Molecular characterization of Echinococcus isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina

Human cystic echinococcosis is a highly endemic zoonotic disease in the province of Neuquén, Patagonia Argentina, although a hydatid control programme has been carried out since 1970. Human infection due to Echinococcus canadensis (G6 genotype) is frequent in Neuquén. However, the reservoir for this species remains undetermined in a region where camels are absent. We investigated the fertility, viability and molecular epidemiology of hydatid cysts obtained from local goats, pigs and sheep in order to identify the possible reservoirs of E. canadensis (G6). We also analyzed isolates from infected dogs. A total of 67 isolates were identified by the DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Cysts from sheep (n = 16), goats (n = 23) and pigs (n = 18) and adult worms from 10 infected dogs were analyzed. The fertility of the hydatid cysts was 78.6%; 90.4% and 94.4% for sheep, goats and pigs, respectively. We detected E. canadensis (G6) in 21 of 23 goat samples and in 1 dog isolate, E. canadensis (G7) in all the pig isolates, E. granulosus sensu stricto (G3) in 1 sheep and the G1 genotype in 15 sheep, 2 goats and 9 dog samples. The G1 haplotypes included the common sheep strain sequence and 2 microvariants of this sequence. E. granulosus sensu stricto (G3) is described for the first time in South America. We conclude that goats act as reservoir for E. canadensis (G6) in Neuquén, and that control strategies may have to be adapted to local molecular epidemiology to improve the control of parasite transmission.     

A refinement and validation of the Monash Canine Personality Questionnaire (MCPQ)

The investigation of canine personality has failed to find strong agreement between studies as to its structure. The area has been hampered by a reliance on human personality models and by a tendency to limit the types of dogs used as subjects. These problems were recently addressed during the development of the Monash Canine Personality Questionnaire (MCPQ). In this follow-up study, over 450 participants provided demographic information about themselves and their dog and completed the MCPQ for their dog. Structural Equation Modelling results necessitated reassessing the original data and reanalysis suggested a more succinct questionnaire, the MCPQ-R, to measure five dimensions of canine personality very similar to those revealed in earlier work (extraversion, motivation, training focus, amicability and neuroticism). Owner reports of personality were generally consistent across demographic variables for all five canine personality subscales. There was no association between any subscale score and owner gender or education level, or dog sex or sexual status (desexed or not desexed). Significant, but generally weak associations were found for owner age and Extraversion (r = 0.17, P < 0.001), owner knowledge of their dog and Training Focus (r = 0.22, P < 0.001), time spent inside and Extraversion (r = ?0.13, P = 0.007), dog age and Extraversion (r = 0.14, P = 0.004), Training Focus and Extraversion (r = 0.13, P = 0.007), dog height and Neuroticism (r = ?0.20, P < 0.001) and dog height and Amicability (r = 0.2, P < 0.001), dog weight and Neuroticism (r = ?0.17, P < 0.01) and dog weight and Amicability (r = 0.19, P < 0.01). There were also few differences in personality ratings across recognised purebred dog breed groups, although Working Dogs and Terriers scored significantly more highly on the Extraverted subscale than other groups (P < 0.001) and Working Dogs and Gundogs scored more highly on the Training Focus subscale (P < 0.001). These results are consistent with the view that the MCPQ-R assesses a construct, canine personality, which is relatively stable and comprised of five dimensions.     

Differences in the proportion of collagen and muscle in the canine lower urinary tract with regard to gonadal status and gender

Gonadectomy not only affects hormonal homeostasis but also alters the turnover of different components of the extracellular matrix in urogenital tissues. Collagen is an important component of the bladder and urethral walls and thus crucial for the mechanical properties of normal lower urinary tract (LUT) functions. This study aimed at investigating the possibility of differences in the proportion of collagen and muscle tissues in the LUT of intact and gonadectomised male and female dogs. Twenty clinically healthy dogs were used including 10 sexually intact dogs (5 males, 5 anoestrus females) and 10 gonadectomised dogs (4 males and 6 females). Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra were collected. The tissue sections were stained with Masson's Trichrome. Quantitative evaluation of the blue-stained area for collagen and red-counterstained area for muscle was performed using colour image analysis. The relative proportion of collagen and muscle significantly differed with the gonadal status, the gender and the region. Overall, gonadectomised dogs had a higher (P < 0.001) proportion of collagen and consequently a lower (P < 0.001) proportion of muscle than intact dogs. Regardless of gonadal statuses, females had a higher (P < 0.05) proportion of collagen and a lower (P < 0.05) proportion of muscle tissues than males. Gender differences were found in all four regions of the LUT in intact dogs but only in proximal urethra in gonadectomised dogs where spayed females had a higher (P < 0.05) proportion of collagen and less muscle (P < 0.05). Regional differences were observed in females; a higher proportion of collagen and therefore less muscle were found in the urethra compared with the bladder. Proportional differences in collagen and muscle between intact and gonadectomised animals suggest a relation of different hormonal statuses to structural changes in the canine LUT. Excessive collagen deposits and less muscular volume may impair structural and functional integrity of the LUT which may associate with the development of post-neutering urinary incontinence in the dog.     

Capillaria plica (syn. Pearsonema plica) infection in a dog with chronic pollakiuria: Challenges in the diagnosis and treatment

Capillaria plica (syn. Pearsonema plica) is a nematode parasite of the urinary tract of canids, felids and mustelids, which can cause cystitis, pollakiuria, dysuria and hematuria. An eight-month-old female crossbred dog from Switzerland presented a six-month history of frequent urination. During the first clinical examination, C. plica eggs were detected in the urine sediment. Three series of treatments with fenbendazole (50 mg/kg body weight [BW]/day, orally) for 10 days each, three single day treatments with moxidectin–imidacloprid (spot-on) and one single administration of ivermectin (0.2 mg/kg BW subcutaneously) were performed within an eight-month period. None of those treatments succeeded in eliminating the C. plica infection or in resolving the clinical signs. An endoscopic examination of the urine bladder still revealed numerous adult viable C. plica worms attached to the bladder mucosa. A two-day treatment with levamisole (7.5 mg/kg BW/day intramuscularly) was subsequently performed. An endoscopic control of the urine bladder two days after this treatment and a urine analysis after two weeks confirmed the elimination of the parasites. The clinical signs disappeared within one month. Levamisole was shown to be effective against C. plica infection in a dog, whereas previous treatments with fenbendazole, moxidectin and ivermectin had failed.     

Cutaneous Distribution and Circadian Rhythm of Onchocerca lupi Microfilariae in Dogs

Background

Among the arthropod-borne nematodes infesting dogs, Onchocerca lupi (Spirurida: Onchocercidae) is of increasing zoonotic concern, with new human cases of infection diagnosed in Turkey, Tunisia, Iran and the USA. Knowledge of the biology of this nematode is meagre. This study aimed at assessing the distribution and periodicity of O. lupi microfilariae from different body regions in naturally infested dogs.

Methodology/Principal Findings

Skin samples were collected from six dogs infested with O. lupi but without apparent clinical signs. Two skin samples were collected from 18 anatomical regions of dog 1 at necropsy. In addition, single skin biopsies were performed from the forehead, inter-scapular and lumbar regions of dogs 2–6, in the morning, afternoon, and at night. Two aliquots of the sediment of each sample were microscopically observed, microfilariae counted and morphologically and molecularly identified. Most of the 1,667 microfilariae retrieved from dog 1 were in the right ear (59.6%), nose (26.5%), left ear (6.7%), forehead (3.0%), and inter-scapular (2.9%) regions. In dogs 2–6, the overall mean number of microfilariae was larger on the head (n = 122.8), followed by the inter-scapular (n = 119.0) and lumbar (n = 12.8) regions. The overall mean number of microfilariae was larger in the afternoon (153.4), followed by night (75.4) and morning (25.8).

Conclusions

Onchocerca lupi microfilariae were more common in the head (i.e., ears and nose) than in the remaining part of the dog''s body, indicating they tend to aggregate in specific body regions, which are the best sites to collect skin samples for diagnostic purposes. The periodicity pattern of microfilariae of O. lupi and their concentration in specific body regions is most likely a result of the co-evolution with their as-yet-unknown vector. The detection of skin microfilariae in asymptomatic animals, suggests the potential role of these animals as carriers and reservoirs of O. lupi.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

The effect of time left alone at home on dog welfare

The aim of this study was to investigate the effect of time left alone on dog behaviour and cardiac activity. Twelve privately owned dogs, with no history of separation related behaviour problems, were video-recorded on three different occasions when left alone in their home environment. The treatments lasted for 0.5 h (T_0.5); 2 h (T₂) and 4 h (T₄). Video-recording started 10 min before the owner left the house and continued until 10 min after the owner returned, so that interactions between dog and owner as well as behaviour during separation could be studied. Data on heart rate (HR) and heart rate variability (HRV) were collected within the same time period in each treatment. In addition to analysing behaviours separately, behaviours were also grouped together and defined as new variables; physically active, attentive behaviour, vocal, interaction initiated by owner and interaction initiated by dog. There were no differences in behaviour between treatments at equivalent time intervals until the owner returned, although a number of differences were observed at reunion with the owner. Dogs showed a higher frequency of physical activity (P < 0.05) and attentive behaviour (P < 0.01) in T₂ (0.37 ± 0.07; 0.52 ± 0.08, mean frequency of occurrence/15 s ± SE) and T₄ (0.48 ± 0.08; 0.48 ± 0.07) compared to T_0.5 (0.20 ± 0.07; 0.21 ± 0.05). They also showed more tail wagging (P < 0.01) and interacted more with their owners (P < 0.01) in T₂ (0.27 ± 0.08; 0.47 ± 0.09) and T₄ (0.26 ± 0.04; 0.42 ± 0.09) compared to T_0.5 (0.09 ± 0.04; 0.14 ± 0.03). After a longer time of separation, the dogs also showed higher frequencies of lip licking (P < 0.05) and body shaking (P < 0.05) at the owner's return (T_0.5 = 0.09 ± 0.05; T₂ = 0.24 ± 0.08; T₄ = 0.27 ± 0.06 and T_0.5 = 0.03 ± 0.01; T₂ = 0.08 ± 0.03; T₄ = 0.07 ± 0.01, respectively). There was a tendency for higher HR (P < 0.1) during the first and second minute after reunion in T₂ (127.6 ± 1.25, mean bpm ± SE; 111.3 ± 1.24) compared to T_0.5 (106.2 ± 1.06; 87.5 ± 1.02). According to the results of this study, the effect of time left alone was shown by a more intense greeting behaviour by the dog towards their owner as well as by a higher frequency of physical activity and attentive behaviour when the owner returned, already after 2 h of separation. Although this study cannot distinguish between whether dogs were aware of the length of time they were alone (but did not signal it) or whether they were unaware until reminded of it by the return of their owner, it does confirm that dogs are affected by the duration of time at home alone.     

Temperature thresholds of opportunistic annelids used as benthic indicators of aquaculture impact in Newfoundland (Canada)

In Newfoundland, Canada, aquaculture sites set up over deep, hard bottom substrates dictate the use of visual indicators to monitor aquaculture footprints on the seafloor. Opportunistic annelids (referred to as opportunistic polychaete complexes, or OPC) are among these indicators. The effect of temperature on the distribution and survival of Ophryotrocha cyclops, the species constituting OPC in Newfoundland aquaculture sites is not known. To address this knowledge gap, this study assesses O. cyclops survival at different temperatures in the laboratory, and describes relationships between O. cyclops presence and seafloor temperatures measured in the field. Results show that worms died within two days at temperatures > 12 °C in the laboratory, and appear restricted to temperatures below 7.9 °C at aquaculture sites, tending to be more frequent at temperatures below 5 °C. We recommend that seafloor temperature be recorded and considered in the assessment of benthic aquaculture impact based on OPC presence.     

An antibiotic selection marker for schistosome transgenesis

Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would be desirable to provide a means to enrich for populations of transgenic worms. We adapted murine leukaemia retrovirus vectors – widely used in human gene therapy research – to transduce schistosomes, leading to integration of transgenes into the genome of the blood fluke. A dose–response kill curve and lethal G418 (geneticin) concentrations were established: 125–1,000 μg/ml G418 were progressively more toxic for schistosomules of Schistosoma mansoni with toxicity increasing with antibiotic concentration and with duration of exposure. By day 6 of exposure to ?500 μg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ?250 μg/ml G418. When schistosomules were transduced with murine leukaemia retrovirus encoding the neomycin resistance (neoR) transgene and cultured in media containing G418, the neoR transgene rescued transgenic schistosomules from the antibiotic; by day 4 in 1,000 μg/ml and by day 8 in 500 μg/ml G418, significantly more transgenic worms survived the toxic effects of the antibiotic. More copies of neoR were detected per nanogram of genomic DNA from populations of transgenic schistosomes cultured in G418 than from transgenic schistosomes cultured without G418. This trend was G418 dose-dependent, demonstrating enrichment of transgenic worms from among the schistosomules exposed to virions. Furthermore, higher expression of neoR was detected in transgenic schistosomes cultured in the presence of G418 than in transgenic worms cultured without antibiotic. The availability of antibiotic selection can be expected to enhance progress with functional genomics research on the helminth parasites responsible for major neglected tropical diseases.     

Bromophenol accumulation and sediment contamination by the marine annelids Notomastus lobatus and Thelepus crispus

The occurrence of bromophenols in Notomastus lobatus and Thelepus crispus and associated sediment and porewater was determined. 4-Bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol were identified in N. lobatus with the highest concentrations occurring in the anterior and posterior abdomen and substantially less in the thorax (345, 209, and 6.6 μg/ml of worm, respectively). The concentration of dibromobenzyl alcohol was highest in the tail (last 15 setigers) of T. crispus (1.02 mg/ml of worm), but did not differ among body parts as the bromophenols did in N. lobatus. Concentrations in sediments were significantly reduced from those in the worms and decreased from the inner burrow lining out to 5 cm from N. lobatus burrows. Similarly for T. crispus, concentrations of dibromobenzyl alcohol in the surrounding sediments and porewater were low and increased with proximity to the burrow. For both species, concentrations in surface sediment (upper 1 cm) did not differ from those at 6 cm depth. Several lines of evidence indicate that these compounds are produced by the worms and hence their presence in sediments is derived from the worms. The contamination of sediment by these bromophenols has important implications for the structure and activities of benthic-associated communities.     

Prevalence,incidence and molecular characterization of tape worms in Al Taif governorate,KSA and the effectiveness of Spirulina platensis as a biological control in vitro

Cestoda is a class of parasitic worms in the flatworm phylum (Platyhelminthes). Most of the species-and the best-known-are those in the subclass Eucestoda; they are ribbon-like worms as adults, known as tapeworms. The results reported that about 9.94% of selected sheep were infected with tape worms in native breed. Concerning the seasonal incidence of tape worms among sheep, the results revealed that the highest percentage was recorded during winter season (11.3%), while the lowest percentage was recorded during the spring (7.72%). The species of tape worms recorded were Moniezea expansa, Moniezea benedene, Avitellina centripunctata, Thysaniezia giardia and Stilesia hepatic. Molecular characterization of Al Taif Moniezia species cox1 gene records four different species Me Taif1, Me Taif2, Me Taif3 and Me Taif4, that have identity ranged from 90 to 99% to species in Senegal and Ethiopia (Me13, Me14 and Me22). In vitro treatment of Monizia spp. with different concentrations (10, 20, 30 and 50 μglml) of Spirulina platensis extract (Blue-green Algae) affects the morphological architecture of tape worms by using scanning electron microscopy (SEM). The results revealed that Spirulina platensis extract treatment, especially for higher doses 30 and 50 μglml, caused the sucker opening to narrow as a result of scolex swelling more than usual, deformation of the proglottids occurred with circular areas of declare swelling along their margins and microtriches covering the whole tegument appears in several directions. In conclusion, as per of our knowledge it is the first time to record new species of Moniezia species in Al Taif governorate and the anthelmintic potential of Spirulina platensis.     

The efficacy of systematic desensitization for treating the separation-related problem behaviour of domestic dogs

The evaluation of systematic desensitization to treat separation-related problem behaviours, such as destruction of property, excessive barking, or house-soiling, has tended to rely on single case-studies. Eight dogs exhibiting separation-related behaviour, and their owners, participated in a controlled experiment using a within-subjects design to evaluate the efficacy of a combination of systematic desensitization and counter-conditioning. Treatment produced significant reductions in both the frequency (T(9)) = 0.0, P = 0.008) and the severity (T(9)) = 0.0, P = 0.008) of separation-related behaviours compared to baseline. Six dogs, for which follow-up data were obtained three months after treatment ended, showed almost complete elimination of the problem behaviour. The use of counter-conditioning, and other behavioural advice, did not appear to be related to the success of the treatment, suggesting that systematic desensitization was the critical element. Speed of progress and final success was not related to the consistency with which the owners applied systematic desensitization, indicating that even when owners apply systematic desensitization haphazardly, it can still be successful in treating separation-related behaviour in dogs.     

Effects of an Enhanced Human Interaction Program on shelter dogs’ behaviour analysed using a novel nonparametric test

The aim of this study was to investigate the effects of introduction and interruption of an Enhanced Human Interaction Program (EHIP) on shelter dogs’ behaviour and welfare and to apply a novel statistical method to analyse the behavioural data. Twenty-two dogs, which were never subjected to similar programs, were studied. The EHIP consisted of walking and petting the dogs for 15 min once a week. Dogs were divided into two groups: dogs participating in the program (EHIP, n = 9) and control dogs receiving no such program but exposed to human visual stimuli (n = 13). EHIP dogs started being walked on day 8 of the study and continued until day 42 when the walking schedule was stopped. Behavioural observations were performed by instantaneous scan sampling every 3 min for 3 h on days 1, 3, 8, 10, 15, 36, 38, 42, 57, 59 and 64. Hence three periods were identified: the first in which all the dogs were not walked, the second in which EHIP dogs were walked (subdivided into early interaction and late interaction phase), the third in which all the dogs were not walked. Data were analysed using the inferential multivariate nonparametric methodology called NPC (Nonparametric Combination) Test. EHIP increased the time the dogs were visible from the front of the pen and the time they were tail wagging (p < 0.05). These changes may make the animals more likely to be re-homed. No behavioural sign of stress was recorded when the program was discontinued. These findings lead to conclude that EHIPs may be useful enrichment programs for shelter dogs. Furthermore, the NPC Test proved to be a suitable and powerful statistical tool since it does not need a modelling for dependence among variables and it is not affected by the problem of loss of degrees of freedom when the number of variables is large compared to sample size as was the case in the present study.     

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.     

Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress

Alterations of plant growth, chlorophyll fluorescence parameters, nodule carbon metabolism and polyols concentration as result of salt stress were examined in alfalfa (Medicago sativa). Plants, in symbiosis with Sinorhizobium meliloti GR4 strain, were grown under controlled conditions for 35 days (DAS) and subjected to 150 mM of NaCl stress. Plant biomass (PDW) and nitrogen fixation rate (NFR) were markedly affected by salt stress conditions; the highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F₀) and increased the optimum quantum yield of PSII (F_v/F_m ratio). The enzyme activities sucrose synthase activity and phosphoenolpyruvate carboxylase, responsible for the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS with the beginning of flowering. The content of total soluble sugars and proline increased under salt stress, and these concentrations were higher in nodule than in leaf. This last result suggests that the nodule is an organ specially protected in order to maintain its functioning, even under stress conditions. Besides, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment. Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf, which supports the central function of this molecule in the adaptive response of nodules to salt stress. The increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant.     

Canine cognitive dysfunction syndrome: Prevalence,clinical signs and treatment with a neuroprotective nutraceutical

Cognitive dysfunction syndrome (CDS) is a progressive neurodegenerative disorder of senior dogs. Since age-related behavioural changes may be useful indicators for early diagnosis and treatment, the first purpose of the present study was to investigate the prevalence of clinical signs of CDS in a general population of aged dogs. The second aim was to evaluate the use of a neuroprotective nutraceutical (Senilife^®, Innovet Italia srl, Rubano, Italy) using an open-label clinical pilot trial.Dogs were recruited from a geriatric population not referred for behavioural consultations. A questionnaire with a checklist of behaviours was filled out to evaluate behavioural items grouped in the following categories: disorientation (D), socio-environmental interaction (I), sleep–wake cycles (S), house soiling (H), general activity (A)—(DISHA). Each owner was asked to rate the frequency of the behavioural signs: never, rarely, often, or always.One hundred and twenty-four dogs were assessed in the first survey; 22 of the 124 dogs tested in the survey were ruled out based on exclusion criteria (clinically and/or sensory severe impairment), 42 dogs had alterations in one category and 33 dogs had signs in 2 or more categories. Consequently 75 dogs had signs consistent with CDS.Among this population eight dogs affected by CDS were enrolled for the second step of the project, an open-label clinical pilot trial with the neuroprotective nutraceutical Senilife^®. Senilife^® contains 25 mg phosphatidylserine, 50 mg of standardized Ginkgo biloba extract, 33.5 mg/d-alpha tocopherol and 20.5 mg pyridoxine per capsule and is dosed at one capsule per 5 kg body weight. The investigator asked the owners to rate the frequency of behaviours referring to DISHA using a four point frequency scale (never, rarely, often, always). Post-treatment, the owners were asked to evaluate all the signs in each category on a five point scale (much better, slightly better, the same, slightly worse, much worse). At the time of the first visit (V₀) the owners were briefed verbally about the procedure; no behavioural advice was given throughout the study time and whenever appropriate therapy with Senilife^® (was started. At V₀, V₁ (28 ± 3 days), V₂ (56 ± 3 days) and V₃ (84 ± 3 days) a control visit was performed and the owners were interviewed. Dogs treated with Senilife^® showed a highly significant difference at V₃ compared to V₀ (p < 0.001).Preliminary results from dogs on Senilife^® showed a marked improvement of CDS related signs, even if the dogs failed to show a complete remission of symptoms.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

Differences in preovulatory follicle dynamics and timing of preovulatory LH surge affect fertility of maiden sheep reared in semi-arid extensive conditions

In the current study follicular dynamics, pituitary function, ovulatory response and luteal activity of 30 maiden Barbarine sheep were analyzed according to oestrus occurrence and lambing outcome after oestrus synchronisation with cloprostenol. Animals were retrospectively classified in three groups named as O? (n = 7, ewes not displaying oestrus), O+L? (n = 7, ewes showing oestrus but failing to lamb) and O+L+ (n = 16; ewes showing oestrus and lambing thereafter). All the sheep ovulated and daily transrectal ultrasonographies revealed that preovulatory follicles were present at cloprostenol injection in all the animals. In sheep O+L+ and O+L?, 50% and 57% of the ovulatory follicles were the largest follicles at cloprostenol treatment (mean size of 4.1 ± 0.26 mm and 4.3 ± 0.74 mm, respectively). In O? ewes, the same percentage was higher (86%, P < 0.05 when compared to group O+L+; mean size of 4.0 ± 0.46 mm). The number of large follicles and the final diameter of the ovulatory follicles at oestrous tended thereafter to be higher in group O+L+ (1.4 ± 0.1 and 6.4 ± 0.2) than in groups O+L? (1 ± 0.2 and 5.7 ± 0.36) and O? (0.9 ± 0.2 and 5.9 ± 0.5, respectively). Conversely, the number of medium follicles at oestrus detection was higher in the group O+L? (2.1 ± 0.3, P < 0.05) than in the other two groups (1 ± 0.2 and 1 ± 0.3 for O+L+ and O? respectively). Timing of preovulatory LH surge was earlier for ewes O? (24.0 ± 4.75, P < 0.05) than for sheep O+L+ and O+L? (37.9 ± 2.45 h and 38.0 ± 4.75 h, respectively) and 94% of O+L+ ewes had a LH surge between 16 h and 64 h after cloprostenol injection compared to 57% in O+L? and O? groups (P < 0.05). Thus, maiden Barbarine sheep failing to display oestrus or conceive showed alterations in their follicular dynamics and, thereafter, pituitary function and ovulatory response.     

Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs

The main purpose of this study was to determine if temporal relationships exist between serum concentrations of free fractions of thyroxin (fT₄) and triiodothyronine (fT₃), follicle-stimulating hormone (FSH) levels, and Sertoli cell differentiation in euthyroid ram lamb testes. Additionally, testicular thyroid hormone (TH) receptors (TRs) were identified using immunohistochemistry and Western blot analysis. Weekly testicular biopsies and jugular blood samples were collected from 12 ram lambs over the 9 weeks of study. Hormone concentrations and the numbers of dividing Sertoli cells per seminiferous tubule (ST) area were analyzed relative to chronological age of animals and the two distinctive stages of Sertoli cell differentiation: (a) tight junction/ST lumen formation and (b) the onset of support mechanisms for the development of multiple germ cell types (presence of primary spermatocytes in >95% STs). Circulating FSH concentrations increased (p < 0.05) immediately after first detection of ST lumen and reached a nadir (p < 0.05) just prior to the end of the first wave of spermatogenesis. A decline in both fT₄ and fT₃ levels (p < 0.05) occurred after Sertoli cells had formed the ST lumen and began supporting germ cell differentiation. There was a positive correlation between the numbers of proliferating Sertoli cells and serum fT₄ (r = 0.51, p < 0.001) and fT₃ (r = 0.52, p < 0.001) concentrations. TRs were expressed throughout the study period; however, prior to the formation of ST lumen, two isoforms were detected while only one TR isoform was present by the end of the first wave of spermatogenesis. Overall, the exit of Sertoli cells from the cell cycle that presages their final differentiation begins when THs and FSH levels are high, suggesting a permissive role of these hormones in the maturation of STs in prepubertal ram lambs.     

Vertical habitat segregation as a mechanism for coexistence in sympatric rodents

Coexistence has been widely studied in small mammals and frequently is assumed to be facilitated by habitat segregation. Using live trapping and spool-and-line experiments, we analyzed habitat selection and segregation across multiples scales for Peromyscus leucopus and Ochrotomys nuttalli. At the habitat scale of a forest stand (∼1–100s ha) P. leucopus co-occurred at all sites where O. nuttalli was found, and we did not detect evidence of positive or negative associations or habitat segregation. However, O. nuttalli was restricted to early successional forests, and P. leucopus had significantly lower abundances in early successional forests than in other habitats. We found similar patterns at the mesohabitat scale of the study site (400 m²). O. nuttalli abundance increased with increasing shrub and tree densities, while increases in P. leucopus abundance were associated with open understories. At the microhabitat scale of the individual movement trail, we found vertical segregation. Movement trails for O. nuttalli were at significantly higher elevation (mean height = 142.93 ± 37.10 cm) than P. leucopus trails (mean height = 15.4 ± 4.98 cm; F-value = 35.29, p < 0.001). We concluded that microhabitat segregation was driven by differential use of vertical space for movement and foraging. We suggest O. nuttalli is superior to P. leucopus in its ability to acquire food especially in shrubby subcanopies where few acorns are available. However, P. leucopus is superior in its ability to forage and avoid predators.