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In brief The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency. Abstract The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus–oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

In Portugal, the relinquishment of dogs and cats is a major problem. Thus, the authors developed a questionnaire to understand the factors influencing the relinquishment of companion animals in Portugal. With a view to analyze the factors behind relinquishment by companion animal guardians, the authors analyzed surveys completed by 72 participants, divided into two groups: REL (relinquisher, n = 36), i.e., guardians who had relinquished a companion animal and NREL (non-relinquisher, n = 36) i.e., guardians who had never relinquished a companion animal. The only significant factors for the REL group were the presence of children and the type of dwelling (apartment). Additionally, the duration of guardianship of the companion animal before relinquishment (REL group) was significantly shorter if there were children in the household than if there were no children.

Rapid antigen diagnostic (RAD) tests have been developed for the identification of the SARS-CoV-2 infection. However, they require nasopharyngeal or nasal swab, which is invasive, uncomfortable, and aerosolising. The use of saliva test was also proposed but has not yet been validated. Trained dogs may efficiently smell the presence of SARS-CoV-2 in biological samples of infected people, but further validation is needed both in laboratory and in field. The present study aimed to (1) assess and validate the stability over a specific time period of COVID-19 detection in humans’ armpit sweat by trained dogs thanks to a double-blind laboratory test–retest design, and (2) assess this ability when sniffing people directly. Dogs were not trained to discriminate against other infections. For all dogs (n. 3), the laboratory test on 360 samples yielded 93% sensitivity and 99% specificity, an 88% agreement with the Rt-PCR, and a moderate to strong test–retest correlation. When sniffing people directly (n. 97), dogs’ (n. 5) overall sensitivity (89%) and specificity (95%) were significantly above chance level. An almost perfect agreement with RAD results was found (kappa 0.83, SE 0.05, p = 0.001). Therefore, sniffer dogs met appropriate criteria (e.g., repeatability) and WHO's target product profiles for COVID-19 diagnostics and produced very promising results in laboratory and field settings, respectively. These findings support the idea that biodetection dogs could help reduce the spread of the virus in high-risk environments, including airports, schools, and public transport.

The adverse health effects of both active and passive tobacco smoke have been well-known in humans for a long time. It is presumable that even pets, which intimately share the owner’s lifestyle, may be exposed to the same risks. This study aimed to detect and quantify cotinine (a metabolite of nicotine) in the serum and hair of dogs using a specific commercial ELISA immunoassay kit. A total of 32 dogs, 16 exposed and 16 unexposed to the owner’s smoke, were enrolled. The cotinine concentration was higher in the exposed than the unexposed group in both matrices (p < 0.001), with greater values in serum than in hair (p < 0.001). Exposed bitches had higher hair cotinine than male dogs (p < 0.001). Conversely, serum and fur cotinine concentrations were lower in female than male dogs of the unexposed group (p < 0.01). The exposure intensity, age, and weight of the dogs did not affect cotinine concentrations. A cut-off value of 2.78 ng/mL and 1.13 ng/mL cotinine concentration in serum and fur, respectively, was estimated to distinguish between the exposed and unexposed dogs. Cotinine was confirmed as a valuable marker of passive smoking also in dogs. Although owners do not perceive secondhand smoke as a risk for their dogs, greater awareness should be advisable, especially in pregnant animals.

BC is a nutraceutical that can modulate intestinal microbiota. This study investigates the effects of BC diet supplementation on luminal and mucosa-associated microbiota in the jejunum, caecum, and colon of rabbits. Twenty-one New Zealand White female rabbits were divided into three experimental groups (n = 7) receiving a commercial feed (CTRL group) and the same diet supplemented with 2.5% and 5% BC (2.5% BC and 5% BC groups, respectively), from 35 (weaning) to 90 days of age (slaughtering). At slaughter, the digestive tract was removed from each animal, then both content and mucosa-associated microbiota of jejunum, caecum, and colon were collected and analysed by Next Generation 16SrRNA Gene Sequencing. Significant differences were found in the microbial composition of the three groups (i.e., beta-diversity: p < 0.01), especially in the caecum and colon of the 2.5% BC group. The relative abundance analysis showed that the families most affected by the BC administration were Clostridia UCG-014, Barnesiellaceae, and Eggerthellaceae. A trend was also found for Lachnospiraceae, Akkermansiaceae, and Bacteroidaceae. A functional prediction has revealed several altered pathways in BC groups, with particular reference to amino acids and lactose metabolism. Firmicutes:Bacteroidetes ratio decreased in caecum luminal samples of the 2.5% BC group. These findings suggest that BC supplementation could positively affect the intestinal microbiota. However, further research is needed to establish the optimal administration dose.

The study provides baseline data regarding 17-β-estradiol (E2), progesterone (P4), and cortisol profile of 30 Nicastrese goats during different physiological periods. Animals were evaluated monthly from the pre-mating period (non-pregnant), during pregnancy, and from 30 to 105 d of lactation. The effects of single or twin births and the kid's sex were also considered. Serum E2, P4, and cortisol concentrations were measured using immunoenzymatic assay kits. The highest concentrations of E2 and P4 (P) were found during pregnancy and their lowest values (P) in the non-pregnant period. E2 was negatively correlated with P4 (r=-0.41; P) during lactation. The mothers with twin kids showed the highest concentration of P4 (P) at > 95–115 d of gestation and the lowest of E2 (P) at > 50–70 d of lactation. Pregnant goats carrying male kid(s) presented the highest E2 concentrations (P) at > 130–150 d of gestation. Different physiological conditions induced a temporal relationship with the endocrine profile in Nicastrese goats. Understanding the effects of single or twin fetuses on the gestation and lactation will also be helpful to improve the managemental approach for the health of mothers and their kids.

IntroductionIntegrating by-products into livestock diet represents a great opportunity for implementing the concept of circular economy while reducing feed costs. Olive cake (OC) is considered an agro-industrial waste, but the high content of valuable metabolites makes it a promising feed integration. Therefore, this study investigated the effect of OC integration in beef cattle diet on different blood parameters.MethodsForty-eight young growing fattening Limousines-−24 bulls (body weight 350 ± 15 kg) and 24 heifers (280 ± 10 kg)—, aged 240 ± 20 days, were randomly allocated to 1 of 3 dietary treatments: concentrate at 0% (Control group: CTR), 10% (Low-olive cake group: L-OC), or 15% (High-olive cake group: H-OC) of OC inclusion. Blood samples and body weights were collected before administrating the supplemented diet (0 d), at the end of the stocker growing phase (56 d), and at the end of the fattening (147 d). After being slaughtered, animal carcasses were weighted. A linear regression model was fitted for each blood parameter with the 0 d as covariate and diet, time, sex, diet × time, and diet × sex as fixed effects.ResultsIn males, body weight was highest in CTR, but carcass weight was similar in all the groups. All the blood parameters were within physiological ranges, independently from the animal diet. CTR group showed the highest alanine aminotransferase (ALT, P = 0.0027) and creatine kinase (P = 0.0119), whereas total bilirubin (P = 0.0023) was higher in H-OC than in CTR. Moreover, ALT was highest in CTR at 56 d, becoming similar in all the groups at 147 d (P = 0.0280). Instead, the increase observed in total cholesterol from 56 to 147 d was lower in H-OC compared with CTR and L-OC (P = 0.0451). A significant effect of diet × sex interaction was observed on triglycerides, urea, liver enzymes, and insulin. These data support the OC inclusion of up to 15% of the concentrate with no detrimental effect on beef cattle metabolic status.DiscussionIn conclusion, OC can be considered as a component in beef diet giving an opportunity to improve agriculture sustainability.

Globally, most people now own a pet. Scales have been developed to understand the impact of pet ownership on people’s lives and to measure specific aspects of the owner–pet relationship. The Cat-/Dog-Owner Relationship Scale (C/DORS) is a tool developed to investigate this relationship in both dog and cat owners. The aim of the study was to refine and validate the C/DORS for cat owners in Italian. Exploratory Factor Analysis and Confirmatory Factor Analysis (CFA) were used to determine the most appropriate factor model. Construct validity was confirmed by correlating the C/DORS subscales with the Lexington Attachment to Pets (LAPS) subscales. Results confirmed the original structure of the English version (i.e., three factors: Pet Owner Interaction = POI, Perceived Emotional Closeness = PEC, Perceived Costs = PC) and CFA confirmed the structure of LAPS and C/DORS scales. Cronbach’s alpha demonstrated the Italian version of the two scales to have good internal reliability for all domains. Owners of cats living exclusively indoors reported higher scores on POI and PEC compared to indoor/outdoor cats. Owning both cats and dogs was correlated with lower scores on POI, and fewer perceived costs (i.e., PC) of cat ownership. Finally, behaviour problems, not being neutered, and lack of previous experience with cat ownership were associated with lower scores on PC.

The latest intrauterine fetal developmental stage and the neonatal period represent the most challenging phases for mammalian offspring. Toward the term of pregnancy, during parturition, and after birth, the hypothalamic–pituitary–adrenal axis (HPA) is a key system regulating several physiologic processes, through the production of cortisol and dehydroepiandrosterone (DHEA). This study was aimed to assess DHEA concentrations in hair and claws of 126 spontaneously dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). The possible influence of newborn sex, breed body size, and timing of death on DHEA concentrations in both matrices was evaluated. Claw DHEA concentrations were higher in the PRE-P group when compared to STILL-P and NEON-P puppies (p < 0.05), whilst no significant differences were found in hair for all the studied factors. The results confirm the hypothesis that higher amounts of DHEA are produced during the intrauterine life in dogs, also in puppies that will die soon after birth.

Quality of life is defined as an individual’s satisfaction with its physical and psychological health, its physical and social environment, and its ability to interact with the environment. Understanding companion dogs’ QOL can help veterinarians and owners know when treatment options have successfully alleviated symptoms of disease in such fields as veterinary physiotherapy. For this study, 20 adult dogs were selected from patients of a physiotherapy referral center with orthopedic, neurological, and/or degenerative conditions. The severity of the medical problem was ranked, and the symptoms, the treatment plan, and demographic data were recorded at the time of the physical examination. In addition, the owner of the dog was asked to fill out a questionnaire on the quality of life of the pet (the Milan Pet Quality of Life scale) at the time of the first consultation as well as the last follow-up after the treatment. The MPQL measures four domains of QOL: physical (signs of medical conditions), psychological (emotional and behavioral well-being), social (quality and extent of social interactions), and environmental (freedom and safety in one’s environment). The results of the study indicated a significant improvement in the psychological QOL domain following physiotherapeutic treatment. The social QOL domain declined with the severity of lameness, while the physical QOL, as reported by the owner, declined with the overall criticality of the medical condition, as ranked by the physiotherapist. The results of the study support the recent evidence of a relationship between pain and canine psychological well-being and highlight the importance of investigating psychological and emotional aspects of dogs’ QOL when treating orthopedic and neurological cases with physiotherapy.

Climate change is a threat to global health and can affect both veterinary and human health. Intense weather events, including sudden and violent thunderstorms or periods of extreme heat, are predicted to rise in frequency and severity and this could lead owners to significantly change their habits and schedules based on the weather, could modify human management and could aggravate pre-existing behavioral problems in pets. The aims of the present study were to identify and quantify possible weather events impact on management, behavior, and behavioral problems of Italian dogs and cats, based on previous owners' experiences with their animals. Two questionnaires were prepared, one for dogs and one for cats, investigating owners' perceptions of the impact of weather events on their pets' behavior. A number of 392 dogs and 426 cats' owners answered the questionnaire. Our study showed that many behaviors in both species were equally modified by environmental temperature. Play and activity increased with cold weather and decreased with heat, and sleep increased with drops in temperature and with hot weather. In particular, the increase in activity in correspondence with the thermic drop was more significant in males, while the increase in playing behavior was statistically greater in the Sheepdogs and Cattle dogs –group1. Weather events did not affect aggressive and house soiling behaviors in both dogs and cats, but weather events, including wild thunderstorms, torrential rains influenced the pets' behavior. Understanding how pets modify their behaviors based on a different owners' schedule and to weather events can help to refine prevention strategies through societal changes and owner education.

Local livestock breeds are pivotal to ensure sustainable agriculture and represent a real genetic resource in the context of genetic variability reduction. This study aimed at broadening the knowledge about hematological values in female Verzaschese and Camosciata delle Alpi goats (an Italian local and a cosmopolitan goat breed, respectively) and investigating factors affecting them (i.e., breed, age, season). Blood samples were collected monthly from 34 Verzaschese and 37 Camosciata delle Alpi female goats kept under the same semi-extensive farming system for a whole year. The main hematological parameters were evaluated, and descriptive as well as inferential statistical analyses were performed. Reference intervals for complete blood cell count were established and several inter-breed differences were found. In particular, most of the red blood cell parameters were higher in Verzaschese than Camosciata delle Alpi (

In June 2014, Albania was granted EU candidate status, thus starting a process of compliance with the membership criteria. In this context, a modern meat inspection approach in line with the European legislation was applied to a pig slaughterhouse in northern Albania in order to investigate the ante-mortem (AM) and post-mortem (PM) conditions and the relationship between these findings. For this purpose, 3930 pigs divided into 35 batches were evaluated over a 3-month period. The most frequent AM conditions recorded were tail lesions and dyspnea (9.1%), followed by skin (8.9%) and ear lesions (8.5%), while in the PM inspections, pleuritis was the most frequently observed condition (10.2%), followed by pneumonia (8.5%), liver alterations (5.7%), milk spot liver (3.8%), and pericarditis (3.3%). With the exception of liver alterations, the other PM lesions mentioned were positively associated with lesions on the ears (OR = 1.036; p < 0.001) and skin (OR = 1.026; p = 0.011) and dyspnea (OR = 1.021; p = 0.005), confirming the link between these variables and the health and welfare conditions of pigs on farms. Overall, the evidence that emerged from this Albanian slaughterhouse can be considered in line with other European contexts, especially in light of the considerable variability in the data present in the literature.

The return to pasture use as an alternative to intensive livestock farming implies some risks with the lack or the excessive presence of potentially toxic elements; in this regard, wild animals have been used as bioindicators for decades. Thus, the purpose of this study is quantifying Cu, Cr, Mn, Zn, Se, As, Cd, Ni, Pb, Al, Fe, and Mg in fur from roe deer and understanding if it is a valid bioindicator tool. Hair was collected from 39 hunted roe deer and divided by age (

The aim of this study was to explore the effects of the Italian COVID-19 lockdown on the cat-owner relationship and on cat behaviour. In order to do so, we used a questionnaire to obtain information on the owner and cat's demographics, living environment, cat behaviour, and possible changes in some aspects of the cat-owner relationship. The questionnaire was distributed online when the lockdown was still ongoing and was completed by 548 cat owners, mainly women. Our findings suggest that the greater amount of time that owners spent at home with their cats, as a consequence of the social restrictions, had a positive effect on the owner's perception of their relationship with their cat (e.g., higher frequency of interaction, greater emotional closeness, reduced perceived costs of caring for the cat). Similarly, the lockdown seemed to have improved the welfare of many cats, as suggested by the positive changes in many of their behaviours, even for those cats with pre-existing behavioural problems. Overall, our findings suggest that the lockdown provided many cats with a more thriving physical and social environment. They also highlight the need to reconsider some cat management practices commonly implemented in non-exceptional circumstances. The restrictive measures implemented to stem the spread of COVID-19 abruptly changed the lives of many cats and their owners. This study explored whether the lockdown in Italy affected the cat-owner relationship, as well as cat behaviour and welfare. A survey that included questions on owner and cat's demographics, living environment, cat behaviour and a modified version of the Cat/Dog Relationship Scale (C/DORS) was distributed online during the lockdown and was completed by 548 cat owners, mainly women (81.6%). With regard to the C/DORS subscales, both emotional closeness and cat-owner interactions increased during confinement, as opposed to a reduction in perceived costs. The effect of the type of job, family role and owner's age on the C/DORS scores suggests that the relationship improved for those owners that, due to the lockdown, increased the time spent with their cats. For 58.8% of respondents, their cat's general behaviour did not change, but when changes occurred, they were mostly positive (20.4%). Attention-seeking and demanding behaviours were the most increased during lockdown (25.7%). Cats with pre-existing problematic behaviours tended to either remain stable or improve during confinement. The overall positive effects of lockdown-related environmental changes on a cat's behaviour suggest that some aspects of commonly implemented cat management practices should be revised to improve cat welfare in normal circumstances.

In the last decades several studies have been investigated the behavior and cognition of dogs (Canis familiars) especially from socio-cognitive aspects. We already gained a solid knowledge on how dogs communicate with humans and conspecific partners, how they are able recognize, interpret and rely on social-communicational cues in their everyday lives, thus continuously perceiving a flow of visual information. However, the fundaments of how they process these visual information are still missing from the literature. The current PhD project have been developed around three independent studies, all of them investigating visual cognition in dogs (and for one case, also in humans) in the domain of perceiving motion.

The aim of this paper was to compare body growth, hematological profile development, and clinical biochemistry in the female progeny of a sire with the female progeny of its clone. Sixteen Friesian female calves, 9 daughters from a tested bull (BULL) and 7 from its somatic cell nuclear transfer clone (CLONE) were monitored from birth to 60 wk of life. Body weight (BW), wither height (WH), hip height (HH), body length (BL), and hearth girth (HG) were measured at birth and 4, 8, 12, 16, 20, 24, 36, and 50 wk. Blood samples were taken from jugular vein at 12 to 48 h from birth and 1, 2, 3, 4, 8, 12, 16, 20, 24, and 36 wks of age, to be analyzed for hematological, serum protein, and metabolic profiles. At the same time, rectal temperature (RT) was recorded. Age at puberty was assessed on surviving heifers by measuring weekly plasma progesterone levels. Data were evaluated using a mixed model, taking into account the repeated measures in time on the calf. For each variable, different covariance structures were tested, choosing the best according to the Akaike's Information Criteria. Significant was set at P < 0.05, and a trend was considered for P < 0.10. At 24 wk of age, WH was lower in CLONE daughters than BULL daughters. Around 20 wk of age, there was a trend for lower BW in CLONE daughters than BULL daughters, confirmed from differences in HG. There was no difference in RT due to sire effect. Blood glucose concentration decreased in both groups during the first 4 wk of life; at birth, only a trend for higher blood glucose in CLONE daughters was recorded, whereas an opposite trend was observed for plasma creatinine. Total leukocyte count did not differ between progenies. Circulating lymphocytes tended to be lower in CLONE than BULL daughters. The neutrophil: lymphocyte ratio tended to be higher in CLONE than BULL calves. No difference was demonstrated for erythrocyte features, whereas mean platelet volume tended to be lower in CLONE than BULL progeny. From these results, there were no differences between progenies from BULL and its clone that suggest welfare problems in the first 6 mo of life.

Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

Concentration of progesterone in milk may be used to predict pregnancy status of dairy cattle by the 21st day after insemination. However, the accuracy of this method may be affected by fat-solubility of progesterone and sample storage conditions. After coagulation of a milk sample with rennet, an alternative method is to quantify progesterone concentration in whey with a novel, validated EIA. In this experiment, a receiver operating characteristic (ROC) analysis was performed to estimate the optimal discrimination point for whey progesterone concentration, using a sample of 991 Friesian cows evaluated between the 42nd and 44th day after insemination. Cows also were diagnosed for pregnancy by rectal palpation at this time. The overall conception rate at palpation was 57%. ROC analysis indicated that 259 pg/mL progesterone in whey was the most effective cutoff to discriminate correctly between pregnant and non-pregnant cows. Using this point for prediction, sensitivity was 98.2%, specificity was 70.9% and the area under ROC curve was 0.859, levels generally considered to denote moderate accuracy. The negative likelihood ratio at the cutoff of 259 pg/mL was 0.02, indicating satisfactory performance in detecting negative subjects, while the positive likelihood ratio (+LR = 3.37) suggested average performance. In conclusion, EIA of progesterone concentration in whey is a viable method for predicting pregnancy status in cows. However, operators should take management objectives for the herd into account in determining the cutoff point and also considering important influencing variables such as conception rate in the herd. This method can provide diagnostic support for efforts to improve reproductive success, especially in low-fertility herds.

Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

Despite some experiments have been carried out to investigate dog’s visual cognitive abilities and to study the mechanisms underlying how they promptly modify their behaviour while interacting with humans, it is still not clear to which features of the stimuli they mainly attend when solving a task. In addition to physical proprieties, there are different factors that modulate the saliency of a visual stimulus in dogs, such as the previous experience and training received. To investigate whether during the associative learning some aspects of visual information have different salience during the associative learning and the role played by previous experiences, the project was developed around three independent studies. The purpose of the first study was to assess the visual processing of global and local levels of hierarchical stimuli in domestic dogs. Fourteen dogs were trained to recognise a compound stimulus in a simultaneous conditioned discrimination procedure and were then tested for their local/global preference in a discrimination test. As a group, dogs showed a non-significant trend for global precedence, although large inter-individual variability was observed. Choices in the test were not affected by either dogs’ sex or type of stimulus used for training. However, the less time a dog took to complete the training, the higher the probability that it chose the global level of test stimulus. Moreover, dogs which showed a clear preference for the global level in the test were significantly less likely to show positional responses during discrimination training. These differences in the speed of acquisition and response patterns may reflect individual differences in the cognitive requirements during discrimination training. The individual variability in global/local precedence suggests that experience with using visual information may be more important than predisposition in determining global/local processing in dogs. The second study, composed by two experiments, investigated the relevance of visual stimuli using more complex images: human face pictures. Dogs exhibit characteristic looking patterns when looking at human faces but little is known about what the underlying cognitive mechanisms are and how much these are influenced by individual experience. In Experiment 1, seven dogs were trained in a simultaneous discrimination procedure to assess whether they could discriminate a) the owner’s face parts (eyes, nose or mouth) presented in isolation and b) whole faces where the same parts were covered. Dogs discriminated all the three parts of the owner’s face presented in isolation, but needed fewer sessions to reach the learning criterion for the eyes than for both nose and mouth. Moreover, covering the eyes region significantly disrupted face discriminability compared to the whole face condition while such difference was not found when the nose or mouth was hidden. In Experiment 2, dogs were presented with manipulated images of the owner’s face (inverted, blurred, scrambled, grey-scale) to test the relative contribution of part-based and configural processing in the discrimination of human faces. Furthermore, by comparing the dogs enrolled in the previous experiment and seven ‘naïve’ dogs we examined if the relative contribution of part-based and configural processing was affected by dogs' experience with the face stimuli. Naïve dogs discriminated the owner only when configural information was provided, whereas expert dogs could discriminate the owner also when part-based processing was necessary. The present study provides the first evidence that dogs can discriminate isolated internal features of a human face and corroborate previous reports of salience of the eyes region for human face processing. Although the reliance on part-perception may be increased by specific experience, our findings suggest that human face discrimination by dogs relies mainly on configural rather than on part-based elaboration. For a more applied comprehension of the phenomenon and to understand what happens in a more natural situation, the third study explored the characteristic of dog’s attention to natural visual stimuli (humans) during a training session in which different type of exercises and levels of difficulty were requested. Moreover, the quality and quantity of training dogs had received during their lives was taken into account to investigate a possible influence on their attention to humans. As in the second study, the specific effect of training emerged also in the third. Having received a specific training modified dogs’ attention towards the caregiver. The variation of attention pattern did not emerge in a baseline condition but only when the caregiver asked the dog to perform some obedience exercises evidencing that the training had a very context-related effect. In conclusion, dogs seem to acquire more quickly and spontaneously global/configural information of a visual stimulus and when an effect of previous experience such as training is present, this effect is specific and strictly context-related.

Environmental temperature affects water turnover and isotope fractionation by causing water evaporation from the body in mammals. This may lead to rearrangement of the water stable isotope equilibrium in body fluids. We propose an approach to detect possible variations in the isotope ratio in different body fluids on the basis of different homoeothermic adaptations in varying reproductive stages. Three different reproductive stages (pregnant heifer, primiparous lactating cow, and pluriparous lactating cow) of two dairy cattle breeds (Italian Friesian and Modenese) were studied in winter and summer. Blood plasma, urine, faecal water, and milk were sampled and the isotope ratios of H (2H/1H) and O (18O/16O) were determined. Deuterium excess and isotope-fractionation factors were calculated for each passage from plasma to faeces, urine and milk. The effects of the season, reproductive stages and breed on δ 2H and δ 18O were significant in all the fluids, with few exceptions. Deuterium excess was affected by season in all the analysed fluids. The correlations between water isotope measurements in bovine body fluids ranged between 0.6936 (urine-milk) and 0.7848 (urine-plasma) for δ 2H, and between 0.8705 (urine-milk) and 0.9602 (plasma-milk) for δ 18O. The increase in both isotopic δ values in all body fluids during summer is representative of a condition in which fractionation took place as a consequence of a different ratio between ingested and excreted water, which leads to an increased presence of the heavy isotopes. The different body water turnover between adult lactating cattle and non-lactating heifers was confirmed by the higher isotopic δ for the latter, with a shift in the isotopic equilibrium towards values more distant from those of drinking water.

The aim of this work was to study: 1) the welfare in breeders of clam (Venerupis philippinarum and Venerupis decussata) using different biomarkers (cortisol, stress oxidative biomarkers, detoxification enzymes); 2) the reproduction and the larval development of these molluscs. The first study evaluated the variation of cortisol in clams subjected to the following stress conditions: anoxia and fasting. The study evidenced a tendency in the increase of cortisol in the animals under anoxia and a decrease in the animals under fasting conditions. In a successive work was evaluated the general morphology and the oxidative stress response in clams sampled at different sites of the Venice Lagoon. The histological evaluation revealed the presence of the parasite Perkinsus olseni in animals from all sites, with an intense immunitary response of the hosts. The hemocytes, that participate at the infiammatory response, showed an intense immunopositivity to several antibodies of the oxidative stress. The bivalve molluscs are often used as biondicator of environmental pollution, so we decided to evaluate the expression of two genes involved in the detoxification process of xenobiotics, in animals treated with different concentrations of benzo(a)pyrene. The expression of GSTr evidenced a significant increase of mRNA in the animals treated with the highest dose, but the analysis of CYP4 expression did not evidence differences among treatments. In treated animals, the histopatological analysis revealed an increase of a particular type of cells involved in the detoxification process of molluscs called brown cells. In the second part of the work we investigated about the reproduction of these bivalves using an histological approach to evaluate the gonad maturation and a radioimmunoassay method to analyse the sex steroids variation. The steroids progesterone, 17β-estradiol and testosterone resulted involved in the gonad maturation of the clams. The histological analysis revealed a different time of maturation of the two species of clams. The study of the larval development revealed useful information about the larval management.

The aim of the present study was to evaluate the relationship between AOPP (Advanced oxidation protein products) and bovine neutrophils 'in vitro'. For this purpose AOPP were produced "in vitro" by oxidizing bovine serum albumin with HOCl (hypochlorous acid) and bovine neutrophils were isolated from whole blood of dairy cattles. AOPP-BSA were incubated with freshly isolated bovine neutrophils, unstimulated and stimulated with PMA a strong activator of the respiratory burst. Neutrophils ROS production and viability were measured by luminol-amplified chemiluminescence and by MTT and lactate dehydrogenase assays (LDH), respectively. Results obtained have shown that AOPP-BSA are able to reduce significantly ROS production of PMA stimulated neutrophil and their viability measured by MTT assay, no cell lysis was detected by LDH assay. On the basis of these results, our work has studied if AOPP are able to trigger apoptotic events. For this purpose, caspase 8-9-3 and DNA laddering were used as markers in order to discriminate between the 'intrinsic' and the 'extrinsic' pathway of apoptosis. The results obtained showed that un-stimulated bovine neutrophils incubated with AOPP-BSA show a higher but not significant production of active caspase 8 in comparison with the incubation with BSA. Also caspase 3 display an increase, but not significant, in un-stimulated neutrophils after 6 hours of incubation with AOPP-BSA, respects the incubation with BSA. No differences were obtained for caspase 9 and for DNA laddering. Therefore, in these experimental conditions is possible to conclude that the 'intrinsic' pathway of apoptosis was not involved in the reduced functionality of neutrophils or in their reduced viability, but bovine neutrophils incubated with AOPP-BSA seem to be "accompanied" to the early phases of the 'extrinsic' pathways of apoptosis. In addition, the present work wanted to evaluate the capacity of triggered neutrophils to generate AOPP in vitro. BSA was incubated with un-stimulated and PMA-stimulated bovine neutrophils for 1-2-3 hours and the production of specific markers of protein oxidation such as AOPP, dityrosines and carbonyls was assessed. BSA incubated with stimulated neutrophils presents a significant higher level of AOPP and dityrosines respects the incubation with un-stimulated neutrophils. Carbonyls don't seem to be produced in these condition, at least at the beginning of the incubation. In parallel, BSA incubated with the same concentration of HOCl produced by PMA-stimulated neutrophils, for 1-2-3 hours, presents a higher level of AOPP, dityrosines and carbonyls. Therefore, it's possible to conclude that bovine neutrophils are able to oxidize BSA in vitro and generate chemical and structural modification such as AOPP and dityrosines, in the experimental condition used. However, carbonyls seem to be a non-specific indicator of neutrophils-mediated protein oxidation. The direct exposure of BSA to HOCl couldn't fully mimic the complex events leading to BSA oxidation and AOPP production by activated neutrophils.

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.