Genomics and New Approaches to Study Complex Traits in Pigs and Other Livestock Species. A Focus on the Investigation of Gene Networks Related to Fat Quality and Deposition in Pigs and Preliminary Research to Study Factors Related to Performances in Piglets and Poultry

The recent development of new technologies and the progress of genetics and genomics have opened new horizons in breeding programs. Genomic selection has been successfully applied to dairy cattle breeding schemes, but had a limited success in other livestock species, such as pigs and chickens. Anyway, the technological advances seen in the last years may represent important tools for the deciphering of complex traits, for which traditional selection has been slowly accelerating. The studies reported in the present thesis are addressed towards the application of different OMICs technologies to the analysis of productive traits in livestock species. The study was mainly focused on the investigation of candidate genes and gene networks associated to porcine fatness traits using genomics, transcriptomics and single gene studies. The results confirmed the important role that ELOVL elongase 6 gene region plays in backfat fatty acid composition, and reported associations between markers on Perilipin genes and fatness traits. Furthermore, a transcriptome analysis performed on backfat samples of pigs divergent for backfat thickness highlighted several expression patterns related to adipose tissue deposition and suggested that Perilipin 2 gene may play an important role in adipose tissue deposition. OMICs technologies were also applied in two preliminary studies performed with the aim of identifying factors and genetic causes involved in variations in pig colostrum metabolome and in the occurrence of myopathy-like defects in breast muscle of broiler chickens. On the whole, the application of genomics, transcriptomics and metabolomics showed to be an effective tool for the study of complex traits in different livestock species, and for the detection of genes involved in phenotypic variations. Further studies are needed, but the found evidences contribute to increase the knowledge about markers useful for the genetic improvement of complex polygenic traits

Molecular Pathways and Key Genes Associated With Breast Width and Protein Content in White Striping and Wooden Breast Chicken Pectoral Muscle

Growth-related abnormalities affecting modern chickens, known as White Striping (WS) and Wooden Breast (WB), have been deeply investigated in the last decade. Nevertheless, their precise etiology remains unclear. The present study aimed at providing new insights into the molecular mechanisms involved in their onset by identifying clusters of co-expressed genes (i.e., modules) and key loci associated with phenotypes highly related to the occurrence of these muscular disorders. The data obtained by a Weighted Gene Co-expression Network Analysis (WGCNA) were investigated to identify hub genes associated with the parameters breast width (W) and total crude protein content (PC) of Pectoralis major muscles (PM) previously harvested from 12 fast-growing broilers (6 normal vs. 6 affected by WS/WB). W and PC can be considered markers of the high breast yield of modern broilers and the impaired composition of abnormal fillets, respectively. Among the identified modules, the turquoise (r = -0.90, p < 0.0001) and yellow2 (r = 0.91, p < 0.0001) were those most significantly related to PC and W, and therefore respectively named “protein content” and “width” modules. Functional analysis of the width module evidenced genes involved in the ubiquitin-mediated proteolysis and inflammatory response. GTPase activator activity, PI3K-Akt signaling pathway, collagen catabolic process, and blood vessel development have been detected among the most significant functional categories of the protein content module. The most interconnected hub genes detected for the width module encode for proteins implicated in the adaptive responses to oxidative stress (i.e., THRAP3 and PRPF40A), and a member of the inhibitor of apoptosis family (i.e., BIRC2) involved in contrasting apoptotic events related to the endoplasmic reticulum (ER)-stress. The protein content module showed hub genes coding for different types of collagens (such as COL6A3 and COL5A2), along with MMP2 and SPARC, which are implicated inCollagen type IV catabolism and biosynthesis. Taken together, the present findings suggested that an ER stress condition may underly the inflammatory responses and apoptotic events taking place within affected PM muscles. Moreover, these results support the hypothesis of a role of the Collagen type IV in the cascade of events leading to the occurrence of WS/WB and identify novel actors probably involved in their onset

Protection of Animals during Transport: Analysis of the Infringements Reported from 2009 to 2013 during On-Road Inspections in Italy

Council Regulation (EC) No 1/2005 requires that vehicles that are transporting animals be subjected to checks conducted by competent authorities. Yearly, each member state sends a report to the European government on the infringements that have been discovered during on-road inspections. The reports that were published by the Italian Ministry of Public Health from 2009 to 2013 were analyzed. Possible associations between the type of infringement (related to animal welfare (AW), vehicle (V) and accompanying documents (D)), year, season, transported species, place of inspection, and competent authorities were identified. A total of 985 infringements were analyzed, with some vehicles receiving more than one (mean: 1.58; max: 9). A score (from 1 to 3) that was related to the severity of the infringements was created. In 2009 and 2010, there was a 50% higher probability of encountering penalties of a lower severity (D or V) than in 2011 (p < 0.0001). Vehicles that were transporting pigs showed the highest probability of committing animal welfare-related infringements (odds ratio (OR) = 3.85, 95% confidence interval (95%CI) = 1.82-8.76, p < 0.0001). Vehicles were four times more likely to suffer animal welfare-related penalties when traffic police worked in synergy with veterinary services (OR = 4.12, 95%CI = 1.70-11.13, p = 0.0005). Vehicles that were transporting Equidae and "other species," including pets, for commercial purposes were more likely to be fined for a lack or incompleteness of the veterinary documents than those transporting cattle (p = 0.002 and p = 0.004, respectively). This study gives statistical evidence of the implementation of EC 1/2005. The training of transporters and drivers on how to manage transport in an animal welfare-friendly manner and a standardized method on how to conduct road inspections among competent authorities are recommended

Time course evaluation of collagen type IV in Pectoralis major muscles of broiler chickens selected for different growth-rates

Collagen type IV (COL4) is one of the major components of animals’ and humans’ basement membranes of several tissues, such as skeletal muscles and vascular endothelia. Alterations in COL4 assembly and secretion are associated to muscular disorders in humans and animals among which growth-related abnormalities such as white striping and wooden breast affecting Pectoralis major muscles (PMs) in modern fast-growing (FG) chickens. Considering the high prevalence of these myopathies in FG broilers and that a worsening is observed as the bird slaughter age is increased, the present study was intended to evaluate the distribution and the expression level of COL4 protein and its coding genes in PMs of FG broilers at different stages of muscle development (i.e., 7, 14, 21, 28, 35, and 42 d of age). Medium-growing (MG) chickens have been considered as the control group in consideration of the lower selection pressure on breast muscle growth rate and hypertrophy. Briefly, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. The normalized expression levels of COL4 coding genes showed an overexpression of COL4A2 in FG than MG at d 28, as well as a significant decrease in its expression over their rearing period. Overall, results obtained through the gene expression analysis suggested that selection for the hypertrophic growth of FG broilers may have led to an altered regulation of fibroblast proliferation and COL4 synthesis. Moreover, western blot and IHC analyses suggested an altered secretion and/or degradation of COL4 protein in FG broilers, as evidenced by the fluctuating trend of 2 bands observed in FG over time. In view of the above, the present research supports the evidence about a potential aberrant synthesis and/or degradation of COL4 and corroborates the hypothesis regarding a likely involvement of COL4 in the series of events underlying the growth-related abnormalities in modern FG broilers

Detection of differentially expressed genes in broiler pectoralis major muscle affected by White Striping – Wooden Breast myopathies

Abstract White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broiler fillets present an impaired visual appearance that negatively affects consumers' acceptability. Previous studies have highlighted in affected fillets a severely damaged muscle, showing profound inflammation, fibrosis, and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation, and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further defining the transcription patterns associated with these myopathies

Equine Transport-Related Problem Behaviors and Injuries: A Survey of Italian Horse Industry Members

An online survey was conducted to determine associations between equine transport management and transport-related injuries and problem behaviors in Italy. The survey was composed of four sections: respondents\u2019 demographic information and background, transport management practices, journey details and vehicle design, and transport injuries experienced by the horse in the previous two-year period. Univariable and multivariable logistic regression with a binary outcome variable was performed to explore associations between variables (respondents\u2019 and journeys\u2019 details and transport practices) and equine transport-related problem behaviors (TRPBs) and injuries. TRPBs were also considered an explanatory variable for injuries. The survey generated 201 responses; only 148 were complete and analyzed. TRPBs were reported by 14.45% of the respondents and the odds of TRPBs was linked to the respondent gender (p = 0.034), the use of tranquilizers prior to transport (p = 0.002), the use of a whip for loading (p = 0.049), the lack of protection equipment (p = 0.050), and shavings (p = 0.025) on the vehicle floor. Horse injuries (11.49%) were reported by more respondents who did not check the brakes of their transport vehicle before traveling (p = 0.043), had vehicles with padding on the chest bar (p = 0.038), and for horses reported to display TRPBs (p = 0.001). Finally, 10 respondents reported they were injured during horse transport (10/140; 7.14%), 50% simultaneously with their horses. The study findings should be interpreted with caution due to small sample size bias and participants\u2019 recall bias. Nevertheless, the results are in concordance with the literature, confirming that horse transport is a risk for the horse\u2019s and handler\u2019s health and well-being. Further studies are needed to identify best management practices to educate equine industry members on how to minimize transport-related problems

An innovative tool for assessing welfare of camels

Whilst there are many tools for the assessment of welfare in livestock, there is none for camels. This study aimed therefore to pilot a method for assessing the welfare status of camels using animal-, resource- and management-based indicators at a camel market in Qatar. Adapting the AWIN protocol, data related to housing, feeding, health, and behaviour were collected at three levels: caretaker, herd, and animal. The Caretaker level was an interview exploring the caretaker’s background, experience, and routine management practices. The Herd level was a check of the herd and of the place (i.e. box/pen) where camels were kept. At the Animal level, BCS, health, and behavioral parameters were recorded from 2 animals/pens, randomly selected. The number of animals/pens varied (average: 7, range: 1–37 animals) with a total population of 528 animals. The size of the pen was variable (26–256 m2), and consequently the space allowance varied from 2.5 to 34 m2/animal. The environmental temperature was high (average: 42 °C, range: 37–50 °C) and when in the paddock there was a shelter (86%) the camels moved into the shade (313/528 animals). In all paddock, there was a water point, but the water was often not available (22%), dirty (41%), or warm (max:42.9 °C); the majority of the camels therefore drunk when clean and fresh water was offered (bucket test latency time: median =8 sec, IQR =3–40 sec). BCS varied and was rarely optimal (median =2, IQR =2–3). Most of the animals (89%, p < .001) were free of movements (1% tied, 10% hobbled). However, many animals were not free from disease (38%), injuries (5%), scars (7%), and cauterization (38%). Skin diseases were the most common health problems (28%; p < .001), followed by respiratory diseases (4%). The majority of the animals showed a good human-animal relationship (friendly, 48%, or neutral, 30%, approach; p < .001), and no stereotypes were noted. However, some animals were aggressive (6%), when they were old, in pain (2%), or distressed (8%). The caretaker came mainly from Sudan (91%; p < .001), with experience in camel handling often learned by father-son tradition (82%; p < .001) and for many of them, animal welfare was ‘treat the animals gently, feeding and watering them’. This was a preliminary study to pilot a tool to assess welfare in camels; further studies are needed to validate this tool in other camel farms worldwide

The evolution of vimentin and desmin in Pectoralis major muscles of broiler chickens supports their essential role in muscle regeneration

Vimentin (VIM) and desmin (DES) are muscle-specific proteins having crucial roles in maintaining the lateral organization and alignment of the sarcomeric structure during myofibrils’ regeneration. The present experiment was designed to ascertain the evolution of VIM and DES in Pectoralis major muscles (PM) of fast-growing (FG) and medium-growing (MG) meat-type chickens both at the protein and gene levels. MG broilers were considered as a control group whereas the evolution of VIM and DES over the growth period was evaluated in FG by collecting samples at different developmental stages (7, 14, 21, 28, 35, and 42 days). After performing a preliminary classification of the samples based on their histological features, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. Overall, the findings obtained at the protein level mirrored those related to their encoding genes, although a potential time lag required to observe the consequences of gene expression was evident. The two- and 3-fold higher level of the VIM-based heterodimer observed in FG at d 21 and d 28 in comparison with MG of the same age might be ascribed to the beginning and progressive development of the regenerative processes. This hypothesis is supported by IHC highlighting the presence of fibers to coexpressing VIM and DES. In addition, gene expression analyses suggested that, unlike VIM common sequence, VIM long isoform may not be directly implicated in muscle regeneration. As for DES content, the fluctuating trends observed for both the native protein and its heterodimer in FG might be ascribed to its importance for maintaining the structural organization of the regenerating fibers. Furthermore, the higher expression level of the DES gene in FG in comparison with MG further supported its potential application as a marker of muscle fibers’ regeneration. In conclusion, the findings of the present research seem to support the existence of a relationship between the occurrence of muscle regeneration and the growth rate of meat-type chickens and corroborate the potential use of VIM and DES as molecular markers of these cellular processes

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 \ub1 5.6 kg to 151.8 \ub1 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

Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age