic activities of phenoloxidase, superoxide dismutase and peroxidase from treated postlarvae were significantly higher than untreated postlarvae. Increased survival was also found after challenge with Aeromonas sp. This is the first study that demonstrates the probiotic effect of Psychrobacter sp. in P. vannamei postlarvae.

Aqueous extract of Ocimum tenuiflorum decreases levels of blood glucose in induced hyperglycemic tilapia (Oreochromis niloticus)

OBJECTIVE To evaluate, in hyperglycemic tilapia [Oreochromis niloticus (O. niloticus)], the effect of this aqueous extract on blood glucose levels. METHODS The hyperglycemia in O. niloticus was induced by adding glucose to fish pond water. An aqueous extract of Ocimum tenuiflorum (O. tenuiflorum) was prepared by boiling fresh leaves and the doses of 0, 40, 80, 200 and 400 mg per liter of pond water were tested. RESULTS The blood sugar concentration for tilapia with hyperglycemic induced was an average of 50% higher than the control group. The blood glucose levels in tilapia after the induction of hyperglycemia were higher than the control group for 90 min after the treatment. The treatment with the aqueous extract of O. tenuiflorum dropped the serum glucose level of hyperglycemic tilapia until it was similar to that of the control group and was dose dependent. CONCLUSIONS The results indicated that O. tenuiflorum was endowed with anti-hyperglycemic activity. To our knowledge, this is the first report on the use of fish as a diabetes model to test natural extracts from plants.

Gene silencing of Dim-1, a member of the disorganized muscle family, in Haemonchus contortus

RNA interference has been widely used in parasites. In Haemonchus contortus, reproducible silencing has been reported; however, in this species an altered phenotype has been observed for only a few genes silenced by RNA interference. The aim of this work was to evaluate the effect of silencing Dim-1, a member of the disorganized muscle family on third stage larvae (L3) of H. contortus. Effective silencing of Dim-1 in L3 led to reduced L3 migration and slowed larval development from L3 to early L4. To our knowledge, this is the first report to demonstrate Dim-1 silencing affecting phenotypic characteristics of this parasite.

Growth Hormone Secretagogue (A233) Improves Growth and Changes the Tissue Fatty Acid Profile in Juvenile Tilapia (Oreochromis niloticus)

Growth hormone (GH) release is a process that is well regulated by several factors, including GH secretagogues. GH can mediate the regulation of the fatty acid level and composition. The aim of this study was to determine the effect of a synthetic GH secretagogue peptide (A233) on the growth and fatty acid composition in tilapia (Oreochromis niloticus). To address this objective, we administrated a diet supplemented with A233 to juvenile tilapia for 60 days. The group fed with a diet supplemented with 600 μg of A233 per kg of feed increased in weight (4.81 ± 0.09 g) and specific growth rate (2.49 ± 0.03%/day) compared to the control diet group (3.63 ± 0.08 g, 2.07 ± 0.04%/day; respectively) (p < 0.001). In the muscle, the total lipids for the control diet group were higher than that in the group fed with 600 μg of A233 per kg feed; however, no differences were detected in the liver. In both tissues, the patterns of fatty acid composition and content were generally similar, with some exceptions. Tilapia fed with 600 μg of A233 per kg of feed showed, in liver and muscle, a significantly higher composition and content of n-3 polyunsaturated fatty acids (such as 20:5n-3, 22:5n-3, 22:6n-3) and n-3/n-6 PUFA than animals fed with the control diet. To our knowledge, this is the first report on the the effects of natural or synthetic GH secretagogues (GHS) on fatty acid composition, implying an increase in the nutritional quality of the tilapia.

Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites

Cyathostomins are the most common and important group of large intestine nematodes, infecting horses worldwide. The current control strategy is associated with the development of anthelmintic resistance, which has been reported worldwide. Therefore, experiments with this family of parasites have become progressively important to provide their monitoring and control strategies. The aim of the present study was to propose a faster and more economic assay for isolation of genomic DNA from the adult stage of Cyathostomin parasites than reported. Adult parasites were collected from a single horse from a farm in Sao Jose dos Pinhais, PR, Brazil, and were identified. Genomic DNA was isolated from ten individual female adult parasites using a standardized procedure developed. Then, extraction from ten individual female was carried out by another DNA extraction method. DNA concentration from both methods were measured and compared. We obtained a good DNA quality with this standardized procedure. As a result of this analysis, we propose a modified phenol-chloroform method, which will contribute to assays that require DNA extraction from adult worms for genomic DNA sequences of cyathostomin, or species-specific identification.

Development and validation of a meat juice ELISA for the diagnosis of Fasciola hepatica in cattle in Cuba

Abstract Objective To establish and validate a home-made ELISA for determination of antibodies against excretory-secretory proteins of Fasciola hepatica in bovine meat juice samples. Methods The validity criteria of the assay were defined based on standards of International Organization for Standardization. The following parameters were evaluated: excretion/secretion antigen concentrations for coating, anti-bovine immunoglobulin G dilution, linearity, accuracy and precision. Results The assay was validated on 126 meat juice samples with known infection status. Using the receiver operating characteristic (n = 126) the optimal cut-off for the ELISA assay was 0.78, above this value the probability for an animal to have fasciolosis was 11 times. And the specificity and sensitivity were 100% and 90.91% respectively. The repeatability of the intra- and inter-assay tests had coefficients of variation lower than 10% and 20% respectively. Conclusions The ELISA is a suitable test for further use in studies towards the epidemiology of Fasciola hepatica in Cuba.