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History of the rural world of Portugal may justifiably begin in the present with the major problems affecting Portuguese agriculture, demographic contraction in the countryside and the uncertainty that prevails in the re-conversion of rural areas. The historical evolution of the Portuguese agricultural sector has given it a particular stamp. Several of the problems affecting Portuguese agriculture today could already be seen as the end of the nineteenth century: for instance, poor productivity, the increase in smallholdings in the north and the existence of latifundia to the south. According to authors, although the rural development prospects they have outlined for the Portugal space are subject to a number of conditioning factors, there is one general and irreversible structural base: the limitations of a society open to change and competition within the European Community and the world system. Portuguese rural space is not necessarily obliged to evolve as a clearly defined outcome of economic activity arising from a urban-industrial continuum, determined by the economic progress and growth of developed Europe. The emergence of new social needs, which prove impossible to achieve in the urban space will cause the evolution of rural space in Portugal to lengthen and deepen, both conceptually and in real life.

Objetivando avaliar o efeito de três diferentes níveis de adubação potássica sobre a produção do maracujazeiro azedo (Passiflora edulis Sims e P. edulis Sims f. flavicarpa Deg.), foi realizado um experimento na Fazenda Água Limpa da Universidade de Brasília de dezembro/1999 a maio/2001, utilizando-se o delineamento em blocos casualizados, em esquema fatorial 9x3, sendo nove genótipos e três níveis de adubação potássica, totalizando 27 tratamentos, quatro repetições e nove plantas úteis/parcela. Os genótipos avaliados foram: Híbrido EC-2-0; Marília Seleção Cerrado; F1 (Roxo Fiji x Marília); Porto Rico; Vermelhão (RC1); F1 (Marília x Roxo australiano); Redondão; IAC-273 e Itaquiraí. Os três níveis de adubação potássica aplicados foram de 0, 640 e 1280 kg de K2O ha-1, utilizando-se como fonte o cloreto de potássio. Ao final de cinco meses de colheita os genótipos híbrido EC-2-0 e Marília Seleção Cerrado foram os que apresentaram as maiores produções (21.675 e 21.577 kg ha-1) e maiores números de frutos por planta (155,28 e 149,24), respectivamente. O peso médio de fruto variou de 88g (Itaquiraí) até 103,42g (F1 - Marília x Roxo Australiano). As doses de adubação potássica influenciaram as seguintes variáveis estudadas: produção de frutos de primeira ha-1; produção frutos totais ha-1; peso médio de frutos de primeira e peso médio de frutos 1A. A interação genótipos x adubação potássica apresentou diferença estatística para o número de frutos de primeira/planta para uma dosagem de 640 kg K2O ha-1.

White striping (WS) is a myopathy characterized by the appearance of white stripes parallel to the muscle fibers in the breast of broiler chickens, composed of adipose and connective tissues. This condition causes economic losses and, although common, its etiology remains poorly understood. Hence, the objective was to identify genes and biological mechanisms involved in the early stages of WS using a paternal broiler line that grows slightly slower than commercial ones, at 35 days of age, through the RNA sequencing of the pectoralis major muscle. Thirty genes were differentially expressed between normal and WS-affected chickens, with 23 upregulated and 7 downregulated in the affected broilers. Of these, 14 genes are novel candidates for WS and are implicated in biological processes related to muscle development ( CEPBD , DUSP8 , METTL21EP , NELL2 , and UBE3D ), lipid metabolism ( PDK4 , DDIT4 , FKBP5 , DGAT2 , LIPG , TDH , and RGCC ), and collagen ( COL4A5 and COL4A6 ). Genes related to changes in muscle fiber type and the processes of apoptosis, autophagy, proliferation, and differentiation are possibly involved with the initial stage of WS development. In contrast, the genes linked to lipid metabolism and collagen may have their expression altered due to the progression of the myopathy.

Background: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue., Methods and Results: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia., Conclusions: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Long non-coding RNAs (lncRNAs) are non-coding transcripts involved in various biological processes. The Y chromosome is known for determining the male sex in mammals. LncRNAs on the Y chromosome may play important regulatory roles. However, knowledge about their action mechanisms is still limited, especially during early fetal development. Therefore, we conducted this exploratory study aiming to identify, characterize, and investigate the differential expression of lncRNAs between male and female swine fetuses at 35 days of gestation. RNA-Seq libraries from 10 fetuses were prepared and sequenced using the Illumina platform. After sequencing, a data quality control was performed using Trimmomatic, alignment with HISAT2, and transcript assembly with StringTie. The differentially expressed lncRNAs were identified using the limma package of the R software (4.3.1). A total of 871 potentially novel lncRNAs were identified and characterized. Considering differential expression, eight lncRNAs were upregulated in male fetuses. One was mapped onto SSC12 and seven were located on the Y chromosome; among them, one lncRNA is potentially novel. These lncRNAs are involved in diverse functions, including the regulation of gene expression and the modulation of chromosomal structure. These discoveries enable future studies on lncRNAs in the fetal stage in pigs.

Ca and P homeostasis across the egg-laying cycle is a complex process involving absorption in the small intestine, reabsorption/excretion in the kidneys, and eggshell gland secretion. Diets with inadequate calcium and phosphorus can interfere with their absorption and digestibility, resulting in eggshell quality losses and reduced productive life, affecting egg production and welfare. A better understanding of gene expression profiles in the kidneys of laying hens during the late egg-laying period could clarify the renal role in mineral metabolism at this late stage. Therefore, the performance, egg quality and bone integrity-related traits, and expression profiles of kidney candidate genes were evaluated in 73-week-old laying hens receiving different Ca and P ratios in their diet: a high Ca/P ratio (HR, 22.43), a low ratio (LR, 6.71), and a medium ratio (MR, 11.43). The laying hens receiving the HR diet had improved egg production and eggshell quality traits compared to the other two groups. Humerus length was shorter in the HR than in the other groups. The CYP24A1 and TRPC3 genes were differentially expressed ( p.adj ≤ 0.05) among the groups. Therefore, their expression profiles could be involved in calcium and phosphate transcellular transport in 73-week-old laying hens as a way to keep mineral absorption at adequate levels.

Introduction: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs., Methods: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization., Results: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway., Conclusion: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS., (© 2023 S. Karger AG, Basel.)

Femoral head necrosis (FHN) is one of the most common conditions in fast growing broilers, being characterized by separation of articular cartilage from epiphysis and classified as femoral head separation (FHS) or FHS with laceration (FHSL) depending on severity. Although molecular mechanisms involved with this disorder have been observed, its etiology is still unclear. Therefore, the expression of 15 candidate genes, chosen based on previous transcriptomic studies, was evaluated in the articular cartilage (AC) of normal and FHS-affected broilers at 21 days of age. Samples were collected based on the absence or presence of FHS for physical-chemical and qPCR analysis. The AvBD2, RHAG, COL28A1, ADA and ANGPTL7 were upregulated in FHS-affected broilers compared to the healthy group. These genes are involved in immune response, defense against pathogens, inflammation, cellular migration and adhesion, indicating different molecular mechanisms to control FHS progression at early age. Our results can contribute to improve the knowledge on FHN etiology in chickens and other species, such as horse and pigs that are severely affected by bone disorders., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Femoral head separation (FHS) is characterized by the detachment of growth plate (GP) and articular cartilage, occurring in tibia and femur. However, the molecular mechanisms involved with this condition are not completely understood. Therefore, genes and biological processes (BP) involved with FHS were identified in 21-day-old broilers through RNA sequencing of the femoral GP. 13,487 genes were expressed in the chicken femoral head transcriptome of normal and FHS-affected broilers. From those, 34 were differentially expressed (DE; FDR ≤0.05) between groups, where all of them were downregulated in FHS-affected broilers. The main BP were enriched in receptor signaling pathways, ossification, bone mineralization and formation, skeletal morphogenesis, and vascularization. RNA-Seq datasets comparison of normal and FHS-affected broilers with 21, 35 and 42 days of age has shown three shared DE genes ( FBN2 , C1QTNF8 , and XYLT1 ) in GP among ages. Twelve genes were exclusively DE at 21 days, where 10 have already been characterized ( SHISA3, FNDC1, ANGPTL7, LEPR, ENSGALG00000049529, OXTR, ENSGALG00000045154, COL16A1, RASD2, BOC, GDF10 , and THSD7B ). Twelve SNPs were associated with FHS ( p < 0.0001). Out of those, 5 were novel and 7 were existing variants located in 7 genes ( RARS, TFPI2, TTI1, MAP4K3, LINK54 , and AREL1) . We have shown that genes related to chondrogenesis and bone differentiation were downregulated in the GP of FHS-affected young broilers. Therefore, these findings evince that candidate genes pointed out in our study are probably related to the onset of FHS in broilers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ibelli, Peixoto, Zanella, Gouveia, Cantão, Coutinho, Marchesi, Pizzol, Marcelino and Ledur.)

Femoral head separation (FHS) is usually a subclinical condition characterized by the detachment of articular cartilage from the bone. In this study, a comprehensive analysis identifying shared and exclusive expression profiles, biological processes (BP) and variants related to FHS in the femoral articular cartilage and growth plate in chickens was performed through RNA sequencing analysis. Thirty-six differentially expressed (DE) genes were shared between femoral articular cartilage (AC) and growth plate (GP) tissues. Out of those, 23 genes were enriched in BP related to ion transport, translation factors and immune response. Seventy genes were DE exclusively in the AC and 288 in the GP. Among the BP of AC, the response against bacteria can be highlighted, and for the GP tissue, the processes related to chondrocyte differentiation and cartilage development stand out. When the chicken DE genes were compared to other datasets, eight genes ( SLC4A1 , RHAG , ANK1 , MKNK2 , SPTB , ADA , C7 and EPB420) were shared between chickens and humans. Furthermore, 89 variants, including missense in the SPATS2L , PRKAB1 and TRIM25 genes, were identified between groups. Therefore, those genes should be more explored to validate them as candidates to FHS/FHN in chickens and humans.

Background: Umbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design., Results: In the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others., Conclusions: We have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated., (© 2021. The Author(s).)

Locomotor problems are among one of the main concerns in the current poultry industry, causing major economic losses and affecting animal welfare. The most common bone anomalies in the femur are dyschondroplasia, femoral head separation (FHS), and bacterial chondronecrosis with osteomyelitis (BCO), also known as femoral head necrosis (FHN). The present study aimed to identify differentially expressed (DE) genes in the articular cartilage (AC) of normal and FHS-affected broilers by RNA-Seq analysis. In the transcriptome analysis, 12,169 genes were expressed in the femur AC. Of those, 107 genes were DE (FDR < 0.05) between normal and affected chickens, of which 9 were downregulated and 98 were upregulated in the affected broilers. In the gene-set enrichment analysis using the DE genes, 79 biological processes (BP) were identified and were grouped into 12 superclusters. The main BP found were involved in the response to biotic stimulus, gas transport, cellular activation, carbohydrate-derived catabolism, multi-organism regulation, immune system, muscle contraction, multi-organism process, cytolysis, leukocytes and cell adhesion. In this study, the first transcriptome analysis of the broilers femur articular cartilage was performed, and a set of candidate genes (AvBD1, AvBD2, ANK1, EPX, ADA, RHAG) that could trigger changes in the broiler´s femoral growth plate was identified. Moreover, these results could be helpful to better understand FHN in chickens and possibly in humans., (© 2021. The Author(s).)

Since pre- and postnatal development are programmed during early prenatal life, studies addressing the complete transcriptional landscape during organogenesis are needed. Therefore, we aimed to disentangle differentially expressed (DE) genes between fetuses (at 35 days old) and embryos (at 25 days old) through RNA-sequencing analysis using the pig as model. In total, 1705 genes were DE, including the top DE IBSP , COL6A6 , HBE1 , HBZ , HBB , and NEUROD6 genes, which are associated with developmental transition from embryos to fetuses, such as ossification, skeletal muscle development, extracellular matrix organization, cardiovascular system, erythrocyte differentiation, and neuronal system. In pathway analysis, embryonic development highlighted those mainly related to morphogenic signaling and cell interactions, which are crucial for transcriptional control during the establishment of the main organs in early prenatal development, while pathways related to myogenesis, neuronal development, and cardiac and striated muscle contraction were enriched for fetal development, according to the greater complexity of organs and body structures at this developmental stage. Our findings provide an exploratory and informative transcriptional landscape of pig organogenesis, which might contribute to further studies addressing specific developmental events in pigs and in other mammals.

Background: Copy number variations (CNVs) are a major type of structural genomic variants that underlie genetic architecture and phenotypic variation of complex traits, not only in humans, but also in livestock animals. We identified CNVs along the chicken genome and analyzed their association with performance traits. Genome-wide CNVs were inferred from Affymetrix® high density SNP-chip data for a broiler population. CNVs were concatenated into segments and association analyses were performed with linear mixed models considering a genomic relationship matrix, for birth weight, body weight at 21, 35, 41 and 42 days, feed intake from 35 to 41 days, feed conversion ratio from 35 to 41 days and, body weight gain from 35 to 41 days of age., Results: We identified 23,214 autosomal CNVs, merged into 5042 distinct CNV regions (CNVRs), covering 12.84% of the chicken autosomal genome. One significant CNV segment was associated with BWG on GGA3 (q-value = 0.00443); one significant CNV segment was associated with BW35 (q-value = 0.00571), BW41 (q-value = 0.00180) and BW42 (q-value = 0.00130) on GGA3, and one significant CNV segment was associated with BW on GGA5 (q-value = 0.00432). All significant CNV segments were verified by qPCR, and a validation rate of 92.59% was observed. These CNV segments are located nearby genes, such as KCNJ11, MyoD1 and SOX6, known to underlie growth and development. Moreover, gene-set analyses revealed terms linked with muscle physiology, cellular processes regulation and potassium channels., Conclusions: Overall, this CNV-based GWAS study unravels potential candidate genes that may regulate performance traits in chickens. Our findings provide a foundation for future functional studies on the role of specific genes in regulating performance in chickens.

Chicken feed efficiency (FE) traits are the most important economic traits in broiler production. Several studies evaluating genetic factors affecting food consumption in chickens are available. However, most of these studies identified genomic regions containing putative quantitative trait loci for each trait separately. It is still a challenge to find common gene networks related to these traits. Therefore, here, a genome-wide association study (GWAS) was conducted to explore candidate genomic regions responsible for Feed Intake (FI), Body Weight Gain (BWG) and Feed Conversion Ratio (FCR) traits and their gene networks. A total of 1430 broilers from an experimental population was genotyped with the high density Affymetrix 600K SNP array. A total of 119 associated SNPs located in 20 chromosomes were identified, where some of them were common in more than one FE trait. In addition, novel genomic regions were prospected considering the SNPs dominance effects and sex interaction, identifying putative candidate genes only when these effects were fit in the model. Relevant candidate genes such as ATRNL1, PIK3C2A, PTPRN2, SORCS3 and gga-mir-1759 were highlighted in this study helping to elucidate the genomic architecture of feed efficiency traits. These results provide new insights on the mechanisms underlying the consumption and utilization of food in chickens.

Hernia is one of the most common defects in pigs. The most prevalent are the scrotal (SH), inguinal (IH) and umbilical (UH) hernias. We compared the inguinal ring transcriptome of normal and SH-affected pigs with the umbilical ring transcriptome of normal and UH-affected pigs to discover genes and pathways involved with the development of both types of hernia. A total of 13,307 transcripts was expressed in the inguinal and 13,302 in the umbilical ring tissues with 94.91% of them present in both tissues. From those, 35 genes were differentially expressed in both groups, participating in 108 biological processes. A total of 67 polymorphisms was identified in the inguinal ring and 76 in the umbilical ring tissue, of which 11 and 14 were novel, respectively. A single nucleotide polymorphism (SNP) with deleterious function was identified in the integrin α M ( ITGAM ) gene. The microtubule associated protein 1 light chain 3 γ ( MAP1LC3C ), vitrin ( VIT ), aggrecan ( ACAN ), alkaline ceramidase 2 ( ACER2 ), potassium calcium-activated channel subfamily M α 1 ( KCNMA1 ) and synaptopodin 2 ( SYNPO2 ) genes are highlighted as candidates to trigger both types of hernia. We generated the first comparative study of the pig umbilical and inguinal ring transcriptomes, contributing to the understanding of the genetic mechanism involved with these two types of hernia in pigs and probably in other mammals.

The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies., Competing Interests: The authors have declared that no competing interests exist.

Dietary l-arginine (ARG) supplementation has been studied as a nutritional strategy to improve reproductive performance of pregnant sows, since arginine is a conditionally essential amino acid. However, reports addressing the molecular mechanisms that mediate supplementation effects on embryos and fetuses development are still scarce. Therefore, we aimed to evaluate the effects of 1.0% ARG supplementation of commercial pregnant gilts on genes and proteins from energy metabolism and antioxidant defense pathways in embryos and fetuses. We also analyzed the global transcriptome profile of 25- and 35-day-old conceptuses. At Day 25, we observed a lower abundance of phospho-AMP-activated protein kinase (phospho-AMPK) protein and downregulation of oxidative phosphorylation system genes in ARG embryos. On the other hand, ARG fetuses showed greater expression of MLST8 and lower expression of MTOR genes, in addition to lower abundance of phospho-AMPK and phospho-mammalian target of rapamycin (phospho-mTOR) proteins. Transcriptome analysis at Day 35 did not present differentially expressed genes. For the antioxidant defense pathway, no differences were found between CON and ARG conceptuses, only trends. In general, supplementation of gilts with 1.0% ARG during early gestation affects energy sensitive pathways in 25- and 35-day conceptuses; however, no effects of supplementation were found on the antioxidative defense pathway in 25-day embryos., (© 2020 Wiley Periodicals LLC.)

Background: Bacterial chondronecrosis with osteomyelitis (BCO) develops in the growth plate (GP) of the proximal femur and tibia and is initiated by damage to the less mineralized chondrocytes followed by colonization of opportunistic bacteria. This condition affects approximately 1% of all birds housed, being considered one of the major causes of lameness in fast growing broilers. Although several studies have been previously performed aiming to understand its pathogenesis, the molecular mechanisms involved with BCO remains to be elucidated. Therefore, this study aimed to generate a profile of global differential gene expression involved with BCO in the tibia of commercial broilers, through RNA sequencing analysis to identity genes and molecular pathways involved with BCO in chickens., Results: Our data showed 192 differentially expressed (DE) genes: 63 upregulated and 129 downregulated in the GP of the tibia proximal epiphysis of BCO-affected broilers. Using all DE genes, six Biological Processes (BP) were associated with bone development (connective tissue development, cartilage development, skeletal system development, organ morphogenesis, system development and skeletal system morphogenesis). The analyses of the upregulated genes did not indicate any significant BP (FDR < 0.05). However, with the downregulated genes, the same BP were identified when using all DE genes in the analysis, with a total of 26 coding genes explaining BCO in the tibia: ACAN, ALDH1A2, CDH7, CHAD, CHADL, COL11A1, COMP, CSGALNACT1, CYR61, FRZB, GAL3ST1, HAPLN1, IHH, KIF26B, LECT1, LPPR1, PDE6B, RBP4A, SERINC5, SFRP1, SOX8, SOX9, TENM2, THBS1, UCHL1 and WFIKKN2. In addition, seven transcription factors were also associated to BCO: NFATC2, MAFB, HIF1A-ARNT, EWSR1-FLI1, NFIC, TCF3 and NF-KAPPAB., Conclusions: Our data show that osteochondral downregulated genes are potential molecular causes of BCO in broilers, and the bacterial process seems to be, in fact, a secondary condition. Sixteen genes responsible for bone and cartilage formation were downregulated in BCO-affected broilers being strong candidate genes to trigger this disorder.

Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation., Competing Interests: The authors have declared that no competing interests exist.

Scrotal hernias (SH) are common congenital defects in commercial pigs, characterized by the presence of abdominal contents in the scrotal sac, leading to considerable production and animal welfare losses. Since the etiology of SH remains obscure, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of SH affected and unaffected pigs' inguinal rings. From the 22,452 genes annotated in the pig reference genome, 13,498 were expressed in the inguinal canal tissue. Of those, 703 genes were differentially expressed (DE, FDR < 0.05) between the two groups analyzed being, respectively, 209 genes upregulated and 494 downregulated in the SH-affected group. Thirty-seven significantly overrepresented GO terms related to SH were enriched, and the most relevant biological processes were muscular system, cell differentiation, sarcome reorganization, and myofibril assembly. The calcium signaling, hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac muscle contraction were the major pathways possibly involved in the occurrence of the scrotal hernias. The expression profile of the DE genes was associated with the reduction of smooth muscle differentiation, followed by low calcium content in the cell, which could lead to a decreased apoptosis ratio and diminished muscle contraction of the inguinal canal region. We have demonstrated that genes involved with musculature are closely linked to the physiological imbalance predisposing to scrotal hernia. According to our study, the genes MYBPC1, BOK, SLC25A4, SLC8A3, DES, TPM2, MAP1CL3C , and FGF1 were considered strong candidates for future evaluation., Competing Interests: The authors declare no conflict of interest.

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

The objective of this study was to estimate variance components for performance and carcass traits in a paternal broiler line. The (co)variance components were estimated by the restricted maximum likelihood method applied to the animal model, including the fixed effect of group (sex and hatch) and additive genetic and residual as random effects. Estimated heritability for performance traits ranged from 0.09 to 0.42. The genetic correlations between traits ranged from -0.50 to 0.97. The heritability estimates of feed intake, weight gain, and feed conversion from 35 to 41 days of age were of low magnitude. The genetic correlations among them were favorable to genetic selection. These results suggest that moderate genetic gain can be obtained to the feed intake and weight gain when the selection criterion is the body weight and prime cuts traits. The feed conversion that had low heritability estimation and low genetic correlation with the body weight and prime cut traits needs to pay greater attention due to the economic importance in the high-meat production lineage breeding programs.

Sexual dimorphism is a relevant factor in animal science, since it can affect the gene expression of economically important traits. Eventually, the interest in the prenatal phase in a transcriptome study may not comprise the period of development in which male and female conceptuses are phenotypically divergent. Therefore, it would be interesting if sex differentiation could be performed using transcriptome data, with no need for extra techniques. In this study, the sex of pig conceptuses (embryos at 25 days-old and fetuses at 35 days-old) was determined by reads counts per million (CPM) of Y chromosome-linked genes that were discrepant among samples. Thus, ten genes were used: DDX3Y , KDM5D , ZFY , EIF2S3Y , EIF1AY , LOC110255320 , LOC110257894, LOC396706, LOC100625207, and LOC110255257 . Conceptuses that presented reads CPM sum for these genes (ΣCPM chrY ) greater than 400 were classified as males and those with ΣCPM chrY below 2 were classified as females. It was demonstrated that the sex identification can be performed at early stages of pig development from RNA-sequencing analysis of genes mapped on Y chromosome. Additionally, these results reinforce that sex determination is a mechanism conserved across mammals, highlighting the importance of using pigs as an animal model to study sex determination during human prenatal development.

Many economic losses occur in the poultry industry due to leg fragility. Knowing the genomic regions that influence traits associated with the growth and composition of the leg's bone can help to improve the selection process leading to increased leg resistance to fracture. The present study aimed to map quantitative trait loci (QTL) for mineral composition and morphometric traits of the tibia in 478 animals from an F2 broiler × layer cross. The measurement of weight, length and width of Tibia was carried out at 42 days of age. Ash, dry matter, levels of calcium (Ca), phosphorus (P), magnesium (Mg), Zinc (Zn) and Calcium:Phosphorus (Ca:P) ratio were also recorded. The population was genotyped for 128 microsatellite markers and one single nucleotide polymorphism, covering 2630 cM of the chicken genome. A likelihood ratio test was performed to find QTLs. Additive and dominance effects of the QTLs were included in the model. In the chromosomes 2 (GGA2), 6 (GGA6), 8 (GGA8), 24 (GGA24) and 26 (GGA26) some suggestive QTLs (P&lt;0.00276) were mapped for tibia weight (GGA2 and GGA26), ash percentage (GGA2 and GGA6), dry matter percentage (GGA2), Ca (GGA8 and GGA24) and Ca:P ratio (GGA8), many of which are close to genes already identified as good candidates for those traits. The suggestive QTL on GGA2 has a pleiotropic effect on ash percentage, dry matter and bone weight, whereas in the GGA8 there seems to be two QTLs, one for Ca and another for Ca:P ratio. Thus, this study identified at least five genomic regions, in different chromosomes, that can be targeted for further research to identify potential mutations influencing the development and composition of leg bones in Gallus gallus.

Single nucleotide polymorphism (SNP) markers are used to study population structure and conservation genetics, which permits assessing similarities regarding the linkage disequilibrium and information about the relationship among individuals. To investigate the population genomic structure of 300 females and 25 males from a commercial maternal pig line we analyzed linkage disequilibrium extent, inbreeding coefficients using genomic and conventional pedigree data, and population stratification. The average linkage disequilibrium (r2) was 0.291 ± 0.312 for all adjacent SNPs, distancing less than 100 Kb (kilobase) between markers. The average inbreeding coefficients obtained from runs of homozygosity (ROH) and pedigree analyses were 0.119 and 0.0001, respectively. Low correlation was observed between the inbreeding coefficients possibly as a result of genetic recombination effect accounted for the ROH estimates or caused by pedigree identification errors. A large number of long ROHs might indicate recent inbreeding events in the studied population. A total of 36 homozygous segments were found in more than 30% of the population and these ROH harbor genes associated with reproductive traits. The population stratification analysis indicated that this population was possibly originated from two distinct populations, which is a result from crossings between the eastern and western breeds used in the formation of the line. Our findings provide support to understand the genetic structure of swine populations and may assist breeding companies to avoid a high level of inbreeding coefficients to maintain genetic diversity, showing the effectiveness of using genome-wide SNP information for quantifying inbreeding when the pedigree was incomplete or incorrect., Competing Interests: The authors have declared that no competing interests exist.

White striping (WS) is one of the most common myopathies identified in broiler chickens leading to substantial production losses, where the incidence reaches 12% in commercial chickens. It occurs primarily in heavier chickens being a modification of the breast muscle characterized by the presence of pale parallel streaks in the same orientation of the muscle fibers. Since the WS etiology remains unclear, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of WS in affected and unaffected chicken breast muscles. A total of 11,177 genes were expressed in the pectoralis major muscle. Out of those, 1,441 genes were differentially expressed (FDR ≤ 0.01) between the two analyzed groups, being, respectively, 772 genes upregulated and 669 downregulated in the WS affected group. A total of 36 significantly overrepresented GO terms related to WS myopathy were enriched, and the most relevant biological processes were activation of immune system, angiogenesis, hypoxia, cell death, and striated muscle contraction. The unbalance of those biological processes may trigger the occurrence of the WS phenotype in broilers. The possible lack of capillary blood supply homogeneously in the muscle triggers the hypoxia, following the activation of glycolysis, calcium signaling and apoptosis related genes facilitating the tissue damage and WS incidence., (© 2018 Poultry Science Association Inc.)

The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue., Competing Interests: The authors have declared that no competing interests exist.

A new methodology is proposed for ractopamine residue analysis in pork. It consists of enzyme-mediated digestion and deconjugation steps; modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction; and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In brief, the samples were digested with protease and then deconjugated with β-glucuronidase enzyme; they were then subjected to extraction and cleanup by QuEChERS and underwent sequential analysis by LC-MS/MS. The method performance was evaluated in accordance to the validation guidelines regulated by the Brazilian Ministry of Agriculture and Food Supply. The limit of detection was 0.15 μg/kg and limit of quantification was 0.5 μg/kg. When the method was applied to real samples, ractopamine residue was found in concentrations (up to 7.86 μg/kg) below international recommendation limits up to 10 μg/kg. The method is sensitive, accurate, quick, simple, and suitable for routine analysis; therefore, it is a monitoring tool that may be adopted by laboratories to achieve compliance levels.

Genomic regions under high selective pressure present specific runs of homozygosity (ROH), which provide valuable information on the genetic mechanisms underlying the adaptation to environment imposed challenges. In broiler chickens, the adaptation to conventional production systems in tropical environments lead the animals with favorable genotypes to be naturally selected, increasing the frequency of these alleles in the next generations. In this study, ~1400 chickens from a paternal broiler line were genotyped with the 600 K Affymetrix® Axiom® high-density (HD) genotyping array for estimation of linkage disequilibrium (LD), effective population size (N e ), inbreeding and ROH. The average LD between adjacent single nucleotide polymorphisms (SNPs) in all autosomes was 0.37, and the LD decay was higher in microchromosomes followed by intermediate and macrochromosomes. The N e of the ancestral population was high and declined over time maintaining a sufficient number of animals to keep the inbreeding coefficient of this population at low levels. The ROH analysis revealed genomic regions that harbor genes associated with homeostasis maintenance and immune system mechanisms, which may have been selected in response to heat stress. Our results give a comprehensive insight into the relationship between shared ROH regions and putative regions related to survival and production traits in a paternal broiler line selected for over 20 years. These findings contribute to the understanding of the effects of environmental and artificial selection in shaping the distribution of functional variants in the chicken genome.

Background: Excess fat content in chickens has a negative impact on poultry production. The discovery of QTL associated with fat deposition in the carcass allows the identification of positional candidate genes (PCGs) that might regulate fat deposition and be useful for selection against excess fat content in chicken's carcass. This study aimed to estimate genomic heritability coefficients and to identify QTLs and PCGs for abdominal fat (ABF) and skin (SKIN) traits in a broiler chicken population, originated from the White Plymouth Rock and White Cornish breeds., Results: ABF and SKIN are moderately heritable traits in our broiler population with estimates ranging from 0.23 to 0.33. Using a high density SNP panel (355,027 informative SNPs), we detected nine unique QTLs that were associated with these fat traits. Among these, four QTL were novel, while five have been previously reported in the literature. Thirteen PCGs were identified that might regulate fat deposition in these QTL regions: JDP2, PLCG1, HNF4A, FITM2, ADIPOR1, PTPN11, MVK, APOA1, APOA4, APOA5, ENSGALG00000000477, ENSGALG00000000483, and ENSGALG00000005043. We used sequence information from founder animals to detect 4843 SNPs in the 13 PCGs. Among those, two were classified as potentially deleterious and two as high impact SNPs., Conclusions: This study generated novel results that can contribute to a better understanding of fat deposition in chickens. The use of high density array of SNPs increases genome coverage and improves QTL resolution than would have been achieved with low density. The identified PCGs were involved in many biological processes that regulate lipid storage. The SNPs identified in the PCGs, especially those predicted as potentially deleterious and high impact, may affect fat deposition. Validation should be undertaken before using these SNPs for selection against carcass fat accumulation and to improve feed efficiency in broiler chicken production.

In this paper, we have used two approaches to detect genetic associations with scrotal hernias in commercial pigs. Firstly, we have investigated the effects of runs of homozygosity (ROH) with the appearance of scrotal hernias, followed by a Genome Wide Association Study (GWAS). The phenotype classification was based on visual appearance of scrotal hernias. Each affected animal was matched to a healthy control from the same pen. In the total, 68 animals were genotyped using the Porcine SNP60 Beadchip, out of those, 41 animals had the presence of hernias and 27 were healthy animals. Fifteen animals were removed from the analysis due to differences in genetic background, leaving 18 healthy animals and 35 piglets with scrotal hernia. Further, the detection of extended haplotypes shared ROH were conducted for health (control) and affected (case) animals and a permutation test was used to test whether the ROH segments were more frequent in case/case pairs than non-case/case pairs. Using the ROH, we have identified an association ( p = 0.019) on chromosome 2(SSC2) being segregated on animals with the presence of scrotal hernias. Using a GWAS, a region composed by 3 SNPs on the sexual chromosome X (SSCX) were associated with scrotal hernias ( p < 1.6 × 10 -5 ), this region harbors the Androgen Receptor Gene ( AR )., Competing Interests: The authors declare that they have no conflict of interest.

Economic losses due to an increase of leg disorders in broilers have become a major concern of the poultry industry. Despite the efforts to reduce skeletal abnormalities in chickens, insufficient progress has been made. Bacterial chondronecrosis with osteomyelitis (BCO) is one of the main disorders that affect bone integrity in broilers. However, the genetic pathways and genes involved in most bone problems, including BCO, remains unclear. In this study, femoral samples from male broilers with 45 days of age affected or not with BCO were used to compare the relative expression with a reverse transcription real time PCR approach of 13 candidate genes: SPP1 (osteopontin), TNFRSF11B (osteoprotegerin), SPARC (osteonectin), CALB1 (calbidin 1), CALM (Calmodulin 2), IBSP (sialoprotein), COL1A2 (collagen, type I, α 2), BMP2 (bone morphogenetic protein 2), BMP3 (bone morphogenetic protein 3), RANKL (κ-B nuclear factor ligand), SMAD1 (SMAD family member 1), LEPR (leptin receptor) and RUNX2 (related transcription factor Runt 2). Differential expression test between affected and non-affected groups was performed using the REST software. The RUNX2 and SPARC genes were downregulated (P<0.05) in the affected group, with reduced expression of fourfold when compared with the non-affected group. This result indicates that the downregulation of RUNX2 and SPARC can contribute to an increased incidence of BCO in broilers.

Intense selection for production traits has improved the genetic gain of important economic traits. However, selection for performance and carcass traits has led to the onset of locomotors problems and decreasing bone strength in broilers. Thus, genes associated with bone integrity traits have become candidates for genetic studies in order to reduce the impact of bone disorders in broilers. This study investigated the association of the RUNX2 and TNFSF11 genes with 79 traits related to performance, carcass composition, organs, and bone integrity in a paternal broiler line. Analyses of genetic association between single-nucleotide polymorphisms (SNPs) and traits were carried out using the maximum likelihood procedures for mixed models. Genetic associations (P < 0.05) were found between SNP g.124,883A>G in the RUNX2 gene and chilled femur weight (additive plus dominance deviation effects within sex) and with performance traits (additive within sex and additive effects). The SNP g.14,862T>C in the TNFSF11 gene presented genetic associations (P < 0.05) with additive plus dominance deviation effects within sex for performance traits. Suggestive genetic associations (P < 0.10) were found with abdominal fat and its yield. Selection based on SNPs g.14,862T>C in TNFSF11 and g.124,883A>G in RUNX2 could be used to improve performance and carcass quality traits in the population studied, although SNP g.14,862T>C was not in Hardy-Weinberg equilibrium because it was not undergoing a selection process. Furthermore, it is important to validate these markers in an unrelated population for use in the selection process.

Domestic turkeys present several color phenotypes controlled by at least five genetic loci, but only one of these has been identified precisely: the bronze locus, which turned out to be the melanocortin-1 receptor (MC1R) gene. MC1R variation is important for breeders interested in maintaining or developing different color varieties. In this study, we sequenced most of the MC1R gene from 16 White Holland (the main commercial turkey variety) and 19 pigmented turkeys from southern Brazil with two purposes. The first was to describe the MC1R diversity in White Holland turkeys, which may serve as reservoirs of genetic diversity at this locus. The second was to test whether the traditional color classification used by Brazilian breeders is related to previously known MC1R alleles. White Holland turkeys had four different haplotypes corresponding to the bronze (b+) and black-winged bronze (b1) alleles. Pigmented turkeys also had four haplotypes corresponding to the b+ and b1 alleles, but different haplotypes represent the most common b+ allele in these two groups. The black (B) allele was absent from our samples. Overall, our results suggest that white and pigmented individuals form two different populations, and that the traditional color classification used by Brazilian breeders cannot accurately predict the genotypes at the bronze locus.