Purification of polymerase chain reaction (PCR)-amplifiable DNA from compost piles containing bovine mortalities

Livestock production systems utilize composting as a method of disposal of livestock mortalities, but there is limited information on the rate and extent of carcass decomposition. Detection of specific DNA fragments by PCR offers a method for investigating the degradation of carcasses and other biological materials during composting. However, the purity of extracted DNA is critical for successful PCR analysis. We applied a method to purify DNA from compost samples and have tested the method by analyzing bovine and plant DNA targets after 0, 4, and 12 month of composting. The concentration of organic matter from composted material posed a particular challenge in obtaining pure DNA for molecular analysis. Initially extracted DNA from composted piles at day 147 was discoloured, and PCR inhibitors prevented amplification of target plant or bovine gene fragments. Bovine serum albumin improved detection by PCR (25–50 μl final volume) through the removal of inhibitors, but only when concentrations of humic acids in extracted DNA were 1.0 ng μl⁻¹ or less. Optimal purification of DNA from compost was achieved by chromatography using Sepharose 4B columns. The described DNA purification protocol enabled molecular monitoring of otherwise cryptic bovine and plant target genes throughout the composting process. The assay could likely be used to obtain PCR-amplifiable DNA that could be used for the detection of microbial pathogens in compost.     

Effects of age of cattle,turning technology and compost environment on disappearance of bone from mortality compost

As residual bones in mortality compost negatively impact subsequent tillage, two studies were performed. For the first study, windrows of mature cattle or calves were placed on a base of barley straw and covered with beef manure. Windrows were divided into two sections and turned at 3-month intervals. Approximately 5000 kg of finished compost per windrow was passed through a 6 mm trommel screen, with bones collected and weighed. Bone weight was 0.66% of mature cattle compost and 0.38% of calf compost on a dry matter basis, but did not differ after adjustment for weights of compost ingredients. In a subsequent study, four windrows were constructed containing mortalities, straw and beef manure (STATC) or straw, manure and slaughter waste (STATW). Also, straw, beef manure and slaughter waste was added to an 850 L rolling drum composter (DRUMW). Fresh bovine long-bones from calves were collected, weighed and embedded in the compost. Bones were retrieved and weighed when windrows were turned, or with DRUMW, after 8 weeks. Temperatures achieved followed the order STATW > STATC > DRUMW (p < 0.05). Rate of bone disappearance followed a pattern identical to temperature, with the weight of bones in STATW declining by 53.7% during 7 weeks of composting. For STATC, temperatures were uniform over three composting periods, but bone disappearance was improved (p < 0.05) when compost dry matter was lower (46%), as compared to 58%. Using a ratio of five parts manure to one part mortalities, results of this study demonstrated that residual bone was <1% of cured cattle compost and may be reduced by maintaining a high compost temperature and moisture content.     

Characterization of compost produced from separated pig manure and a variety of bulking agents at low initial C/N ratios

The aim of this study was to investigate the composting of separated pig manure solids with or without a variety of bulking agents at a low initial C/N ratio (12.5-23.3). Compost stability was investigated using an oxygen uptake rate (OUR) test and compost maturity was investigated using a germination index test. All treatments showed typical patterns of compost temperature. Temperatures above 60 °C were achieved by Day 2, followed by a thermophilic phase (50-60 °C), which lasted for 1 to 2 weeks followed by a cooling phase. The stability of one of treatments which did not contain any bulking agent - OUR of 25 mmol O₂ kg⁻¹ OM hour⁻¹ - was negatively affected by its initial high water content (69%). The addition of a bulking agent and initial water content below 60% were necessary to compost the separated solid fraction of pig manure at a low initial C/N ratio.     

Sample storage for soil enzyme activity and bacterial community profiles

Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (< 20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (> 50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.     

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.     

Effects of heat treatment on hydrogen production potential and microbial community of thermophilic compost enrichment cultures

Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70 °C. Highest cellulose degradation and highest H₂ yield were 57% and 1.4 mol-H₂ mol-hexose⁻¹ (2.4 mol-H₂ mol-hexose-degraded⁻¹), respectively, obtained at 52 °C with the heat-treated (80 °C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation.     

Saturated fatty acids stimulate and insulin suppresses CIDE-A expression in bovine mammary epithelial cells

Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDE-A) was first identified by its sequence homology with the N-terminal domain of DNA fragmentation factor (DFF). CIDE-A negatively regulates the activity of uncoupling protein 1 (UCP1) in brown adipose tissue. CIDE-A and UCP1 mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of CIDE-A mRNA in bMEC. Treatment with insulin (5-10 ng/ml) induced down-regulation of CIDE-A and UCP1. The expression levels of CIDE-A and UCP1 mRNA in bovine mammary glands at various stages of the lactation cycle were determined by quantitative RT-PCR analysis. CIDE-A mRNA expression at peak lactation (2 months after parturition) was significantly higher than at dry off and non-pregnancy but not late lactation. These results suggest that CIDE-A and UCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby play an important role in lipid and energy metabolism.     

Municipal solid waste (MSW) vermicomposting with an epigeic earthworm, Perionyx ceylanensis Mich

Municipal solid waste (MSW) was vermicomposted in combination with cowdung (CD) using Perionyx ceylanensis for 50 days. The decomposition rate of 55-78% was observed in different vermibed substrates, the highest being CD followed by 10:1 ratio of CD + MSW. The C/N ratio was reduced from 41.8 to 17.6 and 38.8 to 15.4 in MSW + CD (10:1) and CD, respectively. The difference in the final C/N ratio between MSW + CD (10:1) and CD vermicompost was not significant (p < 0.05). The important nutrients, NPK showed significantly (p < 0.05) higher contents in vermicomposts than worm-unworked composts. The degradation rate of cellulose and lignin was 37% and 12%, respectively, in 10:1 vermibed mix with P. ceylanensis. The bacterial, fungal and actinomycetes population in vermicompost was significantly higher than in the compost. The biomass, number and cocoons of P. ceylanensis collected after 50 days showed increase with the increase of CD incorporation in MSW.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

The effectiveness of gender determination using polymerase chain reaction and radioimmunoassay methods in cattle

The aim of this study was to use polymerase chain reaction (PCR) by amplifying DNA from bovine (Bos taurus) fetal cells recovered through uterine puncture and subsequent amniotic fluid aspiration and to compare the effectiveness of the PCR method with amniotic dihydrotestosterone (DHT) levels in gender determination. Amniotic DHT levels between sexes were significantly higher in males than in females in all periods except the period 91 to 120 d. The differences among the amniotic DHT levels at different gestation periods (61 to 90, 91 to 120, 121 to 150, 151 to 180, 181 to 210 d) were not significant in females but were significant in males in the period 61 to 90 d compared with three other periods. Sensitivity was equal to 97.8% (95% CI = 88.2% to 99.6%), and specificity was equal to 85.4% (95% CI = 80.0% to 97.6%). These two values correspond with a cutoff of DHT in amniotic fluid. Distributions of the two sex groups were classified according to the 192.1 pg/mL cutoff value. A total of 93 amniotic fluid samples were examined by PCR analysis. The sex determination of 91 samples by PCR and electrophoresis was in agreement with the visual sexes of the fetuses. In two amniotic fluid samples, DNA was not isolated, and thus no sex determination was made. Fetal gender was correctly identified by PCR in 44 of 45 males and in 47 of 48 females. In PCR, one band (at the length of 102 bp) and two bands (at the lengths of 102 and 226 bp) were observed respectively for female and male fetuses. It may be concluded that the levels of amniotic DHT and PCR might be used for embryo sexing in pregnant cows.     

Metabolic conversion of intra-amniotically-injected deuterium-labeled essential fatty acids by fetal rats following maternal n-3 fatty acid deficiency

Accumulation of polyunsaturated fatty acids (PUFA) in the fetal brain is accomplished predominantly via a highly selective flow of docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, AA) through the placenta. Little is known regarding the endogenous capability of the fetus to generate its own DHA and AA from lower homologues such as linolenic (18:3n-3, ALA) and linoleic (18:2n-6, LA) acids, respectively. Deuterium-labeled d5-ALA and d5-LA at millimolar concentrations were injected directly into the amniotic fluid in order to investigate maternal-independent metabolic conversion of the stable isotopes in brain and liver of the fetus near delivery. After 48 h under adequate maternal diet, the levels of d5-ALA metabolites in the fetal brain and fetal liver were 45 ± 2.2 pmol/mg and 86 ± 4 pmol/mg of which 79% and 63.6% were comprised of d5-DHA. At this time point, incorporation of d5-LA metabolites was 103 ± 5 pmol/mg and 772 ± 46 pmol/mg for brain and liver, of which 50% and 30% were comprised of d5-AA. Following sustained maternal dietary ALA deficiency, the levels of total d5-ALA derived metabolites in the fetal brain and fetal liver were increased to 231 pmol/mg and 696 pmol/mg of which 71% and 26% were comprised of d5-DHA. From the time course and relative rates of d5-ALA precursor displacement by d5-DHA in cellular phosphoglycerides, it is concluded that the fetal rat brain can generate its own DHA from its d5-ALA precursors particularly under dietary stress.     

Des-Tyr1-gamma-endorphin (DT gamma E) and des-enkephalin-gamma-endorphin (DE gamma E): plasma profile and brain uptake after systemic administration in the rat

The plasma disappearance, metabolism and uptake in the brain of [3H-Phe4]-DT gamma E and [3H-Lys9]-DE gamma E were investigated following systemic administration of these neuroleptic-like peptides to rats. 3H-DT gamma E, 3H-DE gamma E and their radioactive metabolites in plasma and brain extracts were determined by reversed-phase HPLC. Plasma disappearance of DT gamma E upon intravenous (IV) dosing followed a biphasic pattern with half-lives of 0.7 min (distribution phase) and 5.5 min (elimination phase). For DE gamma E the plasma disappearance curve was best characterized by a one-compartment model since a second elimination phase was hardly detectable by our methods. The corresponding half-life was 0.6 min, probably representative for the initial distribution phase of DE gamma E. Both neuropeptides distributed rapidly over the larger part of the extracellular fluid. Following the IV route of administration, brain uptake of DT gamma E and DE gamma E appeared to be low. Brain levels of DT gamma E decreased from 0.0075% to 0.0031% of the administered dose/g tissue at 2-15.5 min after injection, whereas those of DE gamma E decreased very rapidly from 0.0174% of the dose/g brain tissue to below the detection limit at 2-4.5 min after injection. As compared to the IV route of administration, subcutaneous (SC) injection of DE gamma E resulted into lower but remarkably longer-lasting peptide concentrations in plasma as well as in brain, possibly because of a sustained release from the SC site of injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repression of induced apoptosis in the 2-cell bovine embryo involves DNA methylation and histone deacetylation

Apoptosis in the bovine embryo cannot be induced by activators of the extrinsic apoptosis pathway until the 8-16-cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in 2-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by methylation of DNA and deacetylation of histones. To test this hypothesis, we evaluated whether reducing DNA methylation by 5-aza-2′-deoxycytidine (AZA) or inhibition of histone deacetylation by trichostatin-A (TSA) would make 2-cell embryos susceptible to DNA fragmentation caused by CCCP. The percent of blastomeres positive for TUNEL was affected by a treatment × CCCP interaction (P < 0.0001). CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle but did increase the percent of cells that were TUNEL positive if embryos were pretreated with AZA or TSA. Immunostaining using an antibody against 5-methyl-cytosine antibody revealed that AZA and TSA reduced DNA methylation. In conclusion, disruption of DNA methylation and histone deacetylation removes the block to apoptosis in bovine 2-cell embryos.     

A NeuGc-containing trisialoganglioside of bovine brain.

A N-glycolyneuraminic acid containing trisialoganglioside was isolated from bovine brains ganglioside mixture using Q-Sepharose. Its chemical structure was characterized as IV3NeuAc, II3NeuAc-NeuGc, Gg4Cer by gas-liquid chromatography, a permethylation study, sialidase degradation, TLC/enzyme-immunostaining, fast atom bombardment-mass spectrometry, fluorometric HPLC and proton nuclear magnetic resonance spectroscopy. This was unique in the mixed sialic acid constituents. (formula; see text) This accounted for 0.78% of the gangliosides. The ceramide structure was almost identical with those of major bovine brain ganglioside, as mainly composed of 18:0 fatty acid (90.9%) and d20:0 sphingosine base.     

Regulation of protein degradation by insulin-degrading enzyme: analysis by small interfering RNA-mediated gene silencing

Proteins are vital to the overall structure of cells and to the function of cells in the form of enzymes. Thus the control of protein metabolism is among the most important aspects of cellular metabolism. Insulin’s major effect on protein metabolism in the adult animal is inhibition of protein degradation. This is via inhibition of proteasome activity via an interaction with insulin-degrading enzyme (IDE). IDE is responsible for the majority of cellular insulin degradation. We hypothesized that a reduction in IDE would reduce insulin degradation and insulin’s ability to inhibit protein degradation. HepG2 cells were transfected with siRNA against human IDE and insulin degradation and protein degradation measured. Both IDE mRNA and protein were reduced by >50% in the IDE siRNA transfected cells. Insulin degradation was reduced by approximately 50%. Cells were labeled with [³H]-leucine to investigate protein degradation. Short-lived protein degradation was unchanged in the cells with reduced IDE expression. Long-lived and very-long-lived protein degradation was reduced in the cells with reduced IDE expression (14.0 ± 0.16 vs. 12.5 ± 0.07%/4 h (long-lived), 9.6 ± 2.2% vs. 7.3 ± 0.2%/3 h (very-long-lived), control vs. IDE transfected, respectively, P < 0.005). The inhibition of protein degradation by insulin was reduced 37-76% by a decreased expression of IDE in HepG2 cells. This shows that IDE is involved in cellular insulin metabolism and provides further evidence that insulin inhibits protein degradation via an interaction with IDE.     

Accumulation of mitochondrial DNA deletions is age, tissue and folate-dependent in rats

Folate is essential for the synthesis, repair and methylation of DNA. Folate depletion causes nuclear genetic and epigenetic aberrations in cell culture, rodents and humans. We hypothesized that folate depletion may also damage mitochondrial (Mt) DNA and induce large-scale deletions due to DNA breakage. MtDNA deletions and mutations accumulate during aging and tumorogenesis and may play causative roles in these processes. Weanling and adult (12 months) Sprague Dawley rats consumed folate deplete, replete and supplemented diets (0, 2 and 8 mg/kg folate, respectively) for 20 weeks. The presence of random and common (4.8 kb) MtDNA deletions was measured in colonic mucosa and liver. Six Mt genomes (<16 kb) harboring random deletions were detected in the liver (3.5-7.0 kb) and three in the colon (3.8-8 kb). Older rats had significantly more random hepatic MtDNA deletions than young rats (64 and 3.2% of samples, respectively, P < 0.0001), while age had no effect on these deletions in the colon (3.1 and 7.7% in young and old, respectively). Folate intake had no effect on the frequency of random deletions in either tissue. There was no discrete effect of aging on the common 4.8 kb deletion in the liver or colon. However, in the liver of old rats, increasing amounts of dietary folate reduced the deletion frequency, with replete and supplemented rats having 2.2- and 3.2-fold less deletions than the depleted rats. Our results confirm that random MtDNA deletions accumulate with age in a tissue-specific fashion. Furthermore, in contrast to previous work, we report that the common 4.8 kb deletion was not modulated by age, but is reduced by folate supplementation in the liver of rats.     

Biodegradability of injection molded bioplastic pots containing polylactic acid and poultry feather fiber

The biodegradability of three types of bioplastic pots was evaluated by measuring carbon dioxide produced from lab-scale compost reactors containing mixtures of pot fragments and compost inoculum held at 58 °C for 60 days. Biodegradability of pot type A (composed of 100% polylactic acid (PLA)) was very low (13 ± 3%) compared to literature values for other PLA materials. Near infrared spectroscopy (NIRS) results suggest that the PLA undergoes chemical structural changes during polymer extrusion and injection molding. These changes may be the basis of the low biodegradability value. Biodegradability of pot types B (containing 5% poultry feather, 80% PLA, 15% starch), and C (containing 50% poultry feather, 25% urea, 25% glycerol), were 53 ± 2% and 39 ± 3%, respectively. More than 85% of the total biodegradation of these bioplastics occurred within 38 days. NIRS results revealed that poultry feather was not degraded during composting.     

Improved volatile fatty acids production from proteins of sewage sludge with anthraquinone-2,6-disulfonate (AQDS) under anaerobic condition

Organic matters in sewage sludge can be converted into volatile fatty acids (VFAs) as renewable carbon sources. This work for the first time applied anthraquinone-2,6-disulfonate (AQDS) for enhancing VFA production from sewage sludge. With 0.066 or 0.33 g AQDS g⁻¹ dried solids (DS), the yields for VFAs peak at 403 or 563 mg l⁻¹, 1.9- or 2.7-fold to the control. The accumulated VFAs were principally composed of acetate and propionate. The AQDS enhances degradation rates of model proteins (bovine serum albumin), but had little enhancement on that of model polysaccharides (dextrans). The acidification step is proposed the rate-limiting step for VFA production from sewage sludge, in which the AQDS molecules shuttle electrons to accelerate the redox reactions associated with amino acid degradation. Methanogenic activities are inhibited in the presence of AQDS. The AQDS-assisted VFAs are renewable organic carbon sources, although their direct use for anaerobic digestion is not advised.     

Effects of spawn,supplement and phase II compost additions and time of re-casing second break compost on mushroom (Agaricus bisporus) yield and biological efficiency

Three cropping experiments (0710, 0803 and 0805) were conducted to determine the effect of adding spawn, various levels of delayed release nutrient, and phase II compost to 2nd break mushroom compost (2BkC) on mushroom yield and biological efficiency (BE). We also investigated the effect of delaying time of re-casing non-supplemented and supplemented 2BkC on mushroom yields and BEs. The addition of 14.6% spawn to nutrient-supplemented 2BkC (w.w./d.w) increased yield by 11.1% over the control (no spawn) but did not affect BE. The addition of delayed release supplements to 2BkC increased maximum yields by 29–54%, depending on the treatment. Substitution of 15% phase II compost in 2BkC (15/85) did not significantly affect mushroom yields. However, use of 15% phase II compost in 2BkC increased the response of the mixture to delayed release supplement. Yield response to increasing levels of supplement was greater in the 15/85 mixture compared to 100% 2BkC. Yields also increased as time of re-casing was delayed up to 10 days. Mushroom yields increased approximately 2.1% for each day re-casing was delayed. Overall yields were generally higher from commercial 2BkC compared to 2BkC originating from the Penn State Mushroom Research Center (MRC) probably due to nitrogen (N) content of the 2BkC. Nitrogen content in commercial 2BkC (Crop 0805) was 3% while N content in 2BkC from Crops 0710 and 0803 was 2.2% and 2.1%, respectively. By optimizing supplement levels and adding 15% phase II compost to commercial 2BkC, or by delaying casing by 5–10 days, it was possible to obtain BEs that were equivalent to supplemented phase II compost.     

Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P < 0.05 or P < 0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P < 0.05 or P < 0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.