The use of high expansion foam for stunning and killing pigs and poultry.

The EFSA Panel on Animal Health and Welfare (AHAW) was asked to deliver a scientific opinion on the use of high-expansion foam for stunning and killing pigs and poultry. A dossier was provided by the applicant as the basis for an assessment of the extent to which the method is able to provide a level of animal welfare at least equivalent to that ensured by the currently allowed methods for pigs and poultry. According to legislation, to be approved in the EU, new stunning methods must ensure (1) the absence of pain, distress or suffering until the onset of unconsciousness, and (2) that the animal remains unconscious until death. An ad hoc Working Group set up by EFSA performed the assessment as follows: (1) The data provided were checked against the criteria laid down in the EFSA Guidance (EFSA, 2018), and was found to partially fulfil those criteria; (2) extensive literature search; (3) data extraction for quantitative assessment; (4) qualitative exercise based on non-formal expert elicitation. The assessment led to conclude that it is more likely than not (certainty > 50%-100%) that high-expansion foam for stunning and killing pigs and poultry, named NEFS in container (Nitrogen Expansion Foam Stunning in container), provides a level of welfare at least equivalent to one or more of the currently allowed methods listed in Annex I of Council Regulation (EC) No 1099/2009. The overall assessment of EFSA is valid only under the technical conditions described in this Opinion for laying hens, broiler chickens of all age and pigs weighing 15-41 kg in situations other than slaughter. The overall assessment of EFSA is that NEFS can be suitable for depopulation using containers for pig and poultry farms respecting the technical conditions and the categories and types of animals defined in this Scientific Opinion.

Welfare of ducks, geese and quail on farm.

This Scientific Opinion concerns the welfare of Domestic ducks (Anas platyrhynchos domesticus), Muscovy ducks (Cairina moschata domesticus) and their hybrids (Mule ducks), Domestic geese (Anser anser f. domesticus) and Japanese quail (Coturnix japonica) in relation to the rearing of breeders, birds for meat, Muscovy and Mule ducks and Domestic geese for foie gras and layer Japanese quail for egg production. The most common husbandry systems (HSs) in the European Union are described for each animal species and category. The following welfare consequences are described and assessed for each species: restriction of movement, injuries (bone lesions including fractures and dislocations, soft tissue lesions and integument damage and locomotory disorders including lameness), group stress, inability to perform comfort behaviour, inability to perform exploratory or foraging behaviour and inability to express maternal behaviour (related to prelaying and nesting behaviours). Animal-based measures relevant for the assessment of these welfare consequences were identified and described. The relevant hazards leading to the welfare consequences in the different HSs were identified. Specific factors such as space allowance (including minimum enclosure area and height) per bird, group size, floor quality, characteristics of nesting facilities and enrichment provided (including access to water to fulfil biological needs) were assessed in relation to the welfare consequences and, recommendations on how to prevent the welfare consequences were provided in a quantitative or qualitative way.

Welfare of calves.

This Scientific Opinion addresses a European Commission request on the welfare of calves as part of the Farm to Fork strategy. EFSA was asked to provide a description of common husbandry systems and related welfare consequences, as well as measures to prevent or mitigate the hazards leading to them. In addition, recommendations on three specific issues were requested: welfare of calves reared for white veal (space, group housing, requirements of iron and fibre); risk of limited cow-calf contact; and animal-based measures (ABMs) to monitor on-farm welfare in slaughterhouses. The methodology developed by EFSA to address similar requests was followed. Fifteen highly relevant welfare consequences were identified, with respiratory disorders, inability to perform exploratory or foraging behaviour, gastroenteric disorders and group stress being the most frequent across husbandry systems. Recommendations to improve the welfare of calves include increasing space allowance, keeping calves in stable groups from an early age, ensuring good colostrum management and increasing the amounts of milk fed to dairy calves. In addition, calves should be provided with deformable lying surfaces, water via an open surface and long-cut roughage in racks. Regarding specific recommendations for veal systems, calves should be kept in small groups (2-7 animals) within the first week of life, provided with ~ 20 m2/calf and fed on average 1 kg neutral detergent fibre (NDF) per day, preferably using long-cut hay. Recommendations on cow-calf contact include keeping the calf with the dam for a minimum of 1 day post-partum. Longer contact should progressively be implemented, but research is needed to guide this implementation in practice. The ABMs body condition, carcass condemnations, abomasal lesions, lung lesions, carcass colour and bursa swelling may be collected in slaughterhouses to monitor on-farm welfare but should be complemented with behavioural ABMs collected on farm.

Welfare of broilers on farm.

This Scientific Opinion considers the welfare of domestic fowl (Gallus gallus) related to the production of meat (broilers) and includes the keeping of day-old chicks, broiler breeders, and broiler chickens. Currently used husbandry systems in the EU are described. Overall, 19 highly relevant welfare consequences (WCs) were identified based on severity, duration and frequency of occurrence: 'bone lesions', 'cold stress', 'gastro-enteric disorders', 'group stress', 'handling stress', 'heat stress', 'isolation stress', 'inability to perform comfort behaviour', 'inability to perform exploratory or foraging behaviour', 'inability to avoid unwanted sexual behaviour', 'locomotory disorders', 'prolonged hunger', 'prolonged thirst', 'predation stress', 'restriction of movement', 'resting problems', 'sensory under- and overstimulation', 'soft tissue and integument damage' and 'umbilical disorders'. These WCs and their animal-based measures (ABMs) that can identify them are described in detail. A variety of hazards related to the different husbandry systems were identified as well as ABMs for assessing the different WCs. Measures to prevent or correct the hazards and/or mitigate each of the WCs are listed. Recommendations are provided on quantitative or qualitative criteria to answer specific questions on the welfare of broilers and related to genetic selection, temperature, feed and water restriction, use of cages, light, air quality and mutilations in breeders such as beak trimming, de-toeing and comb dubbing. In addition, minimal requirements (e.g. stocking density, group size, nests, provision of litter, perches and platforms, drinkers and feeders, of covered veranda and outdoor range) for an enclosure for keeping broiler chickens (fast-growing, slower-growing and broiler breeders) are recommended. Finally, 'total mortality', 'wounds', 'carcass condemnation' and 'footpad dermatitis' are proposed as indicators for monitoring at slaughter the welfare of broilers on-farm.

Welfare of pigs on farm.

This scientific opinion focuses on the welfare of pigs on farm, and is based on literature and expert opinion. All pig categories were assessed: gilts and dry sows, farrowing and lactating sows, suckling piglets, weaners, rearing pigs and boars. The most relevant husbandry systems used in Europe are described. For each system, highly relevant welfare consequences were identified, as well as related animal-based measures (ABMs), and hazards leading to the welfare consequences. Moreover, measures to prevent or correct the hazards and/or mitigate the welfare consequences are recommended. Recommendations are also provided on quantitative or qualitative criteria to answer specific questions on the welfare of pigs related to tail biting and related to the European Citizen's Initiative 'End the Cage Age'. For example, the AHAW Panel recommends how to mitigate group stress when dry sows and gilts are grouped immediately after weaning or in early pregnancy. Results of a comparative qualitative assessment suggested that long-stemmed or long-cut straw, hay or haylage is the most suitable material for nest-building. A period of time will be needed for staff and animals to adapt to housing lactating sows and their piglets in farrowing pens (as opposed to crates) before achieving stable welfare outcomes. The panel recommends a minimum available space to the lactating sow to ensure piglet welfare (measured by live-born piglet mortality). Among the main risk factors for tail biting are space allowance, types of flooring, air quality, health status and diet composition, while weaning age was not associated directly with tail biting in later life. The relationship between the availability of space and growth rate, lying behaviour and tail biting in rearing pigs is quantified and presented. Finally, the panel suggests a set of ABMs to use at slaughter for monitoring on-farm welfare of cull sows and rearing pigs.

Methodological guidance for the development of animal welfare mandates in the context of the Farm to Fork Strategy.

This document provides methodological guidance developed by the EFSA Panel on Animal Health and Welfare to produce Scientific Opinions in response to mandates received from the European Commission in the context of the Farm to Fork Strategy. The mandates relate to the welfare of (i) animals during transport, (ii) calves, (iii) laying hens, (iv) broilers, (v) pigs, (vi) ducks, geese and quails, and (vii) dairy cows. This guidance was developed in order to define the methods and strategy to be applied for responding to the Terms of Reference (ToRs) of the mandates. The mandates each consist of a set of General ToRs which refer to the husbandry systems used in the production of each animal species or the current transport practices for free moving animals and animals transported in cages, and a set of specific ToRs for which difficulties in ensuring animal welfare have been identified and where specific scenarios are envisaged. Part I of the guidance includes a description of welfare consequences for the animals. Part II includes a new methodology for providing quantitative recommendations regarding animal welfare. The proposed methodology follows the assumption that the effect of an exposure variable (e.g. space allowance) on animal welfare can be quantified by comparing the expression of an animal-based measure (ABM) under 'unexposed conditions' (e.g. unlimited space) and under high exposure (e.g. restrictive conditions). The level of welfare as assessed through this ABM can be quantified for different levels of the exposure variable (e.g. at increasing space allowances) and quantitative recommendations can thus be provided. The final version of the methodological guidance was endorsed for public consultation, which took place between 14 February 2022 and 31 March 2022. The comments received are integrated in this document.

A molecular insight into the lipid changes of pig Longissimus thoracis muscle following dietary supplementation with functional ingredients.

In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning.

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.

Behavior and Welfare of Undocked Heavy Pigs Raised in Buildings with Different Ventilation Systems.

The present study aimed to evaluate animal welfare of pigs from the same farm, raised with two ventilation systems. The study involved 60 pens of fattening pigs, raised in two buildings: one naturally ventilated (NV) and the other mechanically ventilated (MV). Pigs were assessed on three observation days: at 40 kg (T1), 100 kg (T2), and 160 kg (T3) of live weight. Animal-based measures were used such as qualitative behavioral analysis (QBA), behavioral measures (BMs), and lesion and health measures (LHMs). Housing conditions (HCs) measured at each observation day were the number of pigs per pen, space allowance, temperature, light, and CO2. The association study was performed using a general linear model and analysis of variance. Ventilation effect was analyzed by performing computational fluid dynamics. Results showed that overall pigs raised in the MV were in a more positive affective state. Despite that, with hot temperatures, the higher occurrence of pig soiling indicated heat stress in pigs and consequent welfare impairment. The higher frequency of pigs showing dog sitting behavior at T2 and T3 suggest welfare worsening in the last phases of fattening. The study concludes that ventilation system influences animal behavior and overall animal welfare, especially during the warmer season.

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.

The multivariate approach identifies relationships between pre-slaughter factors, body lesions, ham defects and carcass traits in pigs.

Abattoir meat inspection has been proposed for the collection of welfare outcomes. The identification of suitable animal-based measures (ABM) is still a critical point that needs to be implemented to avoid collinearity among measures. The present study aims to benchmark the presence of ABM such as skin and tail lesions and ham defects in carcasses from 79 batches of Italian Heavy pigs and to identify possible relationships between the assessed ABM and pre-slaughter factors such as the season and the overnight lairage. Furthermore, the study also considers the effect of pre-slaughter conditions and ABM on carcass traits parameters (cold carcass weight and lean meat percentage). Skin and tail lesions were recorded at the slaughter line. The presence of abscesses, muscle tears and veining defects were assessed in the hams at trimming, according to the Parma Ham Consortium. A multivariate analysis was performed to identify relationships between ABM and pre-slaughter factor; therefore, a linear model was built to assess the effect on carcass weight and lean meat percentage. Main welfare issues were represented by skin and tail lesions and muscle tears (prevalence above 10%). Multivariate analysis evidenced that skin lesions and veining defects were mostly associated with the warm season. Abscesses and muscle tears in the hams were more likely related to overnight lairage, while tail lesions contributed equally to both season and lairage. Moreover, lairage related factors showed to affect lean meat percentage. The findings of the present study suggest that ham defects might be useful indicators of pre-slaughter stress. The validation of these findings with physiological parameters could be of interest for further studies.

On-Farm Welfare Assessment Protocol for Suckling Piglets: A Pilot Study.

Piglets experience welfare issues during the nursery phase. This pilot study aimed to test a protocol for identifying the main welfare issues in suckling piglets and to investigate relationships among animal-based indicators and management conditions. Litters (n = 134), composed of undocked and tail-docked piglets, were assessed at two farms. After birth, observations were made at the age of 7 days and 20 days. At each observation, housing conditions (HCs) were measured, and 13 animal-based indicators, modified from Welfare Quality, Classyfarm, Assurewel and others introduced ex novo, were recorded. A generalized linear mixed model was used, considering animal-based indicators as dependent variables and farm, piglets' age, tail docking and HCs as independent variables. The main welfare issues were lesions of the limb (32.6%) and the front area of the body (22.8%), a poor body condition score (BCS) (16.1%), ear lesions (15.5%), and tail lesions (9.7%). Negative social behaviour (e.g., fighting and biting) represented 7.0% of the active behaviour, with tail biting observed in 8.7% of the piglets. While lesions on the front areas of the body were mostly associated with the farm, tail lesions, low BCS, tear staining, and diarrhoea were associated with light and nest temperature (p < 0.05). In particular, tail biting increased with scarce light (p = 0.007). Tail docking did not influence any animal-based indicator except for tear staining which was higher in the tail-docked as compared to the undocked piglets (p = 0.05), increasing awareness on this practice as a source of negative emotion in piglets. The protocol tested may be a promising tool for assessing on-farm piglets' welfare.

In ovo Injection of a Galacto-Oligosaccharide Prebiotic in Broiler Chickens Submitted to Heat-Stress: Impact on Transcriptomic Profile and Plasma Immune Parameters.

This study investigated the effects of a galactooligosaccharide (GOS) prebiotic in ovo injected on intestinal transcriptome and plasma immune parameters of broiler chickens kept under thermoneutral (TN) or heat stress (HS) conditions. Fertilized Ross 308 eggs were injected in ovo with 0.2 mL physiological saline without (control, CON) or with 3.5 mg of GOS (GOS). Three-hundred male chicks/injection treatment (25 birds/pen) were kept in TN or HS (30 °C) conditions during the last growing phase, in a 2 × 2 factorial design. At slaughter, from 20 birds/injection group (half from TN and half from HS), jejunum and cecum were collected for transcriptome analysis, and plasma was collected. No differences in plasma parameters (IgA and IgG, serum amyloid) and no interaction between injection treatment and environment condition were found. GOS-enriched gene sets related to energetic metabolism in jejunum, and to lipid metabolism in cecum, were involved in gut barrier maintenance. A homogeneous reaction to heat stress was determined along the gut, which showed downregulation of the genes related to energy and immunity, irrespective of in ovo treatment. GOS efficacy in counteracting heat stress was scarce after ten days of environmental treatment, but the in ovo supplementation modulates group of genes in jejunum and cecum of broiler chickens.

Functional analysis finds differences on the muscle transcriptome of pigs fed an n-3 PUFA-enriched diet with or without antioxidant supplementations.

Supplementing pig diets with n-3 polyunsaturated fatty acids (n-3 PUFA) may produce meat products with an increased n-3 fatty acid content, and the combined antioxidants addition could prevent lipid oxidation in the feed. However, to date, the effects of these bioactive compounds at the molecular level in porcine skeletal muscle are mostly unknown. This study aimed to analyse changes in the Longissimus thoracis transcriptome of 35 pigs fed three diets supplemented with: linseed (L); linseed, vitamin E and Selenium (LES) or linseed and plant-derived polyphenols (LPE). Pigs were reared from 80.8 ± 5.6 kg to 151.8 ± 9.9 kg. After slaughter, RNA-Seq was performed and 1182 differentially expressed genes (DEGs) were submitted to functional analysis. The L vs LES comparison did not show differences, while L vs LPE showed 1102 DEGs and LES vs LPE 80 DEGs. LPE compared to the other groups showed the highest number of up-regulated genes involved in preserving muscle metabolism and structure. Results enlighten that the combined supplementation of bioactive lipids (n-3 PUFA from linseed) with plant extracts as a source of polyphenols increases, compared to the only addition of linseed, the expression of genes involved in mRNA metabolic processes and transcriptional regulation, glucose uptake and, finally, in supporting muscle development and physiology. These results improve the knowledge of the biological effect of bioactive compounds in Longissimus thoracis muscle, and sustain the growing interest over their use in pig production.

Effect of diets supplemented with linseed alone or combined with vitamin E and selenium or with plant extracts, on Longissimus thoracis transcriptome in growing-finishing Italian Large White pigs.

BACKGROUND: Supplementing farm animals diet with functional ingredients may improve the nutritional quality of meat products. Diet composition has been also demonstrated to influence the gene expression with effect on biological processes and pathways. However, the knowledge on the effect of nutrients at the molecular level is scant. In particular, studies on the effects of antioxidants and polyphenols dietary supplementation have been investigated mainly in rodents, and only scarcely in farm animals so far. RNA-Seq with next-generation sequencing is increasingly the method of choice for studying changes in the transcriptome and it has been recently employed also in pig nutrigenomics studies to identify diet-induced changes in gene expression. The present study aimed to investigate the effect of diets enriched with functional ingredients (linseed, vitamin E and plant extracts) on the transcriptome of pig Longissimus thoracis to elucidate the role of these compounds in influencing genes involved in muscle physiology and metabolism compared to a standard diet. RESULTS: Eight hundred ninety-three significant differentially expressed genes (DEGs) (FDR adjusted P-value ≤ 0.05) were detected by RNA-Seq analysis in the three diet comparisons (D2-D1, D3-D1, D4-D1). The functional analysis of DEGs showed that the diet enriched with n-3 PUFA from linseed (D2) mostly downregulated genes in pathways and biological processes (BPs) related to muscle development, contraction, and glycogen metabolism compared to the standard diet. The diet supplemented with linseed and vitamin E/Selenium (D3) showed to mostly downregulate genes linked to oxidative phosphorylation. Only few genes involved in extracellular matrix (ECM) organization were upregulated by the D3. Finally, the comparison D4-D1 showed that the diet supplemented with linseed and plant extracts (D4) upregulated the majority of genes compared to D1 that were involved in a complex network of pathways and BPs all connected by hub genes. In particular, IGF2 was a hub gene connecting protein metabolism, ECM organization, immune system and lipid biosynthesis pathways. CONCLUSION: The supplementation of pig diet with n-3 PUFA from linseed, antioxidants and plant-derived polyphenols can influence the expression of a relevant number of genes in Longissimus thoracis muscle that are involved in a variety of biochemical pathways linked to muscle function and metabolism.

Effect of dietary polyunsaturated fatty acid and antioxidant supplementation on the transcriptional level of genes involved in lipid and energy metabolism in swine.

Porcine fat traits depend mostly on the interaction between nutritional and genetic factors. However, the pathways and biological processes influenced by this interaction are still poorly known in pigs, although they can have a huge impact on meat quality traits. The present research provides new knowledge insight into the effect of four diets (D1 = standard diet; D2 = linseed supplementation; D3 = linseed, vitamin E and selenium supplementation; D4 = linseed and plant-derived polyphenols supplementation) on the expression of 24 candidate genes selected for their role in lipid and energy metabolism. The data indicated that 10 out of 24 genes were differentially expressed among diets, namely ACACA, ADIPOQ, ADIPOR1, CHREBP (MLXPL), ELOVL6, FASN, G6PD, PLIN2, RXRA and SCD. Results from the univariate analysis displayed an increased expression of ACACA, ADIPOQ, ADIPOR1, CHREBP, ELOVL6, FASN, PLIN2, RXRA and SCD in D4 compared to D2. Similarly, ACACA, ADIPOQ, ADIPOR1, ELOVL6 and SCD were highly expressed in D4 compared to D3, while no differences were observed in D2-D3 comparison. Moreover, an increased expression of G6PD and ELOVL6 genes in D4 compared to D1 was observed. Results from the multivariate analysis confirmed that D2 was not different from D3 and that ACACA, SCD and FASN expression made D4 different from D2 and D3. Comparing D4 and D1, the expression levels of ELOVL6 and ACACA were the most influenced. This research provides evidence that the addition of both n-3 PUFA and polyphenols, derived from linseed, grape-skin and oregano supplementation in the diets, stimulates the expression of genes involved in lipogenesis and in oxidative processes. Results evidenced a greater effect on gene expression of the diet added with both plant extracts and n-3 PUFA, resulting in an increased expression of genes coding for fatty acid synthesis, desaturation and elongation in pig Longissimus thoracis muscle.

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.