Examination of the Lunatic fringe and Uncx4.1 expression by whole-mount in situ hybridization in the embryo of the CKH-Jsr (jumbled spine and ribs) mouse

The CKH-Jsr (jumbled spine and ribs) mouse was found as a spontaneous mutant with malformation of vertebrae, that is, a short trunk and kinky tail. We examined Lunatic Fringe (Lfng) and Uncx4.1 expression in the presomitic mesoderm (PSM) and somites of Jsr-mutant (CKH-Jsr/+) embryos to elucidate pathogenesis of the Jsr mutation. Expression pattern of Lfng in the PSM of Jsr-mutant embryos was similar to that of the normal (C57BL/6) embryos. However, expression pattern of Uncx4.1 in the somites of Jsr-mutant embryos was impaired to be irregular and mosaic, suggesting that the anterior-posterior (A-P) polarity is disordered in the Jsr mutant. These results indicate that the Jsr mutation disrupts the A-P polarity of somites during the somitogenesis without altering Lfng expression pattern in the PSM.     

Seasonal differential expression of KiSS‐1/GPR54 in the striped hamsters (Cricetulus barabensis) among different tissues

Kisspeptins and G protein coupled receptor (GPR54) play significant roles in regulating reproductive activity among seasonally reproductive animals; however, the mechanisms of KiSS‐1 and GPR54 gene affecting the seasonal reproduction in striped hamster are still unknown. In this study, real‐time quantitative polymerase chain reaction was employed to examine the expression profiles of KiSS‐1 and GPR54 in the hypothalamus, ovaries, testes, uterus and epididymis of striped hamsters across 4 different seasons. Our results showed that, across different seasons, the KiSS‐1 expression mode of male striped hamsters and the GPR54 expression mode of female striped hamsters were consistent with the seasonal photoperiod in the hypothalamus. Meanwhile, across different seasons, the expression profile of KiSS‐1 in the testes and the GPR54 expression profile of male striped hamsters in the hypothalamus were consistent with the intensity of their seasonal reproductive activity. Among different tissues, the expression trend for GPR54 is consistent across 4 seasons, while that for KiSS‐1 is tissue‐dependent. The expression trend for GPR54 across 4 seasons is the same regardless of gender, while that for KiSS‐1 is dramatically different and sex‐dependent across different seasons. These results suggest that the expressions of KiSS‐1 and GPR54 in the striped hamsters were regulated by complicated mechanisms, and the regulatory mechanisms in the striped hamsters are seasonal‐dependent and sex‐dependent. This research will provide a theoretical basis for studying how KiSS‐1 and GPR54 affect seasonal reproduction and the mechanisms behind their influence.     

Pax‐3 and Pax‐7 Label Muscle Progenitor Cells During Myotomal Myogenesis in Coregonus lavaretus (Teleostei: Coregonidae)

In Coregonus lavaretus, prior the mesoderm segmentation, in cells adjacent to the notochord called adaxial cells MyoD and slow myosin heavy chain (MyHC‐slow) proteins were observed. After somite formation, adaxial cells migrate towards the lateral part of the myotome and form a layer of red muscles. Deeper cells differentiate into white muscle fibres. In situ hybridization using Pax‐3 molecular probe revealed, that after somitogenesis, Pax‐3 is expressed in a layer of cells superficial to the myotome resembling the “external cells” (found in many teleosts species) or dermomyotome described in Amniota. During later developmental stages Pax‐3 gene is expressed in cells in intermyotomal space and then in myoblasts between myotubes. In these cells Pax‐7 protein was also observed. Pax‐3/7 positive cells which have migrated into the myotomes differentiate into satellite cells/secondary myoblasts and participate in hypertrophic and hyperplastic growth of muscles.     

In ovo carbohydrate supplementation modulates growth and immunity‐related genes in broiler chickens

A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune‐related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth‐related genes: chicken growth hormone (cGH), insulin‐like growth factor‐I & II (IGF‐I & II) and mucin were studied in hepatic and jejunum tissues of late‐term embryo and early post‐hatch chicks. Expression of candidate immune genes: Interleukin‐2, 6, 10 and 12 (IL‐2, IL‐6, IL‐10 and IL‐12), Tumour necrosis factor‐alpha (TNF‐α) and Interferon gamma (IFN‐γ) were studied in peripheral blood monocyte cells of in ovo‐injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con‐A). Glucose injection significantly increased the expression of IGF‐II gene during embryonic period and both cGH and IGF‐II in early post‐hatch period, while ribose‐injected chicks had higher expression of IGF‐II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose‐injected chicks during embryonic age. Glucose‐injected chicks had higher expression of IL‐6 or IL‐10, while those injected with fructose or ribose had higher expression of IL‐2, IL‐12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late‐term embryos and chicks. In ovo glucose could modulate humoral‐related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens.     

Neutral and acidic milk oligosaccharides of the striped skunk (Mephitidae: Mephitis mephitis)

The biological significance of the tremendous variation in proportions of oligosaccharides and lactose among mammalian milks is poorly understood. We investigated milk oligosaccharides of the striped skunk (Mephitis mephitis) and compared these results to other species of the clade Mustelida. Individual oligosaccharides were identified by proton nuclear magnetic resonance spectroscopy. In the striped skunk, six oligosaccharides were identified: isoglobotriose, 2′‐fucosyllactose, A‐tetrasaccharide, Galili pentasaccharide, 3′‐sialyllactose and monosialyl monogalactosyl lacto‐N‐neohexaose. Four of these have been found in related Mustelida and the other two in more distantly related carnivorans. The neutral and acidic oligosaccharides derive from three core structures: lactose (Gal(β1–4)Glc), lacto‐N‐neotetraose (Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc) and lacto‐N‐neohexaose (Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc).     

Utilization of digital differential display to identify differentially expressed genes related to rumen development

This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)‐based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5‐week‐old pre‐weaning: n = 3; 15‐week‐old weaned calves: n = 6). Among the 11 genes, only 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), aldo‐keto reductase family 1, member C1‐like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre‐ and post‐weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both. © 2015 Japanese Society of Animal Science     

Effects of pure plant secondary metabolites on methane production,rumen fermentation and rumen bacteria populations in vitro

In this study, the effects of seven pure plant secondary metabolites (PSM s) on rumen fermentation, methane (CH₄) production and rumen bacterial community composition were determined. Two in vitro trials were conducted. In trial 1, nine concentrations of 8‐hydroxyquinoline, α‐ terpineol, camphor, bornyl acetate, α‐ pinene, thymoquinone and thymol were incubated on separate days using in vitro 24‐hr batch incubations. All compounds tested demonstrated the ability to alter rumen fermentation parameters and decrease CH₄ production. However, effective concentrations differed among individual PSM s. The lowest concentrations that reduced (p  <  .05) CH₄ production were as follows: 8 mg/L of 8‐hydroxyquinoline, 120 mg/L of thymoquinone, 240 mg/L of thymol and 480 mg/L of α‐ terpineol, camphor, bornyl acetate and α‐ pinene. These concentrations were selected for use in trial 2. In trial 2, PSM s were incubated in one run. Methane was decreased (p  <  .05) by all PSM s at selected concentrations. However, only 8‐hydroxyquinoline, bornyl acetate and thymoquinone decreased (p  <  .05) CH₄ relative to volatile fatty acids (VFA s). Based on denaturing gradient gel electrophoresis analysis, different PSM s changed the composition of bacterial communities to different extents. As revealed by Ion Torrent sequencing, the effects of PSM s on relative abundance were most pronounced in the predominant families, especially in Lachnospiraceae , Succinivibrionaceae , Prevotellaceae , unclassified Clostridiales and Ruminococcaceae . The CH ₄ production was correlated negatively (?.72; p  <  .05) with relative abundance of Succinivibrionaceae and positively with relative abundance of Ruminococcaceae (.86; p  <  .05). In summary, this study identified three pure PSM s (8hydroxyquinoline, bornyl acetate and thymoquinone) with potentially promising effects on rumen CH₄ production. The PSM s tested in this study demonstrated considerable impact on rumen bacterial communities even at the lowest concentrations that decreased CH₄ production. The findings from this study may help to elucidate how PSM s affect rumen bacterial fermentation.     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down‐regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real‐time PCR in buffalo blastocysts produced by Hand‐made cloning and compared it with that in blastocyst‐stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c‐Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.     

Salmonella,Campylobacter, Clostridium difficile,and anti‐microbial resistant Escherichia coli in the faeces of sympatric meso‐mammals in southern Ontario,Canada

The role of free‐ranging wildlife in the epidemiology of enteropathogens causing clinical illness in humans and domestic animals is unclear. Salmonella enterica and anti‐microbial resistant bacteria have been detected in the faeces of raccoons (Procyon lotor), but little is known about the carriage of these bacteria in other sympatric meso‐mammals. Our objectives were to: (a) report the prevalence of Salmonella and associated anti‐microbial resistance, Campylobacter spp, Clostridium difficile, and anti‐microbial resistant Escherichia coli in the faeces of striped skunks (Mephitis mephitis) and Virginia opossums (Didelphis virginiana) in southern Ontario; and (b) compare the prevalence of these bacteria in the faeces of these meso‐mammal hosts with raccoons from a previously reported study. Faecal swabs were collected from striped skunks and Virginia opossums on five swine farms and five conservation areas from 2011 to 2013. Salmonella was detected in 41% (9/22) and 5% (5/95) of faecal swabs from Virginia opossums and striped skunks, respectively. None of the Salmonella serovars carried resistance to anti‐microbials. The prevalence of Campylobacter spp., C. difficile, and anti‐microbial resistant E. coli ranged from 6% to 22% in striped skunk and Virginia opossums. Using exact logistic regression, Salmonella was significantly more likely to be detected in faecal swabs of Virginia opossums than skunks and significantly less likely in faecal swabs from skunks than raccoons from a previously reported study. In addition, Campylobacter spp. was significantly more likely to be detected in raccoons than opossums. Salmonella Give was detected in 8/9 (89%) of Salmonella‐positive Virginia opossum faecal swabs. Our results suggest that striped skunks and Virginia opossums have the potential to carry pathogenic enteric bacteria in their faeces. The high prevalence of Salmonella Give in Virginia opossum faecal swabs in this study as well as its common occurrence in other Virginia opossum studies throughout North America suggests Virginia opossums may be reservoirs of this serovar.     

Limited Genetic Diversity and Gene Expression Differences between Egg‐ and Non‐Egg‐Related Salmonella Enteritidis Strains

Salmonella Enteritidis strains of egg‐ and non‐egg‐related origin were characterized molecularly to find markers correlated with the egg‐contaminating property of Salmonella Enteritidis. Isolates were examined by random amplified polymorphic DNA (RAPD), plasmid profiling and phage typing. Furthermore, the presence of 30 virulence genes was tested by PCR. In genetic fingerprinting and gene content, only small differences between the strains were found and no correlation was observed with the origin (egg‐related versus non‐egg‐related). A major RADP group was present in both egg‐ and non‐egg‐related strains, but other smaller RAPD groups were present as well in both categories of strains. Phage types PT4 and PT21 were predominant. Differential mRNA expression levels of fimA and agfA under conditions of growth simulating the conditions during egg formation were determined by real‐time RT‐PCR. Although differences in fimA and agfA expression levels were observed between the strains, these could not be correlated with the origin of the strains (egg‐related versus non‐egg‐related). The highest expression levels of agfA and fimA were only found in two non‐egg‐related strains, which seemed to be correlated with the presence of a 93 kb plasmid instead of the 60 kb virulence plasmid. Our results seem to indicate only a limited role for at least type I fimbriae (encoded by fim operon) in egg contamination by Salmonella Enteritidis.     

Effects of dietary leucine on antioxidant activity and expression of antioxidant and mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets

The study was conducted to investigate the effects of dietary leucine on antioxidant activity and expression of antioxidant‐ and mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets. Three diets were formulated with different levels of supplemented leucine (0%, 0.25%, 0.5%). Results showed that supplementation of 0.25% leucine significantly increased antisuperoxide anion (ASA) and antihydroxyl radical (AHR) levels and activities of total superoxide dismutade (T‐SOD), glutathione peroxidase (GPx), glutathione S‐transferase (GST), and total antioxidant capacity (T‐AOC) in serum, longissimus dorsi muscle and liver of piglets as compared with the control group. The SOD2, catalase (CAT), GPx, GST, glutathione reductase (GR), and nuclear factor erythroid 2‐related factor 2 (Nrf2) mRNA levels in longissimus dorsi muscle and liver were significantly increased by 0.25% leucine supplementation. However, the malondialdehyde (MDA) content and the mRNA level of Kelch‐like ECH‐associated protein 1 (Keap1) exhibited an opposite tendency. Additionally, supplementation of 0.25% leucine significantly increased the mRNA levels of mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets. Results suggested that supplementation of 0.25% leucine improved antioxidant activity and mitochondrial biogenesis and function of piglets, which was related to the increase in antioxidant enzymes activities and upregulation of expression of antioxidant‐ and mitochondrial‐related genes.     

A genomewide association mapping study using ultrasound‐scanned information identifies potential genomic regions and candidate genes affecting carcass traits in Nellore cattle

The aim of this study was to identify candidate genes and genomic regions associated with ultrasound‐derived measurements of the rib‐eye area (REA), backfat thickness (BFT) and rumpfat thickness (RFT) in Nellore cattle. Data from 640 Nellore steers and young bulls with genotypes for 290 863 single nucleotide polymorphisms (SNPs) were used for genomewide association mapping. Significant SNP associations were explored to find possible candidate genes related to physiological processes. Several of the significant markers detected were mapped onto functional candidate genes including ARFGAP3, CLSTN2 and DPYD for REA; OSBPL3 and SUDS3 for BFT; and RARRES1 and VEPH1 for RFT. The physiological pathway related to lipid metabolism (CLSTN2, OSBPL3, RARRES1 and VEPH1) was identified. The significant markers within previously reported QTLs reinforce the importance of the genomic regions, and the other loci offer candidate genes that have not been related to carcass traits in previous investigations.     

Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.     

Photokinesis and Djopsin gene expression analysis during the regeneration of planarian eyes

Planarians provide the ideal model for studying eye development, with their simple eye structure and exceptionally rapid regeneration. Here, we observed the eye morphogenesis, photophobic behavior, spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica. The results showed that: (i) Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution; (ii) planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue (495 nm) and yellow (635 nm); (iii) the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration; and (iv) Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians.