Secondary Stress Responses in Juvenile Brazilian Flounder,Paralichthys orbignyanus,throughout and after Exposure to Sublethal Levels of Ammonia and Nitrite

This study investigated the secondary stress responses of Paralichthys orbignyanus exposed to ammonia and nitrite and after recovery. Fish were exposed to 0.12, 0.28, and 0.57 mg NH₃‐N/L, or 5.72, 10.43, and 15.27 mg NO₂‐N/L for 10 d followed by the same time length for recovery. Ammonia‐ and nitrite‐free water was used as a control treatment. Blood samples were collected after 1, 5, and 10 d of exposure and after recovery. Fish exposed to ammonia presented lower and higher glucose levels after 10 d of exposure and recovery, respectively. Ammonia induced initial and transient ionic disturbances and metabolic alkalosis. Nitrite exposure caused hyperglycemia, increased plasma K⁺ levels, and respiratory alkalosis, whereas metabolic acidosis was observed after recovery. Increased proportion of monocytes and/or granulocytes and reduced number of lymphocytes were demonstrated in fish exposed to 0.28 mg NH₃‐N/L (Day 1) and 10.43 mg NO₂‐N/L (Day 5) and after recovery in the 0.28 and 0.57 mg NH₃‐N/L treatments. Exposure to ammonia decreased the proportion of granulocytes on Day 5. In conclusion, exposure to concentrations at 0.12 mg NH₃‐N/L and 5.72 mg NO₂‐N/L provoked physiological disorders in Brazilian flounder. Nonetheless, fish exposed to 5.72 mg NO₂‐N/L following a 10‐d recovery period showed complete resumption of homeostasis.     

Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Do?ana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Do?ana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.     

Pharmacokinetic disposition of triclabendazole in cattle and sheep; discrimination of the order and the rate of the absorption process of its active metabolite triclabendazole sulfoxide

A comparative pharmacokinetic study was conducted to determine the order and the rate of absorption of triclabendazole (TCBZ) in cattle and sheep. A commercial suspension of TCBZ (Biofasiolex, Biogénesis S.A., Argentina) was administered at a dose rate of 10 mg/kg by the oral route to six Holstein female calves and six Corriedale female sheep. The plasma concentration profiles of the metabolites triclabendazole sulfoxide (TCBZ-SO) and triclabendazole sulfone (TCBZ-SO₂) were analysed by means of the non-compartmental method. The order of the absorption process of the active metabolite, TCBZ-SO, was determined by construction of curves of cumulative absorbed fraction of the drug by means of the Wagner-Nelson method. The appearance of TCBZ-SO in plasma of cattle and sheep resembles the entry of a constant quantity of drug into the organism per unit time. This is explained by the reservoir effect of the rumen, which acts as a biological slow-release system for TCBZ-SO and its precursor TCBZ to the posterior digestive tract where they are absorbed. The plasma concentration profiles of TCBZ-SO in both species were well described by a one-compartment open model with zero-order process of absorption and first-order process of elimination. The values of AUC_0-∞ and C _max of TCBZ-SO did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep. In the case of TCBZ-SO₂, t _max was the only parameter that did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep.     

Validation of a portable device (iSperm®) for the assessment of stallion sperm motility and concentration

The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm^® Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm^® Equine (Aidmics Biotechnology), Androvision^® (Minitube) and NucleoCounter^® SP‐100? (ChemoMetec). The total motility and progressive motility were determined by the iSperm^® Equine and the Androvision^® using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above‐mentioned devices. There was a significant correlation between the concentrations measured with iSperm^® and NucleoCounter^® at all the measured dilutions. Moreover, <10% difference in concentrations was observed between the iSperm^® and NucleoCounter^® using the Bland–Altman test. There was also a significant correlation between iSperm^® and Androvision^® for total and progressive motility. In the second experiment, the parameters used in the Androvision^® were modified to match those of the iSperm^®. Total motility and progressive motility of frozen/thawed semen samples (n = 10) were determined, and the similarity between the Androvision^® and iSperm^® was confirmed by correlation studies and Bland–Altman test. The results of these experiments demonstrate that the iSperm^® offers a reliable and practical alternative for the semi‐automated measurement of concentration and motility of stallion semen in the field. The iSperm^® enables the practitioner to obtain objective and repeatable measurements on a variety of semen types (fresh, cooled and frozen) in the field at the time of insemination and thus acquire more insight into the quantity and quality of the provided insemination doses. This mare‐side diagnostic tool may help practitioners in identifying presumed subfertility problems more rapidly and act accordingly.     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

The effect of weaning method on coccidial infections in beef calves

Weaning is often cited as a stressful event that can precipitate clinical coccidiosis in cattle. Recently, two-stage weaning methods have been investigated as a means to reduce stress in calves. The objective of this study was to investigate the effect of weaning method on fecal oocyst count (FOC) and Eimeria spp. composition. A group of 108 beef calves (190-240 days of age) were allocated to three treatments on day -7: a fenceline weaned group where calves could see and hear their dams, but were physically separated from them by a fence (FL), a noseclipped group where suckling was prevented (NC), and a group left with their dams to be traditionally weaned (TW) by abrupt separation on day 0. On day 0, noseclips were removed from the NC calves and all groups were completely separated from their dams. The steers (n=50) were transported 172 km to another farm, and heifers (n=58) were taken to distant fields on the same farm. Rectal fecal samples were collected from all calves on days -7, 0, and 14. Steers were also sampled on days 7, 28, and 42. A group of steers (n=24) from each of the treatments previously described were fitted with pedometers and intensively observed on days 1-4. The Modified McMaster's test and Wisconsin sugar flotation test were used for quantification and identification of Eimeria spp. oocysts. Clinical coccidiosis was not observed during the study. Fecal oocyst counts did not differ between treatment groups on any sampling day. Differences between steer and heifer FOC were not detected. Treatment had no effect on species composition, but time effects were detected in the steers. The mean percentage of E. bovis was lower (P<0.05) on days 28 and 42 than all other sampling days. The decrease in the percentage of E. bovis was accompanied by an increase (P<0.05) in the percentage of E. canadensis on days 28 and 42. Behavioral observation showed that TW steers exhibited higher levels of stress related behavior (P<0.05) following complete separation, although this was not reflected in FOC.     

Stomatal penetration of cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing latent anthracnose

The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea ( Vigna unguiculata ) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting > 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C . gloeosporioides from Aeschynomene virginica, Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum .     

Genetic variability of rhizobacteria from wild populations of four Lupinus species based on PCR-RAPDs§

The metabolic capacities of rhizosphere bacteria can depend on intraspecific genetic variability at strain level. We sampled bacteria from the rhizosphere of three populations of four different Lupinus species at two growth stages (flowering: GS1 and fruiting: GS2). Isolates were identified to the genus level by classical biochemical tests. The most abundant genera found were Bacillus, Aureobacterium, Cellulomonas, Pseudomonas, and Arthrobacter. Genetic divergence of rhizobacteria was tested by PCR-RAPDs. The genetic distances were low, with mean values of 37 % for Bacillus, 25 % for Aureobacterium, 46 % for Cellulomonas, 16 % for Pseudomonas, and 23 % for Arthrobacter. Aureobacterium, the most abundant genus, predominated in the rhizosphere of all populations and at both growth stages (GS1 and GS2) of L. angustifolius. The Aureobacterium strains consisted of 11 groups with 90 % similarity indexes. The cluster analysis of these groups shows that strains isolated from different lupin species and sampling times have extraordinary low diversity indexes, or are even identical. This fact, together with the low genetic distance detected in the rhizosphere, reveals a clear specificity in the plant-bacteria interaction. This specificity could be related with several aspects of plant physiology.     

Dietary organic acids blend alone or in combination with an essential oil on the survival,growth, gut/liver structure and de hemato‐immunological in Nile tilapia Oreochromis niloticus

This study evaluated the effects of dietary supplementation of organic acids blend (OAB) alone or in combination with essential oil, Lippia origanoides (OAE) for Nile tilapia fed supplemented diets for 30 days. Fish (1.1 ± 0.04 g) were fed control (Control), or OAB 0.5% or OAB 0.5% + essential oil 0.125% (OAE) respectively. At the end of the experiment, samples were collected for de hemato‐immunological, histological analysis of the intestine and liver, as well as microbiology of the intestine. The pH of the diets supplemented with OAB and OAE reduced 0.92 and 0.19 respectively. The growth and FCR were unaffected by the treatments, but survival was significantly higher in the OAB treatment. Fish fed the OAB diet showed reduced concentration of total heterotrophic bacteria and Pseudomonas sp. in the intestine. Increased glucose in fish fed OAB and high number of circulating monocytes in fish fed OAE diet were observed. The anterior intestine of fish fed OAE diet showed larger number of goblet cells and increased villi height. The diet supplemented with OAB, mainly, improved the intestinal health and survival of tilapia juveniles and can be used in juvenile production.