Toward an integrated ethical review process: an animal-centered research framework for the refinement of research procedures.

The involvement of animals in research procedures that can harm them and to which they are deemed unable to consent raises fundamental ethical dilemmas. While current ethical review processes emphasize the application of the 3Rs (Replacement, Reduction, and Refinement), grounded in a human-centered utilitarian ethical approach, a comprehensive ethical review also involves a harm-benefit analysis and the consideration of wider ethical issues. Nevertheless, to our knowledge, approaches are still needed to facilitate the integrative assessment and iterative revision of research designs to improve their ethical value or to identify cases in which using animals is irremediably unethical. Additionally, frameworks are lacking that explicitly include an animal-centered perspective into the ethical review process beyond welfare concerns, failing to cover broader ethical considerations (such as consent). In previous work we proposed an Animal-Centered Research framework (ACRf) comprising four animal-centered research principles (relevance, impartiality, welfare and consent) which could help researchers and ethical review bodies apprise research designs from an animal-centered perspective. This paper builds on and further develops our previous work by contextualizing the ACRf within the bigger picture of animal research ethical review and by illustrating how the ACRf could be operationalized within current ethical review processes. We contribute an extended framework that integrates the application of the ACRf principles within the ethical review process. To this end, we present findings from a theoretical case study focusing on the ethical review of a research protocol on the study of stress response in pigs. We discuss how our extended framework could be easily applied to facilitate a holistic approach to the ethical review process, and inform an iterative process of refinement, to support the development of research designs that are both more ethical and scientifically valid.

Assessing Substrate Utilization and Bioconversion Efficiency of Black Soldier Fly (Hermetia illucens) Larvae: Effect of Diet Composition on Growth and Development Temperature.

Black soldier fly larvae (BSFL) can utilize food by-products or residues for growth, benefiting farm animal's diets' production sustainability. The experiment aimed to assess the effect of different substrate compositions on larval growth, chemical composition, and substrate temperature. BSFL were allocated to one of the four diets (control, vegetable, carnivorous, and omnivorous) for the entire experiment (8 days). The temperature was measured twice daily using a thermal-imaging camera, and the accumulated degree hours (ADH) was calculated. The results showed that the larvae fed the vegetable diet exhibited a significantly reduced growth performance, with a biomass reduction of 26.3% compared to the control diet; furthermore, vegetable-fed larvae showed a lower dry matter content (-30% compared to the average of other diets) due to lower fat content (-65% compared to average of other diets). The nutritional composition of larvae fed an omnivorous diet was similar to larvae fed a high-quality substrate diet (control diet-chicken feed), indicating that the omnivorous diet could be an ideal solution for rearing BSFL larvae; however, the current European legislation prohibits the use of animal meal. The study also revealed that substrate temperatures did not have a discernible influence on larval growth, further emphasizing the importance of diet in BSFL rearing strategies.

Can environmental nebulization of lavender essential oil (L. angustifolia) improve welfare and modulate nasal microbiota of growing pigs?

The use of phytoextracts has been proposed as a method to improve animal welfare, also in pigs, by reducing stress and anxiety and improving performances. Lavandula angustifolia (Miller) essential oil (LaEO) is an interesting calming phytoextract that could be administered by inhalation for prolonged periods of time to help pigs coping with on-farm conditions. The aim of this study was to assess the effects of daily inhalation of vaporized LaEO on pigs' welfare and health indicators, and nasal microbiota, trying to understand whether this phytoextract represents a feasible tool to improve animal welfare under intensive farming conditions. Eighty-four crossbred barrows were randomly divided into 3 experimental groups: control (C); lavender (L): 3 vaporization sessions of 10 min each of a custom made 1% solution of LaEO; sham (S): same vaporization sessions of L group but only using the solution vehicle. Experimental readouts included growth parameters, behavioural traits, tail and skin lesions, hair steroids and nasal microbiota. L group animals did not show altered growth performance and seemed calmer (increased recumbency time), with decreased amount of skin lesions also associated with lower severity class for tail lesions. They also showed decreased CORT/DHEA ratio, potentially suggesting a beneficial effect of LaEO. Inhalation of LaEO significantly affected the nasal pig microbiome by reducing its diversity. Overall, the study suggests how inhalation of Lavender essential oil may be capable of improving welfare in growing pigs, yet it is pivotal to consider the microbial modulatory capabilities of essential oils before exploiting them on larger scale.

Study on the suitability of life cycle assessment for the estimation of donkey milk environmental impact.

In the last decades, western Countries increased their interest in innovative products like donkey milk and other activities carried out with donkeys (onotherapy, onotourism). Donkey milk is considered a high-added-value food and is very similar to human breast milk. It is also used as an ingredient in cosmetics. The growing public interest suggests the need for a pilot study on the sustainability of donkey milk production, according to Life Cycle Assessment (LCA) criteria. Milk was used as the Declared Functional Unit (DFU) and two different models were described, a Real Scenario Model (RSM, i.e. a farm with its declared milk yield), and an Increased Milk Production Model (IMPM, i.e., the same farm with theoretically increased milk yield). Allocation was applied both in RSM and IMPM; thus, different values of impact categories, i.e., Global Warming Potential (GWP, kg CO2 equivalents), Acidification Potential (ACP, g SO2 equivalents) and Eutrophication Potential (EUP, g PO43-) were observed. GWP improved after mass allocation and showed the lowest equivalents in IMPM, compared to economic and reference allocation criterion (P < 0.05). In RSM, allocations affected GWP in a different way: the smaller size of the DFU resulted in the largest estimation of CO2 equivalents (P < 0.05) for reference allocation, whereas the mass allocation estimates were lower than with economic allocation (P < 0.05). ACP and EUP followed the same trends. No differences were found in IMPM results across the three allocation methods used. Moreover, mass allocation values recorded in RSM did not significantly differ from IMPM. ACP and EUP of RSM improved after economic allocation, although they were less sustainable (P < 0.05) than all IMPM values and RSM equivalents after mass allocation (P < 0.05). As expected, the theoretical model with increased milk yield improved the sustainability of the system. Both scenarios were affected by allocation criteria. In RSM, the economic and mass allocations described a representative scenario where donkey meat contributed to subtracting equivalents from milk (the main product). The present paper is a pilot study estimating for the first time the environmental impact of donkey milk production, with the aim to stimulate further research.

Growth Performance and Feed Intake Assessment of Italian Holstein Calves Fed a Hay-Based Total Mixed Ration: Preliminary Steps towards a Prediction Model.

The aim of this study was to evaluate the effects of a complete hay-based total mixed ration (TMR) for calves, focusing on their feed intake, animal growth performance, and fecal output, and to develop a preliminary estimation equation for solid feed intake and body weight in Holstein heifer calves. Twenty female Italian Holstein calves (37.14 ± 2.72 kg) born between February and July were studied from the day of birth until 77 days of age. From the fourth day of life, they were fed 3 L/day of pasteurized milk twice daily and supplemented with the same hay-based TMR. The data on feed intake, fecal characteristics, and growth performances were collected and showed that these calves had adequate parameters. Moreover, the data collected was used to create equations to predict body weight and solid feed intake using a mixed model. The goodness of fit of the developed equations was evaluated by coefficients of determinations (R2). The equation obtained shows high R2 (0.98 for solid feed intake and 0.99 for calf weight), indicating the satisfactory precision and accuracy needed to predict female calves' body weight and solid feed intake.

Effects of Lavender Essential Oil Inhalation on the Welfare and Meat Quality of Fattening Heavy Pigs Intended for Parma Ham Production.

We assessed the effects of inhalation administration of lavender essential oil (LEO) either once (L1) or twice (L2) a day on animal welfare indicators, carcass and meat quality of Italian heavy pigs. Pigs (n = 108) were allotted to three experimental groups (control -C-, L1 and L2) and lavender was administered, via a vaporizer device, to the treated groups during the entire fattening-finishing period (79-160 kg BW). Tail lesion severity was reduced in L1 at the end of the trial compared to the other groups (p < 0.05). Body lesion counts, however, were higher in L2 than in C (p < 0.05), resulting in a more severe overall damage classification (p < 0.01). At slaughter, no differences were observed in carcass traits or blood stress indicators, only minor differences were observed in meat quality, and no LEO residual was found in fat or lean tissues, highlighting the preserved suitability of thighs for the dry curing process. While it was not possible to conclude on the ability to improve animal welfare of vaporized LEO in this production phase, the absence of adverse effects on meat quality and the discrepancies observed regarding the body lesions in L1 and L2 make further studies on behavioral aspects and the method of administration (route, frequency) of the product desirable.

Mortality during Transport of Pigs Subjected to Long Journeys: A Study in a Large European Abattoir.

To date, especially in Europe, few studies have analysed the implications of long journeys on pig welfare and economic losses, expressed in terms of transport mortality. This study retrospectively analysed data collected from slaughtering registers and travel journals in a large Italian abattoir. We focused on pig transports coming from abroad and arriving at the slaughter plant after long journeys (a total of 59,982 pigs over 370 journeys). We explored the relationship between mortality and the following variables: country of origin, journey duration, astronomical season, stocking density on the truck, number of stops, and prolonged stops during the journey (lasting more than 60 min, likely due to traffic jams or truck problems). Overall, the low mortality rate observed (0.09%) was in line with European estimates. The factors with a significant or tendential effect on mortality during transport were the astronomical season (p = 0.0472, with higher mortality in spring) and the presence of prolonged stops during the journey (p = 0.069, tendential effect). Journey duration, stocking density, country of origin, and the number of stops were not statistically significant. In conclusion, based on this case study, using transport mortality combined with data collected during the common routine activity by the Public Veterinary Services in slaughterhouses could be a simple screening method for identifying problematic journeys or transport conditions.

Influence of Increased Freedom of Movement on Welfare and Egg Laying Pattern of Hens Kept in Aviaries.

This work investigates the effects of structural modifications on the welfare level and laying patterns of hens in a three-tier commercial aviary system. Four experimental groups were used: C (control, housed in a traditional aviary); LM (longitudinal movement, in which internal partitions were removed); VM (vertical movement, in which ramps were installed); and FM (freedom of movement, both LM and VM modifications). Hens showed worse body condition scores (p < 0.05) in all the modified aviaries, while plumage condition was improved in FM but worsened in VM (p < 0.05). No significant effect was observed on egg deposition patterns, egg quality or keel bone damage. When ramps were available (VM and FM groups), hens reduced the number of flights and increased the number of walks from 0.52 to 7.7% of the displacements on average (p < 0.05). Apart from some feather pecking concerns in VM (likely due to overcrowding in some favourite aviary areas), LM and FM seemed to facilitate animal movement and promote species−specific behaviour. It is concluded that hen welfare in aviary systems can be improved by means of tailored structural modifications. Producers may therefore adopt some of these modifications (providing ramps and/or removing vertical barriers) to enhance the welfare of hens.

Literature Review on the Pre-Slaughter Welfare of Italian Heavy Pigs.

This work provides a narrative review of the available information on the welfare of Italian heavy pigs in the pre-slaughter phase (transport, lairage, and stunning). The meat from these pigs is used for specific PDO (Protected Designation of Origin) products, and the production rules for these specialties require higher body weight (160-170 kg) and age (in general more than 9 months) at slaughter than in most other countries. This may lead to specific behavioral and physiological needs of pigs. The present paper summarizes the main research findings and knowledge gaps for each of the pre-slaughter phases. Studies are presented according to the four principles of the Welfare Quality assessment protocol (good feeding, good housing, good health, and appropriate behavior). The results of the literature review indicate a lack of knowledge on several aspects. Most of studies were carried out in a single slaughterhouse, making it difficult to identify risk factors and confounding effects. Moreover, animal-based measures were assessed using different protocols, reducing the possibility of comparison across studies. These findings may serve as a basis for the development of specific research studies and policies aimed at enhancing the animal welfare level and the ethical attributes of this renowned production, also in accordance with consumers' expectations.

Update on animal welfare perception by Italian consumers: A descriptive survey.

The aim of this study was to evaluate if and how the perception of animal welfare by Italian consumers and their purchasing behavior of animal-friendly foods have evolved in recent years. The research was carried out through a survey on a representative sample of Italian consumers (n=969). Responses showed that 69% of consumers declared to pay attention to animal welfare at time of purchasing, and they mostly acquired information about animal welfare through the mass media. In accordance with previous surveys, the categories perceived as having the highest welfare level were fish and dairy cows, whereas the lowest welfare level was attributed to broilers and pigs. Compared to previous surveys, Italian consumers seem to have improved their knowledge about the legislation on animal protection and increased their interest toward animal welfare issues. However, they still find it difficult to identify animalfriendly labels, together with a lack of knowledge about non-conventional (e.g. extensive and organic) farming systems.

Knowledge and Perspectives on the Welfare of Italian Heavy Pigs on Farms.

This review aims to give an overview of the most significant available information on welfare issues of Italian heavy pigs on farms. These animals, whose meat is used to produce typical products, are characterized by much higher body weights and age at slaughter (approximately 160-170 kg, and 9 months of age) than most pigs reared in Europe, resulting in peculiar behavioral and rearing-related needs. To highlight the main findings and the areas in which additional research is needed, the paper was organized by allocating the studies on the basis of the Welfare Quality four principles framework, i.e., good feeding, good housing, good health, and appropriate behavior. The results indicate the possibility of improvement with regard to the management of feeding, watering, and some environmental parameters (light, thermal comfort, enrichments) and a lack of knowledge on the actual space requirements (and a corresponding regulatory gap). Moreover, deficiencies concerning the prevalence of injuries and disease and the relationship between lesions observed post-mortem and rearing conditions needs to be addressed. Lastly, the absence of research concerning the evaluation of the emotional state of animals has been highlighted. It is hoped that these findings will, in the future, serve as a basis for the development of specific policies for these animals in order to increase the ethical value of the entire production chain, in accordance with consumers' demand and expectation for high animal welfare standards.

Use of thermography in pigs: relationship between surface and core temperature.

This study aims to assess the correlation between surface temperature estimated by infrared thermography and core temperature measured with rectal thermometer in weaning and fattening pigs. A total of 108 pigs were used in this study. Thermal images of the eye of each animal were recorded with a thermal imaging camera, rectal temperatures were measured using a calibrated digital thermometer. The average rectal temperature was 38.9 ± 0.4 °C (MIN = 37.9 °C; MAX = 40.1 °C) and the average eye temperature was 36.7 ± 0.1 °C (MIN = 34.8 °C; MAX = 38.8 °C). Our results showed that the mean eye temperature estimated by infrared thermography was significantly correlated (r = .581, P < .01) with rectal temperature. The correlation was significant and strong for weaners (r = .739, P < .01), significant although weak for fatteners (r = .236 P < .05). Thermography could be a valid method to estimate the core temperature of pigs under farm condition.

Pre-Slaughter Sources of Fresh Meat Quality Variation: The Case of Heavy Pigs Intended for Protected Designation of Origin Products.

This study focused on loin quality in Italian heavy pigs intended for the production of PDOs (Protected Designation of Origin) products, and investigated the pre-slaughter factors which negatively affect the quality of fresh meat. Data were collected on 44 shipments (loads) of pigs. Shipments were carried out under commercial conditions. Several pre-slaughter parameters were recorded within the entire process (on-farm, during transport, and at the slaughterhouse). On a subset of pigs (10 animals from every load, N = 440), serum cortisol and creatine kinase were measured and loin samples were analyzed for pH, instrumental color, drip loss, cooking loss, shear force, and sensory quality. Cluster analysis of the instrumentally-assessed meat quality parameters allowed the categorization of the shipments into two clusters: lower quality (LQ) and higher quality (HQ). Our results showed that the factors with significant differences between the two clusters were journey duration, ambient temperature, distance traveled, and irregular behaviors (slipping, falling, and overlapping) at unloading (all greater in LQ, p < 0.05). The pre-slaughter conditions associated with lower loin quality were ambient temperatures above 22 °C, distance traveled above 26 km, travel duration between 38-66 min, more than 5.9% of animals showing irregular behaviors at unloading.

Soiling of Pig Pens: A Review of Eliminative Behaviour.

This is a comprehensive review on the pigs' normal eliminatory behaviour (i.e., defaecation and urination) and pen soiling. This review is aimed primarily at solving issues with pen soiling in current systems, and ultimately at the future design of a well-functioning pig toilet, which we intend to elaborate on in a subsequent publication. In this paper, first, normal elimination is described in relation to what is known about its phylogeny, ontogeny, causation, and function, i.e., according to Tinbergen's four why questions concerning animal behaviour. Then, pen soiling is described as if it were a medical disorder, highlighting its importance, aetiology, symptoms, diagnosis, pathogenesis, treatment, and prevention. Due to its negative consequences in terms of animal welfare, health, workload, and environmental emissions, possible methods to address pen soiling in current systems are described. Probably, pigs do not choose a specific place to eliminate but rather choose the most comfortable place for resting, and avoid eliminating there. We identified four main strategies to reduce pen soiling: (1) reducing the suitability of the designated elimination area to be used for other functions, especially resting or thermoregulation; (2) improving the suitability of other functional areas in the pen to be used for their specific function, such as resting and activity; (3) reducing the suitability of other functional areas to be used for elimination; and (4) improving the suitability of the elimination area for elimination. These prevention strategies and the encompassing disease framework provide a structured approach to deal with pen soiling in existing systems and to support the future design, development, and implementation of a well-functioning pig toilet that can help to achieve some of the main goals of modern pig production, namely reducing environmental emissions as well as substantially improving pig welfare.

Health Risk Perception, Consumption Intention, and Willingness to Pay for Pig Products Obtained by Immunocastration.

Surgical castration without the use of anaesthesia and/or analgesia is considered to be detrimental for the welfare of pigs and for this reason its abandonment is advocated. Immunocastration is a more welfare-friendly alternative method; however, stakeholders in the pork sector fear consumer rejection due to perceived safety issues of immunocastrated meat. This work aimed to analyse whether Italian consumers perceive a health risk arising from the use of this technique and, if so, how the perceived risk may influence the purchase choices and the willingness to pay for products derived from immunocastrated animals. To achieve this objective, a survey was carried out on a representative sample of the Italian population. The results highlight that consumers perceive different levels of risk related to the use of immunocastration and that this influences purchasing behaviour and willingness to pay. Moreover, it should be noted that the willingness to pay is also influenced by certain demographic factors, since this is positively associated with younger respondents with lower incomes and less knowledge of farming systems, who live in rural areas and have a greater sensitivity to animal welfare. Given the concerns expressed by consumers, particular attention must be paid to the information transmitted if this technology will be widely implemented in pig husbandry.

Identification of Possible Pre-Slaughter Indicators to Predict Stress and Meat Quality: A Study on Heavy Pigs.

This study aimed at identifying possible pre-slaughter indicators and/or indexes to be used to predict pig stress response and meat quality variation. Data were collected on 44 shipments (loads) of Italian heavy pigs. For each shipment, several pre-slaughter parameters were recorded on farm, during transport, and at the slaughterhouse. Blood and meat samples were taken from 10 pigs from every of the 44 loads included in the study (N = 440). Blood samples were used to assess cortisol and creatine kinase levels, whereas meat samples were used to assess meat quality (pH, instrumental color, tenderness, water-holding capacity, and sensory analysis). Cluster analysis of blood parameters allowed the categorization of the shipments into two main clusters: Lower Stress (LS) and Higher Stress (HS). The variables/indexes statistically differing between the two clusters were: average vehicle speed during transport, welfare index at slaughter (i.e., "slaughter score"), overall transport and slaughter welfare index (TSWI), distance travelled, and behaviors (slips, falls, overlaps) during unloading, which appeared to be the best descriptors of the welfare conditions experienced by Italian heavy pigs during pre-slaughter handling. No consistent effects of the stress level experienced on meat quality was detected, which warrants the need for further studies conducted under more variable pre-slaughter conditions, with the aim of simplifying and improving the TSWI.

Towards the Abandonment of Surgical Castration in Pigs: How is Immunocastration Perceived by Italian Consumers?

Immunocastration of pigs represents an alternative method to surgical castration, being more respectful of animal welfare. However, this new technology may not be accepted by consumers due to their perception of possible risks tied to the use of the product, thus representing a concern for the production sector. The study aimed at verifying the attitude of Italian consumers towards immunocastration and to assess whether their perception can be affected by science-based information on advantages and disadvantages of immunocastration. A total of 969 consumers (divided in three groups representative of the Italian population) were contacted and asked to complete an online questionnaire. Only technical (neutral) information on immunocastration was provided to the first group; the second and the third group received information on the advantages (+) and disadvantages (-) of the technique, shown in reverse order (+/- and -/+, respectively). The level of information did not affect consumers' perception of immunocastration. Overall, immunocastration is perceived in a predominantly positive manner (54.5%), with a relatively low level of risk perception (34.2%), and a good willingness to pay more for meat deriving from immunocastrated pigs (+18.7%).

Effects of increased space allowance on animal welfare, meat and ham quality of heavy pigs slaughtered at 160Kg.

Sixty barrows (Body Weight-BW- range: 23.9-160 kg) were allotted to two experimental groups (6 pens of 5 pigs each): the control group was kept at a space allowance of 1m2/head; the second group was kept at 1.3m2/head. Behaviour, growth parameters, carcass and meat quality were assessed, as well as fat and cured ham quality. Results showed that pigs raised at 1.3m2/head spent more time laying (particularly in lateral recumbency, P<0.01 and P<0.001, respectively) compared to pigs kept at lower space allowance. They also reduced the aimless exploration of the slatted pen floor (P<0.001) and increased overall expression of other, mainly active, behaviors (e.g., drinking, walking and standing, P<0.01). Pigs raised at 1.3m2/head showed higher final BW (P = 0.02), more favourable Average Daily Gain (ADG) and gain-to-Feed ratio (G:F) both during the last period of the trial (P<0.05 for both parameters) and over the entire trial (P = 0.01 for both parameters). No significant difference was observed between groups for carcass traits and the main meat quality attributes. Subcutaneous fat from green hams had higher α-linolenic acid content (P<0.01) in the group reared at greater space allowance. Green hams from this group lost less weight at trimming (P<0.01) and the resulting cured hams received better sensory evaluations (P<0.05). No difference was observed in fatty acid composition and unsaturation levels of the subcutaneous fat from cured hams. Our data suggest that heavy pigs intended for Parma ham would benefit from the adoption of higher individual floor space allowances, both in terms of animal welfare (increased possibility to rest) and of productive parameters, without having any detrimental effect on the suitability of the thighs for dry-curing or on the quality of the final product.

Behavioral Assessment of Vision in Pigs.

Swine (Sus scrofa) are often the 'gold standard' laboratory animal for ophthalmology research due to the anatomic and physiologic similarities between the porcine and human eye and retina. Despite the importance of this model, few tools for behavioral vision assessment in pigs are available. The aim of this study was to identify and validate a feasible and reproducible behavioral test to assess vision in a pig model of photoreceptor degeneration. In addition, a robust behavioral test will reduce stress and enhance enrichment by allowing animals opportunities for environmental exploration and by reducing the number of invasive experimental procedures. Two distinct behavioral approaches were tested: the obstacle-course test and temperament test. In the obstacle-course test, pigs were challenged (after an initial training period) to navigate a 10-object obstacle course; time and the number of collisions with the objects were recorded. In the temperament test, the time needed for pigs to complete 3 different tasks (human-approach, novel-object, and open-door tests) was recorded. The obstacle-course test revealed significant differences in time and number of collisions between swine with vision impairment and control animals, and the training period proved to be pivotal to avoid bias due to individual animal characteristics. In contrast, the temperament test was not altered by vision impairment but was validated to measure stress and behavioral alterations in laboratory pigs undergoing experimental procedures, thus achieving marked refinement of the study.