Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS‐PCR). The expression levels of these imprinted genes were also investigated by quantitative real‐time PCR (QRT‐PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.     

Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.     

牛印记基因的研究进展

基因组印记是一种表观调控机制,在哺乳动物的发育中具有重要作用。印记基因是仅一方亲本来源的同源基因表达,而来自另一亲本不表达的一种基因。近年来,印记基因被广泛研究。印记基因分为父系印记基因和母系印记基因,其表达具有组织特异性,而且在胚胎不同发育阶段的表达也具有一定差异,胚胎期的营养水平也影响印记基因的表达。DNA甲基化在调控印记基因的表达中起重要作用,影响细胞核移植过程细胞的表观重编程,并影响胎盘及内脏器官的正常发育;基因印记模式的改变也可以引起甲基化的改变进而导致基因印记的丢失等。本文综述了近年来关于牛的印记基因的研究进展情况,为印记基因的后续相关研究工作提供借鉴。     

甘氨酸对延边黄牛体细胞克隆重组胚发育的影响

为了提高延边黄牛体细胞克隆重组胚的发育率,研究了在体外培养液中添加不同浓度的甘氨酸对重组胚发育的影响。分别在体外培养液中添加0、7、14、21mmol/L的甘氨酸,结果表明,在体外培养液中添加甘氨酸能够显著提高延边黄牛体细胞克隆重组胚的发育率,且以7mmol/L浓度的甘氨酸囊胚的发育率最高。     

Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

延边黄牛卵母细胞与体细胞克隆胚胎内脂滴变化规律的研究

用苏丹Ⅳ和苏丹黑B 2种染色方法,对体外培养的延边黄牛各发育阶段卵母细胞和体细胞克隆胚胎内脂滴变化进行研究,并对2种染色方法进行了比较。结果表明,随着细胞发育阶段的不同,脂滴的含量也随之变化,从未经成熟培养的卵母细胞到8细胞期胚胎内脂滴不断增多,脂滴直径不断增大;而从8细胞期到囊胚期胚胎内脂滴不断减少,脂滴直径也不断减小。也显示苏丹IV染色效果较好,可作为测定活体细胞中脂滴的检测方法。     

Evaluation of the global DNA methylation in canine mast cell tumour samples by immunostaining of 5‐methyl cytosine

Cutaneous mast cell tumours (MCT) are the most common skin tumour in dogs, and to our knowledge, there are no previous studies regarding the global methylation of these tumours. DNA hypomethylation and hypermethylation have been described in several tumours and both mechanisms can lead to carcinogenesis. The purpose of this study was to evaluate the global DNA methylation in canine MCT. A total of 48 MCT samples were classified in grades 1, 2 and 3 or high‐grade or low‐grade. Monoclonal antibodies were used for the immunohistochemical detection of the 5‐methylcytosine. The immunostained nuclei were classified in strong, weak or negative pattern, and these were quantified in five distinct microscopic fields (40× objective) in each slide. The results showed that global DNA hypomethylation was predominant in grade 3, high‐grade, less differentiated MCT. These epigenetic changes in neoplastic mast cells warrant further detailed investigation aiming the establishment of tumour epigenetic therapies.     

Parent‐of‐origin effects on carcass traits in Japanese Black cattle

Variances caused by the differential expression of paternally and maternally imprinted genes controlling carcass traits in Japanese Black cattle were estimated in this study. Data on marbling score (BMS), carcass weight, rib thickness, rib‐eye area (REA) and subcutaneous fat thickness (SFT) were collected from a total of 13,115 feedlot steers and heifers in a commercial population. A sire–maternal grandsire model was used to analyse the data, and then, imprinting parameters were derived by replacing the genetic effect of the dam with the effect of the maternal grandsire in the imprinting model to calculate the genetic parameter estimates. The proportions of the total genetic variance attributable to imprinted genes ranged from 8.7% (SFT) to 35.2% (BMS). The remarkably large imprinting variance of BMS was mainly contributed by maternally expressed inheritance because the maternal contribution of the trait was much larger than that of the paternal trait. The parent‐of‐origin effect originating from maternal gene expression was also observed for REA. The results suggested the existence of genomic imprinting effects on the traits of the Japanese Black cattle. Hence, the parent‐of‐origin effect should be considered for the genetic evaluation of Japanese Black cattle in breeding programmes.     

Identification of genes responsible for hereditary diseases in Japanese beef cattle

In the breeding of domestic animals, selection of economically desired traits has been the most important consideration for the improvement of animals, but excluding negative factors in animal production, such as causative genes for hereditary diseases, is also required for the genetic improvement of domestic animals. The incidence of various hereditary diseases has been reported in Japanese beef cattle and these diseases have caused serious problems in the breeding and raising of healthy beef cattle. This article reviews the identification of causative genes for the following three hereditary diseases in Japanese beef cattle: (i) Chediak–Higashi syndrome; (ii) renal tubular dysplasia; and (iii) bovine chondrodysplastic dwarfism. Chediak–Higashi syndrome is a hereditary bleeding disorder reported in Japanese black cattle. To identify the cause of this disease, we cloned and sequenced the bovine LYST gene, which has been found to be involved in Chediak–Higashi syndrome in humans, and found that an amino acid substitution of histidine to arginine at amino acid residue 2015 is the causative mutation in the cattle disease. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation. We mapped the locus for this disease to the 4 cM region of bovine chromosome 1 by linkage analysis and found a large deletion in this region. The deleted region contained the PCLN1 gene encoding a tight‐junction protein of renal epithelial cells, and we concluded that deletion of the PCLN1 gene is responsible for the disease. Bovine chondrodysplastic dwarfism is a hereditary disease of Japanese brown cattle, displayed by short limbs and joint abnormalities. We mapped the locus for the disease to a region of bovine chromosome 6 by linkage analysis. By constructing YAC and BAC contigs covering this region and sequence analysis, we identified a novel gene (LIMBIN), which plays an essential role in bone formation in this region, and found two mutations responsible for the disease. The identification of these mutations provided the basis for DNA‐based diagnostic systems for these three diseases, and after development of the diagnosis systems, the incidences of these hereditary diseases have dramatically decreased.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Ultrasound‐guided proximal paravertebral anaesthesia in cattle

ObjectiveTo develop and evaluate a method for ultrasound-guidance in performing the proximal paravertebral block for flank anaesthesia in cattle through a cadaveric study, followed by clinical application.Study designprospective experimental cadaveric study and clinical series.AnimalsPreviously frozen lumbar sections of cows without known spinal abnormalities were used. The clinical case group comprised of ten animals for which a right flank laparotomy was indicated.MethodsTwenty cow cadavers were used to perform ultrasound-guided bilateral injections of 1.0 mL dye (1.0 mL 1% Toluidine Blue in 1% Borax) at the intervertebral foramen at the level of T13, L1 and L2 spinal nerves. Distance and depth of injection, staining of the dorsal and ventral nerve branches, and deviation from the target were evaluated. The investigator’s confidence as to visualisation and expected success at staining the nerve was assessed. Ten clinical cases received the ultrasound-guided proximal paravertebral anaesthesia. Analgesic success was evaluated using a 4-grade scoring system at 10 minutes after the injection and during surgery, respectively. Categorical variables were described using frequencies and proportions.ResultsBoth dorsal and ventral branches of the spinal nerves T13, L1 or L2 were at least partially stained in 41% of injections, while in 77% of injections one of the branches was stained. Five out of ten clinical cases had a satisfactory anaesthesia. There was no significant association between confidence at injection and either staining or analgesic success.ConclusionResults from the cadaveric and clinical study suggest no significant improvement using ultrasound guidance to perform proximal paravertebral block in cows compared to our previous clinical experience and to references in the literature using the blind method.Clinical relevanceFurther research should be conducted to improve the ultrasound-guided technique described in this study.     

Effects of high‐sulphur water on hepatic gene expression of steers fed fibre‐based diets

Sulphur‐induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is frequently associated with the consumption of high‐sulphur (S) water and subsequent poor performance. Currently, there is no economical method for S removal from surface water sources, and alternative water sources are typically neither readily available nor cost‐effective. Determination of genes differentially expressed in response to high‐S water consumption may provide a better understanding of the physiology corresponding to high dietary S and ultimately lead to the development of treatment and prevention strategies. The objective of this study was to determine changes in gene expression in the liver, an organ important for S metabolism, of fibre‐fed steers consuming high‐S water. For this study, liver tissues were collected on the final day of a trial from yearling steers randomly assigned to low‐S water control (566 mg/kg SO₄; n = 24), high‐S water (3651 mg/kg SO₄; n = 24) or high‐S water plus clinoptilolite supplemented at either 2.5% (n = 24) or 5.0% (n = 24) of diet dry matter (DM). Microarray analyses on randomly selected healthy low‐S control (n = 4) and high‐S (n = 4; no clinoptilolite) steers using the Affymetrix GeneChip Bovine Genome Array revealed 488 genes upregulated (p < 0.05) and 154 genes downregulated (p < 0.05) in response to the high‐ vs. low‐S water consumption. Real‐time RT‐PCR confirmed the upregulation (p < 0.10) of seven genes involved in inflammatory response and immune functions. Changes in such genes suggest that ruminant animals administered high‐S water may be undergoing an inflammation or immune response, even if signs of sPEM or compromised health are not readily observed. Further study of these, and other affected genes, may deliver new insights into the physiology underlying the response to high dietary S, ultimately leading to the development of treatments for high S–affected ruminant livestock.     

牛源大肠杆菌O157：H7的分离及毒力基因鉴定

从2个牛场采集新鲜粪便,增菌后,免疫磁珠富集,涂布筛选性培养基,挑取可疑菌落用rfbE/fliC二重PCR和血清学方法鉴定。设计毒力基因stx1、stx2、eae、hlyA和tccp相应引物,针对O157：H7对分离株进行PCR鉴定。口服攻毒链霉素处理的BALB/c小鼠明确分离株致病性。结果显示,成功分离到7株出血性大肠杆菌O157：H7,并且有1株迟缓性发酵山梨醇麦康凯培养基。毒力基因检测显示,其中6株毒力因子表型为stx1-stx2＋eae＋hlyA＋tccp＋,另有1株表现型为stx1＋stx2＋eae＋hlyA＋tccp＋,各分离株tccp基因均为阳性,但携带的重复片段数量有差异。所采集样品中肠出血性大肠杆菌O157：H7的检出率高达12%。1×1010 CFU同剂量口服接种经PBS洗涤的5株O157：H7分离株全菌,小鼠存活率有差异分别为40%,50%,60%,20%,50%,各分离株在小鼠体内排菌时间也有差异分别为攻毒后7,9,13,13,15d。     

Attribute selection and model evaluation for the maternal and paternal imprinted genes in bovine (Bos Taurus) using supervised machine learning algorithms

Imprinted genes display biased expression of paternal and maternal alleles in mammals. They are marked through epigenetic process during gametogenesis. Characterization of imprinted genes has expanded our understanding of the regulation and function of genes. In the current study, 22 experimentally validated imprinted genes in bovine (Bos Taurus) were analysed. Several supervised machine learning algorithms and attribute weighting methods were used to find characteristics of different types of imprinted genes and suggest a classification method for finding maternally and paternally expressed genes in bovine. For assessing the best model and comparing attributes in other organisms, we have also conducted a comparative analysis for human and sheep imprinted genes. According to the results of the present study, GC contents 10 and 100 kb upstream, Gly and Gln amino acids, Ile/ATC codon usage, LINE and SINE in 100kbup and length of first intron were significantly different between the maternal and paternal genes in cattle. Considering all species together, we found that GC content 100 kb up, LINE 100 kb up and the frequency of amino acids like Gly, Gln and Met were the most important attributes for identifying the paternal and maternal imprinted genes. These findings could imply conservation pattern in the attributes among these species.     

表观遗传学:家畜遗传育种的新挑战

表观遗传变异是在DNA序列并没有发生改变的情况下,基因表达调控发生了可遗传性的变化,并最终导致了基因功能甚至个体表型的变化。表观遗传修饰主要包括DNA甲基化修饰、组蛋白修饰和非编码RNA调控等。目前,表观遗传学正渗透到家畜遗传育种领域,论文着重探讨了表观遗传学在家畜遗传育种中的研究进展及其所面临的新挑战。     

DNA甲基化在哺乳动物卵母细胞和早期胚胎发育中的研究进展

DNA甲基化(DNA methylation)是一种动态、可逆并可以遗传的表观遗传修饰模式,主要发生在哺乳动物原始生殖细胞和早期胚胎发育过程中,能够通过高动态和协同的核酶网络附着在DNA的CpG区域,同时还通过改变调控区域的功能状态进而调控基因表达且不影响DNA序列所携带的遗传信息。DNA甲基化主要涉及基因组印迹、转座元件沉默、X染色体失活和衰老等多种关键生理过程,在哺乳动物卵母细胞和胚胎发育中发挥着重要作用。本文介绍了DNA甲基化的建立与去除机制及其生物学功能,重点阐述了DNA甲基化在哺乳动物卵母细胞和胚胎发育过程中精准生成、维持、读取和删除等动态变化过程,为进一步研究哺乳动物表观遗传调控提供参考依据。