Myostatin gene role in regulating traits of poultry species for potential industrial applications.

The myostatin (MSTN) gene is considered a potential genetic marker to improve economically important traits in livestock, since the discovery of its function using the MSTN knockout mice. The anti-myogenic function of the MSTN gene was further demonstrated in farm animal species with natural or induced mutations. In poultry species, myogenesis in cell culture was regulated by modulation of the MSTN gene. Also, different expression levels of the MSTN gene in poultry models with different muscle mass have been reported, indicating the conserved myogenic function of the MSTN gene between mammalian and avian species. Recent advances of CRISPR/Cas9-mediated genome editing techniques have led to development of genome-edited poultry species targeting the MSTN gene to clearly demonstrate its anti-myogenic function and further investigate other potential functions in poultry species. This review summarizes research conducted to understand the function of the MSTN gene in various poultry models from cells to whole organisms. Furthermore, the genome-edited poultry models targeting the MSTN gene are reviewed to integrate diverse effects of the MSTN gene on different traits of poultry species.

Research Note: Sexual dimorphic effect of myostatin mutation on fat accumulation in Japanese quail.

As an anti-myogenic factor, the myostatin (MSTN) gene was mainly considered as a genetic marker to improve meat production. Moreover, an additional effect of the MSTN mutation on reducing fat deposition in various farm animals suggested a potential application of the MSTN gene on regulating fat deposition in poultry species. Although increase in muscle mass resulted from muscle hyperplasia in the MSTN mutant quail, cellular mechanism behind the decrease in fat deposition was not investigated in the quail model. In the current study, to investigate sexual dimorphic association between fat deposition and Mstn mutation in quail, leg and abdominal fat pads from 4-month-old male and female quail were histologically analyzed. Interestingly, abdominal and leg fat pad weights were significantly decreased by the MSTN mutation only in female quail, but not in male quail, showing sexual dimorphism in regulating fat deposition by the MSTN mutation in quail. Histological analysis also revealed that fat cell sizes of leg and abdominal fats were significantly reduced only in female groups aligning with the decreased fat pad weights. Sexual dimorphic effect of the MSTN mutation on fat cell hypotrophy and reduced fat pad weights in quail provided an important scientific finding to be considered on the usage of the MSTN gene as a genetic marker to reduce fat deposition in poultry species.

Transcriptomic analysis of sheep hypothalamus discloses regulatory genes potentially involved in sex-dependent differences in body weight of progeny born to dams supplemented with omega-3 fatty acids or methionine during late-gestation.

Fetal programming research conducted in sheep has reported sexually dimorphic responses on growth of the progeny born to in-utero methionine or omega-3 fatty acids supplementation. However, the biological mechanism behind the nutrient by sex interaction as a source of variation in offspring body weight is still unknown. A high-throughput RNA sequencing data of hypothalamus samples from 17 lambs were used in the current study to identify differentially expressed genes (DEGs) between males and females born to dams supplemented with different nutrients during late-gestation. Ewes received a basal diet without omega-3 fatty acids or methionine supplementation as the control (CONT); omega-3 fatty acids supplementation (FAS), or methionine supplementation (METS). A list of regulated genes was generated. Data were compared as CONT vs. FAS and CONT vs. METS. For CONT vs. METS, a treatment by sex interaction was found (adjusted P-value < 0.05) on 121 DEGs (112 upregulated and 9 downregulated) on female lambs born to METS compared with METS males. Importantly, with the sex interaction term, more than 100 genes were upregulated in female lamb's hypothalamuses born to METS. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were performed using the DEGs from female lambs. Terms under biological process (related to morphogenesis, organism, and tissue development), cellular component (related to chromatin, extracellular components), and molecular function (involved in chromatin structure and transcription and factors linked to binding DNA) were presented (adjusted P-value < 0.05) for GO. For the IPA, the top-scoring network was developmental disorder, endocrine system development and function, and organ morphology. Only a few differences were observed in the comparison between the interaction of sex and treatment for the CONT vs. FAS comparison. The markedly increased number of DEGs substantially involved in developmental and growth processes indicates the extent to which maternal methionine supplementation causes the sexually dimorphic effects observed in the offspring.

Feeding dams during gestation affects the development of the offspring for their entire life. The objective of the current experiment was to evaluate the changes of the transcriptome in the hypothalamus of the offspring lambs born from dams supplemented with (i) a control diet (without lipids or methionine supplementation), (ii) an omega-3 fatty acid supplementation, or (iii) a methionine supplementation. The supplementation took place in the last third of gestation and the hypothalamus of male and female offspring was collected after being on a fattening diet for 54 d. Hypothalamus samples were used to extract RNA and analyzed using RNA sequencing. There was an interaction due to sex and methionine supplementation. The pathways that were modified were chromatin structure, developmental processes, and organ morphology. The modification observed on these pathways could explain the sex by treatment interaction differences previously observed in growth. There were few sex by omega-3 fatty acid interactions on the hypothalamus transcriptome. Therefore, the sexual dimorphism observed by methionine supplementation may be regulated by the hypothalamus.

Melanophilin regulates dendritogenesis in melanocytes for feather pigmentation.

Limited studies using animal models with a few natural mutations in melanophilin (Mlph) provided partial functions of Mlph in melanosome trafficking. To investigate cellular functions of Mlph, especially ZnF motif of Mlph, we analyzed all three Mlph knockout (KO) quail lines, one and two base pair (bp) deletions as models for total KO, and three bp deletion causing deletion of one Cysteine (C84del) in the ZnF motif. All quail lines had diluted feather pigmentation with impaired dendritogenesis and melanosome transport in melanocytes. In vitro studies revealed capability of binding of the ZnF motif to PIP3, and impairment of PI3P binding and mislocalization of MLPH proteins with ZnF motif mutations. The shortened melanocyte dendrites by the C84del mutation were rescued by introducing WT Mlph in vitro. These results revealed the diluted feather pigmentation by Mlph mutations resulted from congregation of melanosomes in the cell bodies with impairment of the dendritogenesis and the transport of melanosomes to the cell periphery.

Bisphenol Analogs Downregulate the Self-Renewal Potential of Spermatogonial Stem Cells.

PURPOSE: In this study, we investigated the effect of bisphenol-A (BPA) and its major analogs, bisphenol-F (BPF), and bisphenol-S (BPS), on spermatogonial stem cells (SSCs) populations using in vitro SSC culture and in vivo transplantation models. MATERIALS AND METHODS: SSCs enriched from 6- to 8-day-old C57BL/6-eGFP+ male mice testes were treated with varying concentrations of bisphenols for 7 days to examine bisphenol-derived cytotoxicity and changes in SSC characteristics. We utilized flow cytometry, immunocytochemistry, real-time quantitative reverse transcription-PCR, and western blot analysis. The functional alteration of SSCs was further investigated by examining donor SSC-derived spermatogenesis evaluation through in vivo transplantation and subsequent testis analysis. RESULTS: BPF exhibited a similar inhibitory effect on SSCs as BPA, demonstrating a significant decrease in SSC survival, inhibition of proliferation, and induction of apoptosis. On the other hand, while BPS was comparatively weaker than BPA and BPF, it still showed significant SSC cytotoxicity. Importantly, SSCs exposed to BPA, BPF, and BPS exhibited a significant reduction in donor SSC-derived germ cell colonies per total number of cultured cells, indicating that, like BPA, BPF, and BPS can induce a comparable reduction in functional SSCs in the recipient animals. However, the progress of spermatogenesis, as evidenced by histochemistry and the expressions of PCNA and SSC specific markers, collectively indicates that BPA, BPF, and BPS may not adversely affect the spermatogenesis. CONCLUSIONS: Our findings indicate that the major BPA substitutes, BPF and BPS, have significant cytotoxic effects on SSCs, similar to BPA. These effects may lead to a reduction in the functional self-renewal stem cell population and potential impacts on male fertility.

Research Note: Muscle hypertrophy is associated with reversed sexual dimorphism in body size of quail.

Sexual dimorphism is phenotypic differences between males and females in the same species. In general, males in most animals are larger than females at the same age, however, in quail, females have a larger body size with greater muscle mass than males. To understand what characteristics in muscle growth play roles in reversed sexual dimorphism in quail, the weights and the characteristics of the pectoralis major and gastrocnemius muscles (PM and GM, respectively) of male and female quail were compared in the current study. The data showed that 15-wk-old female quail have significantly heavier bodies, and PM and GM weights compared to male quail (1.27-folds, 1.29-folds, and 1.16-folds, respectively). To compare muscle characteristics such as hypertrophy (increased size) and hyperplasia (increased cell number), the PM and GM were stained using hematoxylin and eosin, and then histological characteristics such as total cross-sectional area (CSA), number and size of myofibers, and muscle bundle of the muscles were measured and analyzed. In both PM and GM, there were no differences in total numbers of myofibers and muscle bundles as well as the average numbers of myofibers per bundle between sexes. However, the sizes of myofiber and the bundle were significantly increased in female compared to male (1.33-folds and 1.28-folds in PM, and both 1.23-folds in GM, respectively). The findings of the current study suggest that muscle hypertrophy in female quail, not hyperplasia, can be attributed to the sexual dimorphism in quail size.

The AIRN lncRNA is imprinted and paternally expressed in pigs.

Genomic imprinting plays critical roles during the development of mammalian species and underlying epigenetic mechanisms frequently involve long non-coding RNAs (lncRNAs). The paternal transcription of the antisense Igf2r RNA noncoding (Airn) is responsible for paternal silencing of the mouse insulin-like growth factor 2 receptor (Igf2r) gene and maternal Igf2r expression. Although the corresponding maternal DNA methylation imprint is conserved in humans and pigs, the orthologous AIRN lncRNA has been identified in humans but not in pigs. Here, we aimed to examine imprinted allelic expression of the porcine AIRN lncRNA along with a corresponding differentially methylated region (DMR) and to analyze allelic expression of AIRN and IGF2R in pigs. By comparing parthenogenetic and control porcine embryos, we identified a maternally methylated DMR and a significantly higher expression of AIRN lncRNA in control embryos (P < 0.05) indicating its paternal expression. Further analyses revealed that the expression of AIRN lncRNA was enriched in the pig brain and its subregions, and it was monoallelically expressed; whereas, IGF2R was expressed biallelically suggesting an absence of allele-specific transcriptional regulation. Our findings will lead to further investigations into the role of the imprinted porcine AIRN lncRNA during pig development.

Genomic imprinting is important for the development of mammals and long non-coding RNAs are often involved in the imprinting process. In mice, Airn encodes a long non-coding RNA that is imprinted, and therefore, transcribed only from the paternal allele. This paternal transcription of Airn interferes with the adjacent Igf2r promoter, leading to maternal expression of Igf2r. In pigs, the orthologous AIRN has not been identified as well as its imprinting. In the current study, we report porcine AIRN and allelic expression of both AIRN and IGF2R using our parthenogenetic embryo models and various normal pig tissues.

Greater numbers and sizes of muscle bundles in the breast and leg muscles of broilers compared to layer chickens.

Meat-type (broiler) and egg-type (layer) chickens were bred by intensive selection over the years, resulting in more numbers and larger sizes of myofibers. Although the characteristics are important parameters in muscle growth and meat quality, muscle bundle characteristics have not been studied in poultry. Therefore, this study aimed to compare the histological characteristics of myofibers and muscle bundles in muscles between male broiler (Ross broiler breed) chickens and layer (Hy-Line) chickens. Chicken muscles, pectoralis major (PM) and gastrocnemius (GM), were sampled at the age of 49 days and stained to analyze histological characteristics. Expectedly, body weights (BWs) and weights of PM and GM muscles in 49-day-old broilers were significantly heavier than those in layers. Within PM, broilers exhibited greater number and cross-sectional area (CSA) of myofibers than layers (3.3- and 3.3-fold, respectively). The total number and CSA of PM muscle bundles were approximately 1.5 and 6.6 times greater, respectively, in broilers than layers. Moreover, broilers exhibited 2 times greater number of myofibers per bundle of PM muscle than layers. Within GM, myofiber number and CSA were 2.3- and 2.4-fold greater, respectively, in broilers than layers. In addition, the total number of muscle bundles and bundle CSA were 2.5- and 2.1-fold greater, respectively, in broilers than in the layers. The novel findings of the current study provide evidence that greater muscle mass of broilers occurs by both hyperplasia and hypertrophy of muscle bundles and myofibers.

Imprinting at the KBTBD6 locus involves species-specific maternal methylation and monoallelic expression in livestock animals.

BACKGROUND: The primary differentially methylated regions (DMRs) which are maternally hypermethylated serve as imprinting control regions (ICRs) that drive monoallelic gene expression, and these ICRs have been investigated due to their implications in mammalian development. Although a subset of genes has been identified as imprinted, in-depth comparative approach needs to be developed for identification of species-specific imprinted genes. Here, we examined DNA methylation status and allelic expression at the KBTBD6 locus across species and tissues and explored potential mechanisms of imprinting. RESULTS: Using whole-genome bisulfite sequencing and RNA-sequencing on parthenogenetic and normal porcine embryos, we identified a maternally hypermethylated DMR between the embryos at the KBTBD6 promoter CpG island and paternal monoallelic expression of KBTBD6. Also, in analyzed domesticated mammals but not in humans, non-human primates and mice, the KBTBD6 promoter CpG islands were methylated in oocytes and/or allelically methylated in tissues, and monoallelic KBTBD6 expression was observed, indicating livestock-specific imprinting. Further analysis revealed that these CpG islands were embedded within transcripts in porcine and bovine oocytes which coexisted with an active transcription mark and DNA methylation, implying the presence of transcription-dependent imprinting. CONCLUSIONS: In this study, our comparative approach revealed an imprinted expression of the KBTBD6 gene in domesticated mammals, but not in humans, non-human primates, and mice which implicates species-specific evolution of genomic imprinting.

Research Note: Injection of adenoviral CRISPR/Cas9 system targeting melanophilin gene into different sites of embryos induced regional feather color changes in posthatch quail.

Poultry species is an important animal model in both avian research and the poultry industry. To advance our understanding of genetic factors and benefit both fields, a gene of interest can be genetically edited, and consequential phenotypic changes can be investigated. Injection of adenovirus containing the CRISPR/Cas9 system into avian blastoderm induced genome editing in blastodermal cells randomly, including primordial germ cells, which results in generation of whole-body knockout in the offspring of the virus-injected quail. However, to observe phenotypic and functional changes in whole-body, homozygous knockout of genes using this genome editing technology requires at least 2 generations of breeding of chimeric, and heterozygotes birds. In the current study, we developed a strategy to investigate the gene function in 1-generation by inducing regional genome editing around the injection sites with CRISPR/Cas9 adenovirus. The adenoviral CRISPR/Cas9 vector targeting the melanophilin (Mlph) gene, regulating feather pigmentation, was injected into 2 different regions of embryos, the cervical flexure of quail embryos at HH stage 13 to 15 and the tip of the upper limb bud of embryos at HH stage 22 to 24, to induce genome editing in those regions. Indel mutations in the target loci of the Mlph gene were detected by extracting genomic DNA from the embryonic tissues, and consequential phenotypes, feather color changes, were analyzed at 1 mo after hatch. Injection of the adenovirus into the cervical flexure and the tip of the upper limb bud of embryos resulted in 8 to 21% efficiency of indel mutation in the embryonic cells of the injected regions. In the posthatch quail, gray feathers were shown on their upper back and primary wing feathers, corresponding to the injection sites at embryos. Successful validation of this strategy for inducing genome editing in parts of tissues within 1-generation will accelerate studies on genetic functions with advantages of less time and cost, facilitating avian research and providing foundations for future application for the poultry industry.

Research Note: Distribution of nanospheres in the cuticle layer of the eggshell in major poultry species.

Mineralized eggshell is a unique and protective structure in an avian egg. Among different eggshell layers, the cuticle layer is an outermost layer and plays a critical role in protection against bacterial infection. Although the importance of nanosphere in the cuticle layer on the antimicrobial function has been widely accepted, the detailed nanostructure of the cuticle layer in the major poultry species has not been investigated. In the current study, eggs from Japanese quail, commercial layer chickens, mixed breed turkeys, and White Pekin ducks were collected. To investigate the nanostructure throughout the cuticle layer, images of the cross-sectional cuticle layer were taken using a scanning electron microscope (SEM). Unlike the cuticle layer in ducks showing deformed bunched nanospheres, clearly separated nanospheres were present throughout the cuticle layer in quail, chickens, and turkeys. The average size of the nanosphere was the biggest in turkeys and similar between quail and chickens. Most importantly, the size of nanospheres was increased as they ascended from the bottom of the cuticle layer in quail, showing a positive correlation between the size and distance of the nanospheres. However, different sizes of nanospheres were randomly distributed throughout the cuticle layer in chickens and turkeys, showing a weak correlation in chickens and no correlation in turkeys between the size and distance of nanospheres. These new findings in different nanostructures of the cuticle layers in quail, chickens, turkeys, and ducks will serve as a new foundation to better relate their structures with functions.

The effects of myostatin mutation on the tibia bone quality in female Japanese quail before and after sexual maturation.

In the modern layer industry, improvement of bone quality is one of the prior tasks to solve from economic and welfare standpoints. In addition to nutritional and environmental factors, genetic factors have been considered major factors regulating bone quality in layers but are yet to be fully investigated due to limitations on available animal models. Initially, the myostatin (MSTN) gene was genetically edited in quail to investigate the effect of MSTN mutation on economic traits in meat producing poultry species. In the current study, the function of the MSTN gene on regulation of bone quality in layers was investigated using MSTN mutant female quail as an animal model. Tibia bones were collected from wild-type (WT) and MSTN mutant female quail at 5 wk old and 4 mo old, representing prelaying and actively laying stages, respectively. Left tibia bones were analyzed by microcomputed tomography scanning to evaluate the architectural characteristics, while bone breaking strength (BBS) was measured using right tibia bones. At 5 wk of age, MSTN mutant female quail showed higher BBS and values on parameters related to bone quality such as bone mineral contents (BMC), bone mineral density (BMD), bone volume (BV), and/or trabecular bone thickness in whole diaphysis, whole metaphysis, and metaphyseal trabecular bone, compared to WT female quail. Although BBS and BMD became similar between the 2 groups at 4 mo of age, higher TV and TS in whole metaphysis and higher BMC and TV in whole diaphysis of MSTN mutant group compared to those of WT group suggested that the improved tibia bone quality by MSTN mutation before sexual maturation lasted to a certain degree even after sexual maturation. The use of the MSTN mutant female model provided new insights into genetic regulation on female quail bone quality depending on physiological changes.

TLR3 and MDA5 Knockout DF-1 cells Enhance Replication of Avian Orthoavulavirus 1.

Despite the essential role of innate immunity in defining the outcome of viral infections, the roles played by different components of the avian innate immune system are poorly delineated. Here, we investigated the potential implication of avian toll-like receptor (TLR) 3 (TLR3) and melanoma differentiation-associated (MDA) gene 5 (MDA5) receptors of double-stranded RNA (dsRNA) in induction of the interferon pathway and avian orthoavulavirus 1 (AOAV-1) replication in chicken-origin DF-1 fibroblast cells. TLR3 and MDA5 knockout (KO) DF-1 cells were generated using our avian-specific CRISPR/Cas9 system and stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or infected with AOAV-1 (previously known as Newcastle disease virus). Poly(I:C) treatment in cell culture media resulted in significant upregulation of interferon (IFN)α, IFNß, and Mx1 gene expression in wild type (WT) DF-1 cells but not in TLR3-MDA5 double KO cells. Interestingly, poly(I:C) treatment induced rapid cell degeneration in WT and MDA5 KO cells, but not in TLR3 knockout or TRL3-MDA5 double knockout (DKO) cells, directly linking poly(I:C)-induced cell degeneration to TLR3-mediated host response. The double knockout cells supported significantly higher replication of AOAV-1 virus than did the WT cells. However, no correlation between the level of virus replication and type I IFN response was observed. Our study suggests that innate immune response is host- and pathogen specific, and further investigation is needed to understand the relevance of dsRNA receptor-mediated immune responses in viral replication and pathogenesis in avian species.

Nota de investigación- En bloqueo de los genes TLR3 y MDA5 en las células DF-1 mejoran la replicación de Ortoavulavirus aviar 1. A pesar del papel esencial de la inmunidad innata en la definición del resultado de las infecciones virales, las funciones que desempeñan los diferentes componentes del sistema inmunitario innato aviar no están completamente definidas. En este estudio se investigó el posible papel del receptor aviar tipo toll (TLR) número 3 (TLR3) y los receptores de ARN de doble cadena (dsRNA del gene asociado a la diferenciación de melanoma (MDA) número 5 (MDA5) en la inducción de la vía del interferón y en la replicación del Ortoavulavirus 1 (AOAV-1) en células de fibroblastos DF-1 de origen en pollo. Las células DF-1 con los genes TLR3 y MDA5 bloqueado (KO) se generaron utilizando nuestro sistema CRISPR/Cas9 específico para aves y se estimularon con un ligando de dsRNA sintético poliinosínico: ácido policitidílico [poli(I:C)] o se infectaron con AOAV-1 (anteriormente conocido como el virus de la enfermedad de Newcastle). El tratamiento con poli(I:C) en medios de cultivo celular resultó en una regulación positiva significativa de la expresión génica de interferón (IFN)α, IFNß y Mx1 en células DF-1 de tipo silvestre (WT) pero no en células con doble bloqueo TLR3-MDA5 (DKO). Curiosamente, el tratamiento con poli(I:C) indujo una rápida degeneración celular en las células silvestres (WT) y las células con el gene MDA5 bloqueado, pero no en las células con bloqueo del gene TLR3 o con las células con doble bloqueo de TRL3-MDA5, lo que vincula directamente la degeneración celular inducida por poli(I:C) con la respuesta de la huésped mediada por TLR3. Las células con doble bloqueo soportaron una replicación significativamente mayor del Ortoavulavirus 1 que las células silvestres. Sin embargo, no se observó correlación entre el nivel de replicación del virus y la respuesta de IFN tipo I. Este estudio sugiere que la respuesta inmune innata es específica del huésped y del patógeno, y se necesita más investigación para comprender la relevancia de las respuestas inmunes mediadas por el receptor dsRNA en la replicación viral y en la patogénesis en las especies aviares.

Effects of a myostatin mutation in Japanese quail (Coturnix japonica) on the physicochemical and histochemical characteristics of the pectoralis major muscle.

The aim of this study was to compare the carcass, meat quality, and histochemical characteristics of pectoralis major (PM) muscle between wild type (WT) and myostatin (Mstn) homozygous mutant (HO) quail lines. The HO quail line exhibited significantly heavier body weight (HO vs. WT, 115.7 g vs. 106.2 g, approximately 110%) and PM muscle weight (HO vs. WT, 18.0 g vs. 15.2 g, approximately 120%) compared to the WT (p < 0.001). However, the two groups had similar traits (pH, redness, yellowness, and drip loss) for meat quality, although slightly higher lightness and cooking loss were observed in the mutant quail (103% and 141%, respectively, p < 0.05). For histochemical traits of PM muscle, Mstn mutant quail exhibited lower type IIA and higher type IIB percentage in the deep region than WT quail (p < 0.05), indicating a fiber conversion from the type IIA to IIB. However, the two quail lines had comparable histochemical traits in the superficial region (p > 0.05). These data suggest that Mstn mutation greatly increases muscle mass without significantly affecting meat quality.

Short communication: In ovo injection of all-trans retinoic acid causes adipocyte hypertrophy in embryos but lost its effect in posthatch chickens.

The regulation of adipose deposition in broiler chickens is an important factor for production efficiency to poultry producers and health concerns to customers. Although vitamin A and its metabolite [all-trans retinoic acid (atRA)] have been used for studies on adipogenesis in mammals and avian, effects of embryonic atRA on adipose development in embryonic (E) and posthatch (D) ages in broiler chickens have not been studied yet. Different concentrations of atRA (0 M-2 µM) were injected in broiler eggs at E10, and adipose tissues were sampled at E16. Percentages of adipose tissues in chicken embryos were significantly increased in the group injected with 500 nM of atRA compared to the 0 M group (P < 0.05). In addition, the adipocyte cross-sectional area (CSA) was significantly greater by in ovo injection of 500 nM atRA compared to the injection of 0 M (P < 0.01). Moreover, in ovo atRA-injected embryos were hatched and BWs were measured at D0, D7, and D14. BWs were not different from those of the 0 M group. Percentages of adipose tissues and CSA of the in ovo atRA-injected group (500 nM) were not different from those of the 0 M group at D14. Taken together, the current study clearly showed that in ovo injection of atRA promoted adipose deposition with hypertrophy during embryonic development, but its effects were not maintained in early posthatch age in broiler chickens, implying that embryonic atRA has an important role in the regulation of adipose development in chicken embryos.

Chordin-like 1, a Novel Adipokine, Markedly Promotes Adipogenesis and Lipid Accumulation.

White adipose tissue serves as a metabolically dynamic organ that can synthesize and secrete biologically active compounds such as adipokines as well as a caloric reservoir for maintaining energy homeostasis. Adipokines are involved in diverse biological and physiological processes and there have been extensive attempts to characterize the effects of over two dozen adipokines. However, many of these adipokines are produced by not only adipose tissue, but also other tissues. Therefore, investigations into the effects of adipokines on physiological functions have been challenged. In this regard, we aimed to identify a new secreted protein that is encoded by genes specifically expressed in white adipose tissue through analysis of multi-tissue transcriptome and protein expression. As a result, we report a novel adipokine that is encoded by the adipose-specific gene, chordin-like 1 (Chrdl1), which is specifically expressed in white adipose tissue in mice; this expression pattern was conserved in the human orthologous CHRDL1 gene. The expression of Chrdl1 was enriched in fat cells and developmentally regulated in vitro and in vivo, and moreover, its retrovirus-mediated overexpression and recombinant protein treatment led to markedly increased adipogenesis. Further pathway enrichment analysis revealed enriched pathways related to lipogenesis and adipogenic signaling. Our findings support a pro-adipogenic role of CHRDL1 as a new adipokine and pave the way toward animal studies and future research on its clinical implications and development of anti-obesity therapy.

Lipid metabolism mRNA expression and cellularity of intramuscular adipocytes within the Longissimus muscle of Angus- and Wagyu-sired cattle fed for a similar days on feed or body weight endpoint.

This study investigates intramuscular (IM) adipocyte development in the Longissimus muscle (LM) between Wagyu- and Angus-sired steers compared at a similar age and days on feed (D) endpoint or similar body weight (B) endpoint by measuring IM adipocyte cell area and lipid metabolism mRNA expression. Angus-sired steers (AN, n = 6) were compared with steers from two different Wagyu sires (WA), selected for either growth (G) or marbling (M), to be compared at a similar days on feed (DOF; 258 ± 26.7 d; WA-GD, n = 5 and WA-MD, n = 5) in Exp. 1 or body weight (BW; 613 ± 18.0 kg; WA-GB, n = 4 and WA-MB, n = 5) in Exp. 2, respectively. In Exp. 1, WA-MD steers had a greater (P ≤ 0.01) percentage of IM fat in the LM compared with AN and WA-GD steers. In Exp. 2, WA-MB steers had a greater (P ≤ 0.01) percentage of IM fat in the LM compared with AN and WA-GB steers. The distribution of IM adipocyte area was unimodal at all biopsy collections, with IM adipocyte area becoming progressively larger as cattle age (P ≤ 0.01) and BW increased (P ≤ 0.01). Peroxisome proliferator activated receptor delta (PPARd) was upregulated earlier for WA-MD and WA-MB cattle compared with other steers at a similar DOF and BW (P ≤ 0.02; treatment × biopsy interaction). Peroxisome proliferator activated receptor gamma was upregulated (PPARg) at a lesser BW for WA-MB steers (P = 0.09; treatment × biopsy interaction), while WA-MD steers had a greater (P ≤ 0.04) overall mean PPARg mRNA expression compared with other steers. Glycerol-3-phosphate acyltransferase, lipin 1, and hormone sensitive lipase demonstrated mRNA expression patterns similar to PPARg and PPARd or CCAAT enhancer binding protein beta, which emphasizes their importance in marbling development and growth. Additionally, WA-MD and WA-MB steers often had a greater early mRNA expression of fatty acid transporters (fatty acid transport protein 1; P < 0.02; treatment × biopsy interaction) and binding proteins (fatty acid binding protein 4) compared with other steers. Cattle with a greater marbling propensity appear to upregulate adipogenesis at a younger chronological and physiological maturity through PPARd, PPARg, and possibly adipogenic regulating compounds, lysophosphatidic acid, and diacylglycerol. These genes and compounds could be used as potential markers for marbling propensity of cattle in the future.

This study investigates intramuscular (IM) adipocyte development in the Longissimus muscle (LM) between Wagyu- and Angus-sired steers compared at a similar age and days on feed (D) or similar body weight (B) endpoint by measuring IM adipocyte cell area and lipid metabolism mRNA expression. Angus-sired steers (AN) were compared with steers from two different Wagyu sires (WA), selected for either growth (G) or marbling (M), to be compared at a similar days on feed (DOF; WA-GD, and WA-MD) in Exp. 1 or body weight (BW; WA-GB, and WA-MB) in Exp. 2. The WA-MD and WA-MB steers had a greater percentage of IM fat in the LM compared with AN and WA-GD and WA-GB steers. Intramuscular adipocyte cellularity analysis indicated few differences due to adipocyte size; therefore, marbling differences were likely due to adipocyte number. The WA-MD and WA-MB steers typically experienced an earlier or a greater mRNA upregulation for adipogenic genes that regulate fatty acid transport and triglyceride synthesis compared with other steers. The pattern of mRNA expression was very similar between steers compared at either a similar age and DOF or BW. Indicating that the timing of energy intake above maintenance requirements may be more influential than age for the upregulation of lipid metabolism in cattle.

Research Note: Expression levels of collagen-related genes in PSE conditions and white striping features of broiler pectoralis major muscle.

The objective of this study was to compare the meat quality characteristics and collagen-related gene expression levels in the broiler pectoralis major (PM) muscle among the meat quality groups, including normal, pale, soft, and exudative (PSE), and white striping (WS) groups. The group was classified by their WS degree (moderate or severe striping) and quality traits including pH and lightness values at 24 h postmortem (normal group: pH24 h ≥5.7, 48≤L*≤53, without WS features; PSE group: pH24 h <5.7, L* >53, without WS features; WS group: pH24 h ≥5.7, 48≤L*≤53, with moderate or severe striping). The WS group revealed no differences in all measured meat quality traits compared to the normal group (P > 0.05). PM muscles exhibiting PSE conditions without WS indicated lower pH15 min and pH24 h values (P < 0.05). Whereas, lower lightness and cooking loss values were observed in the normal and WS groups compared to the PSE group (P < 0.05). No significant difference was observed in the level of type I collagen among the groups (P > 0.05), whereas a higher type III collagen level was observed in the WS group than in the other groups (P < 0.05). Additionally, the WS group showed a higher type IV collagen level compared to the normal group (P < 0.05) and a level not different from that of the PSE group (P > 0.05). In contrast, the expression levels of matrix metalloproteinase (MMP) 2, involved in type IV collagen degradation, and angiopoietin-like protein 7, associated with collagen accumulation, were higher in the WS group compared to the normal group (P < 0.05). However, no difference was detected in the MMP1 level among the all groups (P > 0.05). These results suggest that the occurrence of WS features in broiler PM muscle, unlike PSE and normal conditions, can be influenced by the expression levels of collagen-related genes associated with abnormalities in extracellular matrix components.

Research Note: Growth promoting potential in Mstn mutant quail dependent and independent of increased egg size.

In avian species, positive relationships between egg weight (EW) and body weight (BW) have been reported. However, the correlation between the body growth rate and different weights of eggs from genetically mutated avian species was not studied yet. Myostatin (Mstn), an anti-myogenic factor, mutant quail were recently developed, and it was reported that EW produced from Mstn homozygous mutant quail (HO) was heavier compared to those from wild-type quail (WT). In the current study, distributions of pre-incubated EW and associations between EW and BW were compared between the Mstn mutant and WT quail lines. Average egg weight for the HO group was significantly heavier than the WT (P < 0.001) and the number of eggs having heavier EW (over 11 g) was higher in the HO compared to the WT (P < 0.01). BWs at wk (W) 0, 4, and 6 after hatch were also significantly greater in the HO (P < 0.001 in all groups). In addition, linear regression analyses revealed positive relationships between EW and BW from W0 to W6, regardless of sexes and genotypes. Furthermore, Mstn mutant quail were a heavier BW compared to the WT quail originated from eggs with similar weights. These data indicate that increased BW by Mstn mutation is contributed by increased EW and/or growth promoting activity of Mstn mutation independent of increasing egg sizes. These findings provide Mstn as a desirable genetic factor for selection of poultry breeds with superior growth. In addition, the knowledge gained from this study could inspire similar proof-of-concept studies involving standard and commercial lines of poultry.

Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing.

Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.