Animal & Avian Sciences Research Works

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Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins
(MDPI, 2016-05-26) Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M.; Telugu, Bhanu P.
The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.
Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs
(MDPI, 2016-12-03) Sheets, Timothy P.; Park, Chi-Hun; Park, Ki-Eun; Powell, Anne; Donovan, David M.; Telugu, Bhanu P.
The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.
Characterization of Copy Number Variation’s Potential Role in Marek’s Disease
(MDPI, 2017-05-09) Xu, Lingyang; He, Yanghua; Ding, Yi; Sun, Guirong; Carrillo, Jose Adrian; Li, Yaokun; Ghaly, Mona M.; Ma, Li; Zhang, Huanmin; Liu, George E.; Song, Jiuzhou
Marek’s Disease (MD) is a highly contagious pathogenic and oncogenic disease primarily affecting chickens. Chicken Lines 63 and 72, as well as their recombinant congenic strains (RCS) with varied susceptibility to MD, are ideal models to study the complex mechanisms of genetic resistance to MD. In this study, we investigated copy number variation (CNV) in these inbred chicken lines using the Affymetrix Axiom HD 600 K SNP genotyping array. We detected 393 CNV segments across all ten chicken lines, of which 12 CNVs were specifically identified in Line 72. We then assessed genetic structure based on CNV and observed markedly different patterns. Finally, we validated two deletion events in Line 72 and correlated them with genes expression using qPCR and RNA-seq, respectively. Our combined results indicated that these two CNV deletions were likely to contribute to MD susceptibility.
Antimicrobial Effect and Probiotic Potential of Phage Resistant Lactobacillus plantarum and its Interactions with Zoonotic Bacterial Pathogens
(MDPI, 2019-06-05) Nagarajan, Vinod; Peng, Mengfei; Tabashsum, Zajeba; Salaheen, Serajus; Padilla, Joselyn; Biswas, Debabrata
Development of phage-resistant probiotic particularly Lactobacillus is an alternative approach to enhance their beneficial effects as in animal feed supplements. In this study, we developed phage-resistant Lactobacillus plantarum (LP+PR) mutant and compared their antimicrobial effects and probiotic potential against zoonotic bacterial pathogens including Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli (EHEC), Staphylococcus aureus, and Listeria monocytogenes with phage-sensitive L. plantarum (LP) strain. LP+PR strain showed markedly higher growth rate than wild-type LP strain. In co-culture with LP+PR and in the presence of cell-free cultural supernatants (CFCSs) of LP+PR, the growth of S. Typhimurium, EHEC, S. aureus, and L. monocytogenes were reduced significantly (P < 0.05). The adhesion ability of LP+PR was slightly higher than the LP on human epithelial INT-407 cells. Most importantly, LP+PR strain significantly inhibited the adhesive and invasive abilities of all four zoonotic pathogens to INT-407 cells (P < 0.05). Moreover, real-time qPCR revealed that in the presence of LP+PR strain or its CFCSs, expression of virulence genes of these zoonotic bacterial pathogens were suppressed significantly (P < 0.05). These findings suggest that the LP+PR strain is capable of inhibiting major zoonotic bacterial pathogens efficiently and would be a potential candidate for industrial usage in animal production or fermentation.
Marek’s Disease Virus Infection Induced Mitochondria Changes in Chickens
(MDPI, 2019-06-27) Chu, Qin; Ding, Yi; Cai, Wentao; Liu, Lei; Zhang, Huanmin; Song, Jiuzhou
Mitochondria are crucial cellular organelles in eukaryotes and participate in many cell processes including immune response, growth development, and tumorigenesis. Marek’s disease (MD), caused by an avian alpha-herpesvirus Marek’s disease virus (MDV), is characterized with lymphomas and immunosuppression. In this research, we hypothesize that mitochondria may play roles in response to MDV infection. To test it, mitochondrial DNA (mtDNA) abundance and gene expression in immune organs were examined in two well-defined and highly inbred lines of chickens, the MD-susceptible line 72 and the MD-resistant line 63. We found that mitochondrial DNA contents decreased significantly at the transformation phase in spleen of the MD-susceptible line 72 birds in contrast to the MD-resistant line 63. The mtDNA-genes and the nucleus-genes relevant to mtDNA maintenance and transcription, however, were significantly up-regulated. Interestingly, we found that POLG2 might play a potential role that led to the imbalance of mtDNA copy number and gene expression alteration. MDV infection induced imbalance of mitochondrial contents and gene expression, demonstrating the indispensability of mitochondria in virus-induced cell transformation and subsequent lymphoma formation, such as MD development in chicken. This is the first report on relationship between virus infection and mitochondria in chicken, which provides important insights into the understanding on pathogenesis and tumorigenesis due to viral infection.
Allele-Specific Expression of CD4+ T Cells in Response to Marek’s Disease Virus Infection
(MDPI, 2019-09-17) Bai, Hao; He, Yanghua; Ding, Yi; Carrillo, José A.; Selvaraj, Ramesh K.; Zhang, Huanmin; Chen, Jilan; Song, Jiuzhou
Marek’s disease (MD) is a T cell lymphoma disease induced by Marek’s disease virus (MDV), a highly oncogenic α herpesvirus primarily affecting chickens. MD is a chronic infectious disease that threatens the poultry industry. However, the mechanisms of genetic resistance for MD are complex and not completely understood. In this study, to identify high-confidence candidate genes of MD genetic resistance, high throughput sequencing (RNA-seq) was used to obtain transcriptomic data of CD4+ T cells isolated from MDV-infected and non-infected groups of two reciprocal crosses of individuals mating by two highly inbred chicken lines (63 MD-resistant and 72 MD-susceptible). After RNA-seq analysis with two biological replicates in each group, we identified 61 and 123 single nucleotide polymorphisms (SNPs) (false discovery rate (FDR) < 0.05) annotated in 39 and 132 genes in intercrosses 63 × 72 and 72 × 63, respectively, which exhibited allele-specific expression (ASE) in response to MDV infection. Similarly, we identified 62 and 79 SNPs annotated in 66 and 96 genes in infected and non-infected groups, respectively. We identified 534 and 1543 differentially expressed genes (DEGs) (FDR < 0.05) related to MDV infection in intercrosses 63 × 72 and 72 × 63, respectively. We also identified 328 and 20 DEGs in infected and non-infected groups, respectively. The qRT-PCR using seven DEGs further verified our results of RNA-seq analysis. The qRT-PCR of 11 important ASE genes was performed for gene functional validation in CD4+ T cells and tumors. Combining the analyses, six genes (MCL1, SLC43A2, PDE3B, ADAM33, BLB1, and DMB2), especially MCL1, were highlighted as the candidate genes with the potential to be involved in MDV infection. Gene-set enrichment analysis revealed that many ASE genes are linked to T cell activation, T cell receptor (TCR), B cell receptor (BCR), ERK/MAPK, and PI3K/AKT-mTOR signaling pathways, which play potentially important roles in MDV infection. Our approach underlines the importance of comprehensive functional studies for gaining valuable biological insight into the genetic factors behind MD and other complex traits, and our findings provide additional insights into the mechanisms of MD and disease resistance breeding in poultry.
Environmental Influences of High-Density Agricultural Animal Operation on Human Forearm Skin Microflora
(MDPI, 2020-09-26) Peng, Mengfei; Biswas, Debabrata
The human forearm skin microbiome ecosystem contains rich and diverse microbes, which are influenced by environmental exposures. The microbial representatives can be exchanged between human and environment, specifically animals, by which they share certain or similar epidermal microbes. Livestock and poultry are the microbial sources that are associated with the transmission of community-based pathogenic infections. Here, in this study, we proposed investigating the environmental influences introduced by livestock/poultry operations on forearm skin microflora of on-site farm workers. A total of 30 human skin swab samples were collected from 20 animal workers in dairy or integrated farms and 10 healthy volunteer controls. The skin microbiome was 16S metagenomics that were sequenced with Illumina MiSeq system. For skin microbial community analysis, the abundance of major phyla and genera as well as alpha and beta diversities were compared across groups. We identified distinctive microbial compositional patterns on skin of workers in farm with different animal commodities. Workers in integrated farms containing various animals were associated with higher abundances of epidermal Proteobacteria, especially Pseudomonas and Acinetobacter, but lower Actinobacteria, especially Corynebacterium and Propionibacterium. For those workers with frequent dairy cattle operations, their Firmicutes in the forearm skin microbiota were enriched. Furthermore, farm animal operations also reduced Staphylococcus and Streptococcus, as well as modulated the microbial biodiversity in farm workers’ skin microbiome. The alterations of forearm skin microflora in farm workers, influenced by their frequent farm animal operations, may increase their risk in skin infections with unusual pathogens and epidermal diseases.
Dietary Macronutrient Composition Differentially Modulates the Remodeling of Mitochondrial Oxidative Metabolism during NAFLD
(MDPI, 2021-04-26) Kattapuram, Nathan; Zhang, Christine; Muyyarikkandy, Muhammed S.; Surugihalli, Chaitra; Muralidaran, Vaishna; Gregory, Tabitha; Sunny, Nishanth E.
Diets rich in fats and carbohydrates aggravate non-alcoholic fatty liver disease (NAFLD), of which mitochondrial dysfunction is a central feature. It is not clear whether a high-carbohydrate driven ‘lipogenic’ diet differentially affects mitochondrial oxidative remodeling compared to a high-fat driven ‘oxidative’ environment. We hypothesized that the high-fat driven ‘oxidative’ environment will chronically sustain mitochondrial oxidative function, hastening metabolic dysfunction during NAFLD. Mice (C57BL/6NJ) were reared on a low-fat (LF; 10% fat calories), high-fat (HF; 60% fat calories), or high-fructose/high-fat (HFr/HF; 25% fat and 34.9% fructose calories) diet for 10 weeks. De novo lipogenesis was determined by measuring the incorporation of deuterium from D2O into newly synthesized liver lipids using nuclear magnetic resonance (NMR) spectroscopy. Hepatic mitochondrial metabolism was profiled under fed and fasted states by the incubation of isolated mitochondria with [13C3]pyruvate, targeted metabolomics of tricarboxylic acid (TCA) cycle intermediates, estimates of oxidative phosphorylation (OXPHOS), and hepatic gene and protein expression. De novo lipogenesis was higher in the HFr/HF mice compared to their HF counterparts. Contrary to our expectations, hepatic oxidative function after fasting was induced in the HFr/HF group. This differential induction of mitochondrial oxidative function by the high fructose-driven ‘lipogenic’ environment could influence the progressive severity of hepatic insulin resistance.
Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo
(MDPI, 2021-05-18) Asfor, Amin S.; Nazki, Salik; Reddy, Vishwanatha R.A.P.; Campbell, Elle; Dulwich, Katherine L.; Giotis, Efstathios S.; Skinner, Michael A.; Broadbent, Andrew J.
In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.
Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12
(MDPI, 2021-06-03) Xiao, Zhengguo; Kandel, Anmol; Li, Lei
CD4+ T cell activation requires inflammatory cytokines to provide a third signal (3SI), such as interleukin-12 (IL-12). We recently reported that bovine neutrophils can enhance the activation of bovine CD4+ T cells. To explore the interactions between neutrophils and third signal cytokines in bovine CD4+ T cell activation, naïve CD4+ T cells were isolated from cattle lymph nodes and stimulated for 3.5 days with anti-bovine CD3 (first signal; 1SI), anti-bovine CD28 (second signal; 2SI), and recombinant human IL-12 (3SI) in the presence or absence of neutrophils harvested from the same animals. Indeed, the strongest activation was achieved in the presence of all three signals, as demonstrated by CD25 upregulation, IFNγ production in CD4+ T cells, and secretion of IFNγ and IL-2 in cell supernatants. More importantly, 1SI plus neutrophils led to enhanced CD25 expression that was further increased by IL-12, suggesting synergistic action by IL-12 and neutrophils. Consistently, neutrophils significantly increased IFNγ production in 1SI plus IL-12-stimulated CD4+ T cells. Our data suggest the synergy of neutrophils and IL-12 as a novel regulator on bovine CD4+ T cell activation in addition to three signals. This knowledge could assist the development of immune interventions for the control of infectious diseases in cattle.
Insights from Initial Variant Detection by Sequencing Single Sperm in Cattle
(MDPI, 2021-11-15) Yang, Liu; Gao, Yahui; Boschiero, Clarissa; Li, Li; Zhang, Hongping; Ma, Li; Liu, George E.
Meiotic de novo mutation (DNM) is one of the important phenomena contributing to gamete genome diversity. However, except for humans and a few model organisms, they are not well studied in livestock, including cattle. Moreover, bulk sperm samples have been routinely utilized in experiments, which include millions of single sperm cells and only report high-frequency variants. In this study, we isolated and sequenced 143 single sperms from two Holstein bulls and identified hundreds of candidate DNM events in ten sperms with deep sequencing coverage. We estimated DNM rates ranging from 1.08 × 10−8 to 3.78 × 10−8 per nucleotide per generation. We further validated 12 out of 14 selected DNM events using Sanger sequencing. To our knowledge, this is the first single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of point mutations and male fertility. Our preliminary results pointed out future research directions and highlighted the importance of uniform whole genome amplification, deep sequence coverage, and dedicated software pipelines for genetic variant detection using single-cell sequencing data.
Modulation of Plasma and Milk Sphingolipids in Dairy Cows Fed High-Starch Diets
(MDPI, 2021-10-19) Rico, Jorge Eduardo; Sandri, Eveline C.; Sarmiento, Anrea Celemin; Lévesque, Janie; Kenéz, Ákos; Rico, Daniel C.
Bovine milk is a significant source of sphingolipids, dietary compounds that can exert anti-inflammatory actions, and which can modulate the host’s microbiome. Because sphingolipid synthesis can be modified by diet, we hypothesized that dietary conditions which reduced FFA availability may result in reduced sphingolipid synthesis. Twelve ruminally cannulated cows (120 ± 52 DIM; 35.5 ± 8.9 kg of milk/d; mean ± SD) were randomly assigned to treatment in a crossover design with 21-d periods. Treatments were (1) High starch (HS), (2) Control. The HS diet contained 29% starch, 24% NDF, and 2.8% fatty acids (FA), whereas the Control diet contained 20% starch, 31% NDF, and 2.3% FA. Plasma and milk samples were obtained on d 21 of each period and sphingolipids were quantified using targeted metabolomics. Univariate and multivariate analyses of generalized log-transformed and Pareto-scaled data included ANOVA (fixed effects of treatment) and discriminant analysis. The lipidomics analysis detected 71 sphingolipids across plasma and milk fat, including sphinganines (n = 3), dihydro-ceramides (n = 8), ceramides (Cer; n = 15), sphingomyelins (SM; n = 17), and glycosylated ceramides (n = 28). Followed by Cer, SM were the most abundant sphingolipids detected in milk and plasma, with a preponderance of 16:0-, 23:0-, and 24:0-carbon sidechains. Although no effects of HS diets were observed on plasma sphingolipids, we detected consistent reductions in the concentrations of several milk Cer (e.g., 22:0- and 24:0-Cer) and SM (17:0- and 23:0-SM) in response to HS. Discriminant analysis revealed distinct metabolite separation of HS and Control groups, with several Cer and SM being distinctively predictive of dietary treatment. We conclude that HS diets can reduce the secretion of milk Cer and SM, even in the absence of changes in circulating sphingolipids.
Tributyrin, a Butyrate Pro-Drug, Primes Satellite Cells for Differentiation by Altering the Epigenetic Landscape
(MDPI, 2021-12-09) Murray, Robert L.; Zhang, Wei; Liu, Jianan; Cooper, Jason; Mitchell, Alex; Buman, Maria; Song, Jiuzhou; Stahl, Chad H.
Satellite cells (SC) are a population of muscle resident stem cells that are responsible for postnatal muscle growth and repair. With investigation into the genomic regulation of SC fate, the role of the epigenome in governing SC myogenesis is becoming clearer. Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective at enhancing the myogenic program of SC, but their role in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how an HDAC inhibitor, butyrate, promotes myogenic differentiation. SC from tributyrin treated neonatal piglets showed a decrease relative to SC from control animals in the expression of enhance of zeste homologue-2 (EZH2), a chromatin modifier, ex vivo. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis of SC isolated from tributyrin treated pigs showed a global reduction of the tri-methylation of lysine 27 of histone H3 (H3K27me3) repressive chromatin mark. To determine if reductions in EZH2 was the primary mechanism through which butyrate affects SC behavior, SC were transfected with siRNA targeting EZH2, treated with 0.5 mM butyrate, or both. Treatment with butyrate reduced paired-box-7 (Pax7) and myogenic differentiation-1 (MyoD) gene expression, while siRNA caused reductions in EZH2 had no effect on their expression. EZH2 depletion did result in an increase in differentiating SC, but not in myotube hypertrophy. These results indicate that while EZH2 reduction may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance the myogenic program.
Influence of Manure Application on the Soil Bacterial Microbiome in Integrated Crop-Livestock Farms in Maryland
(MDPI, 2021-12-15) Peng, Mengfei; Tabashsum, Zajeba; Millner, Patricia; Parveen, Salina; Biswas, Debabrata
As a traditional agricultural system, integrated crop-livestock farms (ICLFs) involve the production of animals and crops in a shared environment. The ICLFs in the mid-Atlantic region of the United States practice sustainable manure aging or composting processes to provide an on-farm source of soil amendment for use as natural fertilizer and soil conditioner for crop production. However, crop fertilization by soil incorporation of aged manure or compost may introduce different microbes and alter the soil microbial community. The aim of this study was to characterize the influence of aged or composted manure application on the diversity of soil bacterial community in ICLFs. Soil samples from six ICLFs in Maryland were collected before (pre-crop) and during the season (2020–2021) and used to analyze soil bacterial microbiome by 16S rDNA sequencing. Results showed that both phylum- and genus-level alterations of soil bacterial communities were associated with amendment of aged or composted manure. Particularly, Proteobacteria and Actinobacteria were enriched, while Acidobacteria, Bacteroidetes, Planctomycetes, Firmicutes, and Chloroflexi were reduced after manure product application. Meanwhile, the relative abundance of Bacillus was decreased, while two zoonotic pathogens, Salmonella and Listeria, were enriched by manure amendments. Overall, animal manure amendment of soil increased the phylogenetic diversity, but reduced the richness and evenness of the soil bacterial communities. Although manure composting management in ICLFs benefits agricultural sustainable production, the amendments altered the soil bacterial communities and were associated with the finding of two major zoonotic bacterial pathogens, which raises the possibility of their potential transfer to fresh horticultural produce crops that may be produced on the manured soils and then subsequently consumed without cooking.
Differential Expression of CD45RO and CD45RA in Bovine T Cells
(MDPI, 2022-06-04) Kandel, Anmol; Li, Lei; Hada, Akanksha; Xiao, Zhengguo
Effective vaccination induces immune memory to protect animals upon pathogen re-encounter. Despite contradictory reports, bovine memory T cells are identified based on two isoforms of CD45, expression of CD45RO plus exclusion of CD45RA. In this report, we contrasted CD45RA/RO expression on circulatory T cells with IFNγ and IL4 expression induced by a conventional method. To our surprise, 20% of cattle from an enclosed herd did not express CD45RO on T cells without any significant difference on CD45RA expression and IFNγ or IL4 induction. In CD45RO expressing cattle, CD45RA and CD45RO expressions excluded each other, with dominant CD45RO (>90%) expression on gamma delta (γδ) followed by CD4+ (60%) but significantly higher CD45RA expression on CD8+ T cells (about 80%). Importantly, more than 80% of CD45RO expressing CD4+ and CD8+ T cells failed to produce IFNγ and IL-4; however, within the cytokine inducing cells, CD4+ T cells highly expressed CD45RO but those within CD8+ T cells mostly expressed CD45RA. Hence, CD45RO is not ubiquitously expressed in cattle, and rather than with memory phenotype, CD45RA/RO expression are more associated with distinct T cell subtypes.
Weighted Single-Step GWAS Identifies Genes Influencing Fillet Color in Rainbow Trout
(MDPI, 2022-07-26) Ahmed, Ridwan O.; Ali, Ali; Al-Tobasei, Rafet; Leeds, Tim; Kenney, Brett; Salem, Mohamed
The visual appearance of the fish fillet is a significant determinant of consumers’ purchase decisions. Depending on the rainbow trout diet, a uniform bright white or reddish/pink fillet color is desirable. Factors affecting fillet color are complex, ranging from the ability of live fish to accumulate carotenoids in the muscle to preharvest environmental conditions, early postmortem muscle metabolism, and storage conditions. Identifying genetic markers of fillet color is a desirable goal but a challenging task for the aquaculture industry. This study used weighted, single-step GWAS to explore the genetic basis of fillet color variation in rainbow trout. We identified several SNP windows explaining up to 3.5%, 2.5%, and 1.6% of the additive genetic variance for fillet redness, yellowness, and whiteness, respectively. SNPs are located within genes implicated in carotenoid metabolism (β,β-carotene 15,15′-dioxygenase, retinol dehydrogenase) and myoglobin homeostasis (ATP synthase subunit β, mitochondrial (ATP5F1B)). These genes are involved in processes that influence muscle pigmentation and postmortem flesh coloration. Other identified genes are involved in the maintenance of muscle structural integrity (kelch protein 41b (klh41b), collagen α-1(XXVIII) chain (COL28A1), and cathepsin K (CTSK)) and protection against lipid oxidation (peroxiredoxin, superoxide dismutase 2 (SOD2), sestrin-1, Ubiquitin carboxyl-terminal hydrolase-10 (USP10)). A-to-G single-nucleotide polymorphism in β,β-carotene 15,15′-dioxygenase, and USP10 result in isoleucine-to-valine and proline-to-leucine non-synonymous amino acid substitutions, respectively. Our observation confirms that fillet color is a complex trait regulated by many genes involved in carotenoid metabolism, myoglobin homeostasis, protection against lipid oxidation, and maintenance of muscle structural integrity. The significant SNPs identified in this study could be prioritized via genomic selection in breeding programs to improve fillet color in rainbow trout.
Integrated Analyses of DNA Methylation and Gene Expression of Rainbow Trout Muscle under Variable Ploidy and Muscle Atrophy Conditions
(MDPI, 2022-06-26) Salem, Mohamed; Al-Tobasei, Rafet; Ali, Ali; Kenney, Brett
Rainbow trout, Oncorhynchus mykiss, is an important cool, freshwater aquaculture species used as a model for biological research. However, its genome reference has not been annotated for epigenetic markers affecting various biological processes, including muscle growth/atrophy. Increased energetic demands during gonadogenesis/reproduction provoke muscle atrophy in rainbow trout. We described DNA methylation and its associated gene expression in atrophying muscle by comparing gravid, diploid females to sterile, triploid females. Methyl Mini-seq and RNA-Seq were simultaneously used to characterize genome-wide DNA methylation and its association with gene expression in rainbow trout muscle. Genome-wide enrichment in the number of CpGs, accompanied by depleted methylation levels, was noticed around the gene transcription start site (TSS). Hypermethylation of CpG sites within ±1 kb on both sides of TSS (promoter and gene body) was weakly/moderately associated with reduced gene expression. Conversely, hypermethylation of the CpG sites in downstream regions of the gene body +2 to +10 kb was weakly associated with increased gene expression. Unlike mammalian genomes, rainbow trout gene promotors are poor in CpG islands, at <1% compared to 60%. No signs of genome-wide, differentially methylated (DM) CpGs were observed due to the polyploidy effect; only 1206 CpGs (0.03%) were differentially methylated, and these were primarily associated with muscle atrophy. Twenty-eight genes exhibited differential gene expression consistent with methylation levels of 31 DM CpGs. These 31 DM CpGs represent potential epigenetic markers of muscle atrophy in rainbow trout. The DM CpG-harboring genes are involved in apoptosis, epigenetic regulation, autophagy, collagen metabolism, cell membrane functions, and Homeobox proteins. Our study also identified genes explaining higher water content and modulated glycolysis previously shown as characteristic biochemical signs of rainbow trout muscle atrophy associated with sexual maturation. This study characterized DNA methylation in the rainbow trout genome and its correlation with gene expression. This work also identified novel epigenetic markers associated with muscle atrophy in fish/lower vertebrates.
Coding and Noncoding Genes Involved in Atrophy and Compensatory Muscle Growth in Nile Tilapia
(MDPI, 2022-08-12) Ali, Ali; Shaalan, Walaa M.; Al-Tobasei, Rafet; Salem, Mohamed
Improvements in growth-related traits reduce fish time and production costs to reach market size. Feed deprivation and refeeding cycles have been introduced to maximize aquaculture profits through compensatory growth. However, the molecular compensatory growth signature is still uncertain in Nile tilapia. In this study, fish were subjected to two weeks of fasting followed by two weeks of refeeding. The growth curve in refed tilapia was suggestive of a partial compensatory response. Transcriptome profiling of starved and refed fish was conducted to identify genes regulating muscle atrophy and compensatory growth. Pairwise comparisons revealed 5009 and 478 differentially expressed (differential) transcripts during muscle atrophy and recovery, respectively. Muscle atrophy appears to be mediated by the ubiquitin-proteasome and autophagy/lysosome systems. Autophagy-related 2A, F-box and WD repeat domain containing 7, F-box only protein 32, miR-137, and miR-153 showed exceptional high expression suggesting them as master regulators of muscle atrophy. On the other hand, the muscle compensatory growth response appears to be mediated by the continuous stimulation of muscle hypertrophy which exceeded normal levels found in control fish. For instance, genes promoting ribosome biogenesis or enhancing the efficiency of translational machinery were upregulated in compensatory muscle growth. Additionally, myogenic microRNAs (e.g., miR-1 and miR-206), and hypertrophy-associated microRNAs (e.g., miR-27a-3p, miR-29c, and miR-29c) were reciprocally expressed to favor hypertrophy during muscle recovery. Overall, the present study provided insights into the molecular mechanisms regulating muscle mass in fish. The study pinpoints extensive growth-related gene networks that could be used to inform breeding programs and also serve as valuable genomic resources for future mechanistic studies.
Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro
(MDPI, 2022-12-31) Asfor, Amin S.; Reddy, Vishwanatha R. A. P.; Nazki, Salik; Urbaniec, Joanna; Brodrick, Andrew J.; Broadbent, Andrew J.
Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics.
Coding and noncoding genes involved in atrophy and compensatory muscle growth in Nile Tilapia
(MDPI, 2022-08-12) Ali, Ali; Shaalan, Walaa M.; Al-Tobasei, Rafet; Salem, Mohamed
Improvements in growth-related traits reduce fish time and production costs to reach market size. Feed deprivation and refeeding cycles have been introduced to maximize aquaculture profits through compensatory growth. However, the molecular compensatory growth signature is still uncertain in Nile tilapia. In this study, fish were subjected to two weeks of fasting followed by two weeks of refeeding. The growth curve in refed tilapia was suggestive of a partial compensatory response. Transcriptome profiling of starved and refed fish was conducted to identify genes regulating muscle atrophy and compensatory growth. Pairwise comparisons revealed 5009 and 478 differentially expressed (differential) transcripts during muscle atrophy and recovery, respectively. Muscle atrophy appears to be mediated by the ubiquitin-proteasome and autophagy/lysosome systems. Autophagy-related 2A, F-box and WD repeat domain containing 7, F-box only protein 32, miR-137, and miR-153 showed exceptional high expression suggesting them as master regulators of muscle atrophy. On the other hand, the muscle compensatory growth response appears to be mediated by the continuous stimulation of muscle hypertrophy which exceeded normal levels found in control fish. For instance, genes promoting ribosome biogenesis or enhancing the efficiency of translational machinery were upregulated in compensatory muscle growth. Additionally, myogenic microRNAs (e.g., miR-1 and miR-206), and hypertrophy-associated microRNAs (e.g., miR-27a-3p, miR-29c, and miR-29c) were reciprocally expressed to favor hypertrophy during muscle recovery. Overall, the present study provided insights into the molecular mechanisms regulating muscle mass in fish. The study pinpoints extensive growth-related gene networks that could be used to inform breeding programs and also serve as valuable genomic resources for future mechanistic studies.

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