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。     

Basics of gametic imprinting

The fundamental assumption of Mendelian genetics is that behavior of an allele is identical whether it arrives to a zygote through paternal or maternal germline pathway. Gametic imprinting phenomena discovered and studied in mammals show limitations of the classical view in special cases. Two sources of evidence were essential to describe gametic imprinting. The first approach based on genetic evidence demonstrated that some maternally and paternally derived regions of certain chromosomes were not equivalent. Paternal or maternal disomy of the regions containing particular genes caused significant effects on viability and development of progeny. The second set of data was obtained by nuclear transplantations and parthenogenetic activation of mammalian oocytes. These data suggested that the contribution of parental genomes was not equivalent and differential imprinting of nuclear genes during gametogenesis was very likely. The number of loci found in mice, which show gametic imprinting, is 34 and continues to grow. It is generally accepted that gametic imprinting is a mammalian invention and there are differences in imprinting pattern between species. Most hypotheses propose involvement of imprinted genes in the control of fetal growth and fetal-maternal interactions, thus keeping a balance between contradictory fetal and maternal requirements. Molecular mechanisms responsible for gametic imprinting still remain to be studied, but for several genes it was shown that imprinting marks are imposed by a parent-specific methylation process during gametogenesis. These marks are resistant to global demethylation during cleavage and to global de novo methylation after implantation and maintain different methylation patterns in paternal and maternal alleles of imprinted genes. About 20 to 25% of all transgene loci studied demonstrate similarities with imprinted genes. For instance, methylation of some transgenes is dependent on parental gametic pathway and reversible in the next generation. There are data indicating that selection of modifier genes may change the effects of gametic imprinting. It is possible that future selection and crossbreeding programs may take gametic imprinting into consideration.     

新城疫病毒(长春株)F蛋白基因的克隆和序列分析

新城疫病毒（ＮＤＶ）长春株在鸡胚增殖后纯化,提取ＲＮＡ,然后利用特异性引物,经ＲＴ－ＰＣＲ一次性扩增出了ＮＤＶ长春株的全长Ｆ基因。将该Ｆ基因插入ｐＫＳ（－）后,进行了序列测定。序列分析表明,该Ｆ基因核苷酸长度为１７５８ｂｐ,编码５５３个氨基酸,序列中有６个糖基化位点,１３个半胱氨酸残基,裂解位点区（１１２～１１７）氨基酸序列为Ｇｌｙ－Ａｒｇ－Ｇｌｎ－Ｇｌｙ－Ａｒｇ－Ｌｅｕ,与所有弱毒株在这一区域的序列（Ｇｌｙ－Ａｒｇ／Ｌｙｓ－Ｇｌｎ－Ｇｌｙ／Ｓｅｒ－Ａｒｇ－Ｌｅｕ）相符,证明长春株为弱毒株。同源性分析表明,长春株Ｆ基因与目前国外发表的其他ＮＤＶＦ基因相比,核苷酸序列同源性在８８％～９９％之间,推导的氨基酸序列同源性在９０％～９８％之间     

The effect of maternal obesity on fatty acid transporter expression and lipid metabolism in the full‐term placenta of lean breed swine

This study was conducted to evaluate the influence of back‐fat thickness (BF), at mating of sows, on the maternal and newborn circulating lipids, expression of placental fatty acids (FA) transporters and lipid accumulation in placenta. Full‐term placentas were obtained by vaginal delivery from BFI (9–14 mm; n = 37), BFII (15–19 mm; n = 43) and BFIII (20–27 mm; n = 38) sows according to BF at mating, and frozen placental sections were analysed for fat accumulation. Blood samples were collected from the sows of day 105 pregnancy and from cord blood at delivery. mRNA and protein expression levels were evaluated with real‐time RT‐PCR and Western blotting. Our results demonstrated that BFII females had significantly increased litter weight and placental efficiency, decreased maternal triglyceride (TG) and non‐esterified fatty acids (NEFA) levels, decreased maternal IL‐6, TNFα and leptin levels compared to BFIII females (p < .05). BFIII sows were associated with significantly decreased newborn TG levels, increased newborn glucose, IL‐6 and TNFα levels compared to BFI or BFII sows (p < .05). BFI and BFII females had significantly decreased placental TG, NEFA and cholesterol (CHOL) contents compared to BFIII females (p < .05). Moreover, decreased CD36, FATP1, FABP4, and FABP1 mRNA and protein and FATP4 protein expression, and increased LPL activity were also observed in BFIII group compared with BFII group (p < .05). PPARγ mRNA and protein and lipogenic genes such as SREBP‐1c, ACSL1, ACCα, FAS and SCD mRNA expression were downregulated or upregulated, respectively, in the placentas of BFIII sows compared to BFI or BFII sows (p < .05). Overall, this study demonstrated that there is no advantage, in terms of litter live size, litter weight and placental FA transport and metabolism, in performing the mating of sows with BF>19 mm.     

杜洛克与二花脸杂交猪群体SNP偏分离分析

偏分离（TRD）是生物中普遍存在的现象,本研究拟在高密度基因芯片判断基因型的大规模群体中挖掘猪基因组标记位点的偏分离位点并探究其潜在的遗传机制。本研究用60K Illumina Porcine SNP芯片对1 020头F2资源家系（杜洛克与二花脸杂交F0-F2群体）进行基因型分型,用偏分离分析软件TRDscan （BF>100）和TDT （P<0.01）方法在单个位点上检测偏分离信号,并对二者共有的显著信号位点附近100 kb窗口的基因进行功能分析。此外,本研究还利用单倍型分型的软件包PHASEBOOK采用多位点连锁分析的策略分析了父源和母源配子中的偏分离区域,并在该区域内搜寻潜在的QTLs。结果表明：1）在单位点的偏分离分析中共鉴定到44个显著的偏分离位点,筛选到23个相关基因;对父本和母本特异性偏分离效应进行分析时,分别鉴定到27和35个显著偏分离位点,其中,分别有11和25个位点的100 kb附近有已注释的功能基因。2）基于单倍型多位点连锁的偏分离结果显示,父本显著偏分离位点位于5和13号染色体上,在该偏分离区域内搜寻功能相关的QTL,共搜寻到3个与繁殖性状（木乃伊数、产活仔数、黄体数）有关的QTLs;分析母本偏分离位点时,在4、6和12号染色体上定位到显著偏分离区域,结合已知的猪QTL数据库,共找到了5个与繁殖性状相关的QTLs。本研究利用大规模猪家系数据系统地鉴别了猪基因组的偏分离位点,为解析猪中的偏分离现象和进一步探究其生物学机制提供了一定的参考作用。     

Linkage disequilibrium and effective population size in Gir cattle selected for yearling weight

Linkage disequilibrium (LD) plays an important role in genomic selection and mapping of quantitative trait loci (QTL). This study investigated the pattern of LD and effective population size (N_e) in Gir cattle selected for yearling weight. For this purpose, 173 animals with imputed genotypes (from 18 animals genotyped with the Illumina BovineHD BeadChip and 155 animals genotyped with the Bovine LDv4 panel) were analysed. The LD was evaluated at distances of 25–50 kb, 50–100 kb, 100–500 kb and 0.5–1 Mb. The N_e was estimated based on 5 past generations. The r² values (a measure of LD) were, respectively, .35, .29, .18 and .032 for the distances evaluated. The LD estimates decreased with increasing distance of SNP pairs and LD persisted up to a distance of 100 kb (r² = .29). The N_e was greater in generations 4 and 5 (24 and 30 animals, respectively) and declined drastically after the last generation (12 animals). The results showed high levels of LD and low N_e, which were probably due to the loss of genetic variability as a consequence of the structure of the Gir population studied.     

Genetic variances due to imprinted genes in cattle

The effect of paternally expressed, i.e. maternally imprinted, genes on slaughter records from 2744 German Gelbvieh finishing bulls were estimated. Significant effects of paternal gametes were found for two fatness traits and an estimate of meat content. Paternally expressed genes explained 14 and 16% of the phenotypic variances for pelvic fat and kidney fat, respectively. Ignoring paternal gametic effects resulted in inflated estimates of the additive genetic variances. The heritabilities of pelvic and kidney fat dropped from 0.31 to 0.16 and from 0.59 to 0.28, respectively, when paternal gametes were fitted. A 15% influence of paternally expressed genes and a reduction in heritability of 20% were also found for estimated meat content. Simulation studies demonstrated that the uncorrelated random effect of the sire is a useful indicator for the presence of paternal gametic effects in variance component estimations. The presented results correspond well with findings in swine, where a paternally expressed QTL at the Igf2 gene influences similar trait complexes. A viable speculation could therefore be that an imprinted bovine Igf2 gene caused the effects described here.     

Porcine intestinal yeast species,Kazachstania slooffiae,a new potential protein source with favourable amino acid composition for animals

There is little information about Kazachstania slooffiae which dominates among other yeasts in the pigs’ intestine. Therefore, the aims of this study were to characterise the yeast cell contents and to investigate which nitrogen sources, organic acids and alcohols may be utilised or produced by this species. The results showed that, K. slooffiae could use urea, ammonium sulphate, peptides and single amino acids and produce thereby ethanol and formic acid. However, this yeast did not metabolise amino acids, lactic, butyric, propionic and acetic acids as sole carbon source. Using a global metabolite profiling approach employing gas chromatography and high‐resolution liquid chromatography mass spectrometry, was found that the amount of peptides and dehydroascorbic acid considerably increased in the fermentation residues after yeast cultivation. It is noteworthy that the cells of K. slooffiae had higher contents of nitrogen and total amino acids (especially lysine) than the cells of nutritional yeast (Saccharomyces cerevisiae). This study indicates that due to potential production of peptides and formic acid in the intestinal tract, K. slooffiae might have an impact on the gut health. Moreover, from a nutritional standpoint, the cells of this yeast can be a good source of protein with useful amino acid composition for animal.     

Correlation between skeletal muscle fiber type and free amino acid levels in Japanese Black steers

Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26‐month‐old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow‐twitch fibers.     

Correlations between carcass traits and mRNA levels of CGI-105 and CCAAT/enhancer protein α genes in steers of Korean cattle

To understand the molecular mechanisms that regulate intramuscular fat deposition (marbling), cDNA clones expressed in adipose tissues of Korean cattle were identified and characterized. One clone had a total length of 1262 nucleotides coding for 314 amino acids. It was identified as one encoding bovine homolog of human CGI-105 mRNA. CGI-105 is a member of fumarylacetoacetate hydrolase family. Comparison of the deduced amino acid sequences of bovine CGI-105 with those of human revealed more than 89% identity. High levels of CGI-105 mRNA expression were detected in muscle, heart, and kidney tissues among various bovine tissues. Carcass traits, including backfat thickness, rib eye area, yield index, marbling score, and quality grade were analyzed in steer of Korean cattle. A CCAAT/enhancer binding protein alpha (C/EBPα) is one of adipocyte differentiation factors that may affect deposition of fat in muscle. mRNA levels of CGI-105 and C/EBPα genes were determined in the loin muscle tissues of steers. Correlation between carcass traits and mRNA levels of the genes was estimated by Pearson's correlation coefficient. The mRNA levels of C/EBPα showed strong positive correlation (r = 0.83, p < 0.01) with marbling scores. The results of the present study indicate that the manipulation of the expression of the C/EBPα gene may contribute to the development of a method for enhancing intramuscular fat deposition in beef.     

Variations in protein and fat contents and their fractions in milk from two species fed different forages

This study aimed at determining the variations in milk constituents which could be varied by feed and animal species. To achieve this goal, two groups of homoparity Baladi cows and Egyptian buffaloes (n = 20 per species) were used. Each group was divided into two subgroups (n = 10): subgroup I received legume forage (Egyptian clover) and subgroup II received grass forage (sorghum forage). All experimental animals were fed the diet consisting of concentrate, forage and rice straw as 50, 25 and 25% of dry matter intake respectively. Milk samples were taken for analysis. The trial lasted until the 3rd month of parturition. The main results indicated that lactating cattle fed legume forage significantly (p ≤ 0.01) had more content of casein nitrogen (513 mg/100 ml milk), lower content of glutamic acid (23.56 g/100 g milk protein) and more content of cis‐9, trans‐11 18:2 conjugated linoleic acid (CLA) (0.77 g/100 g milk fat) compared with 433, 26.67 and 0.53, respectively, for cattle fed grass forage. With regard to the species effect, results showed that buffalo milk appeared to contain significantly higher (p ≤ 0.01) contents of casein nitrogen, phenylalanine, glutamic and arachidonic acid compared with cow's milk. However, the latter was significantly (p ≤ 0.01) more in the cis‐9, trans‐11CLA (0.59 g/100 g milk fat) than that in buffalo milk (0.47 g/100 g milk fat). The results revealed that not only forage type played a critical role in determining the variations of milk nitrogen distribution, milk amino acids and fatty acids but also animal species had a significant effect on these parameters.     

Differential susceptibility to tetracycline,oxytetracycline and doxycycline of the calf pathogens Mannheimia haemolytica and Pasteurella multocida in three growth media

For clinical isolates of bovine Mannheimia haemolytica and Pasteurella multocida, this study reports minimum inhibitory concentration (MIC) differences for tetracycline, oxytetracycline and doxycycline between cation‐adjusted Mueller‐Hinton broth (CAMHB), foetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) medium. MICs were determined according to CLSI standards and additionally using five overlapping sets of twofold dilutions. Matrix effect: (a) free drug MICs and minimum bactericidal concentrations (MBC) for all drugs were significantly higher in FBS than in CAMHB for both pathogens (p < 0.001); (b) MICs and MBCs were higher for CAMHB and FBS compared to RPMI for P. multocida only. Net growth rate for P. multocida in CAMHB was significantly slower than in FBS and higher than in RPMI, correlating to MIC and MBC ranking. Drug effect: doxycycline MICs and MBCs were significantly lower (p < 0.001) in both CAMHB and FBS than tetracycline and oxytetracycline for both pathogens. Only for M. haemolytica were oxytetracycline MIC and MBC significantly lower than tetracycline, precluding the use of tetracycline to predict oxytetracycline susceptibility in this species. Determining potencies of tetracyclines in a physiological medium, such as FBS, is proposed, when the objective is correlation with pharmacokinetic data for dosage determination.     

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.     

One year investigation of the prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido

We investigated the seasonal prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido. Samples of raw milk were collected quarterly from May 2013 through February 2014. The mean clostridial spore count for the raw milk from 336 milk tankers was 27.6 CFU/100 ml. The clostridial species isolated most frequently from raw milk samples was Clostridium tyrobutyricum. The dominant species was C. tyrobutyricum regardless of the season. The percentage of samples with low spore counts (<10 CFU/100 ml) was highest (60.9%) during winter (February) and lowest (34.5%) in autumn (November). In comparison, the percentage of samples with high spore counts (>100 CFU/100 ml) was highest (5.7%) in autumn (November) and lowest (1.1%) during spring (May).     

Total nutrient digestibility and small intestine starch digestion in Nellore and Angus young bulls fed a whole shelled corn diet

Eighteen Nellore and 18 Angus young bulls with BW of 381 ± 12 kg were randomly assigned into two feeding groups (whole shelled corn [WSC] or ground corn with silage [GC]) to evaluate the interaction of breed and diet on total nutrient digestibility, pancreatic α‐amylase, and maltase activity and SLC5A1expression in the small intestine. Experimental diets (DM basis) included (a) a diet containing 30% corn silage and 70% GC and soya bean meal‐based concentrate and (b) a diet containing 85% WSC and 15% of a soya bean meal‐ and mineral‐based pelleted supplement. The treatments were Nellore fed GC diet; Nellore fed WSC diet; Angus fed GC diet; and Angus fed WSC diet. Total faecal collection for the digestibility trial occurred from day 48 until day 50 of the experimental period. Feeding the WSC diet reduced DM and NDF intake (p < 0.01). Angus had greater DM and nutrient intake in kg/day (p < 0.01). However, there was no breed effect on DM and nutrient intakes based on percentage of BW (p > 0.19). Angus had greater starch digestibility (p = 0.03) than Nellore. Cattle fed the WSC diet had greater DM, NDF and starch digestibility (p < 0.01) compared with those fed the GC diet. The activity of pancreatic α‐amylase (U/g of protein) was greater in Nellore (p < 0.01) and was not affected by diet (p = 0.52). In duodenum, maltase activity (U/g of protein) was greater in bulls fed GC diet (p = 0.02). Expression of the gene SLC5A1was not affected by breed or diet (p > 0.05). In conclusion, Nellore had less capacity to digest starch. However, they did not have less pancreatic α‐amylase and duodenal maltase activity compared to Angus. The use of the WSC diet increases DM and total nutrient digestibility.     

Influence of protein fermentation and carbohydrate source on in vitro methane production

Incubations were carried out with batch cultures of ruminal micro‐organisms from sheep to analyse the influence of the N source on in vitro CH₄ production. The two substrates were mixtures of maize starch and cellulose in proportions of 75:25 and 25:75 (STAR and CEL substrates, respectively), and the three nitrogen (N) sources were ammonia (NH₄Cl), casein (CA) and isolated soya bean protein (SP). Five isonitrogenous treatments were made by replacing non‐protein‐N (NPN) with CA or SP at levels of 0 (NPN), 50 (CA50 and SP50, respectively) and 100% (CA100 and SP100) of total N. All N treatments were applied at a rate of 35 mg of N/g of substrate organic matter and incubations lasted 16.5 h. With both proteins, N source × substrate interactions (p = 0.065 to 0.002) were detected for CH₄ production and CH₄/total VFA ratio. The increases in CH₄ production observed by replacing the NPN with protein‐N were higher (p < 0.05) for STAR than for CEL substrate, but the opposite was observed for the increases in volatile fatty acid (VFA) production. As a consequence, replacing the NPN by increased levels of CA or SP led to linear increases (p < 0.05) in CH₄/total VFA ratio with STAR, whereas CH₄/total VFA ratio tended (p < 0.10) to be decreased with CEL substrate. Increasing the amount of both proteins decreased linearly (p < 0.05) ammonia‐N concentrations, which may indicate an incorporation of amino acids and peptides into microbial protein without being first deaminated into ammonia‐N. In incubations with the tested N sources as the only substrate, the fermentation of 1 mg of CA or SP produced 1.24 and 0.60 μmol of CH₄ respectively. The results indicate the generation of CH₄ from protein fermentation, and that the response of CH₄ production to protein‐N supply may differ with the basal substrate.     

Maternal dietary linoleic acid supplementation promotes muscle fibre type transformation in suckling piglets

As meat quality is basically dependent on muscle fibre characteristics, it is important to know how muscle fibres are regulated and transformed. This study aimed to investigate the effect of maternal dietary supplementation on muscle fibre types using 3% saturated fatty acid (palmitic acid, PA) or 3% unsaturated fatty acid (linoleic acid, LA) from 80 days of gestation to the weaning of offspring (25 days post‐natal). The results indicated that higher mRNA levels of MyHCI type genes were found in the soleus muscles of piglets that suckled from LA‐supplemented sows than from PA‐supplemented sows. In addition, LA treatment increased the gene expression of the type I muscle fibre marker troponin I (p < 0.01), suggesting that LA promoted muscle fibre type transformation to type I fibres. Moreover, PGC‐1α (p < 0.01) and MEF2c (p < 0.05) mRNA levels were higher in the piglets from the LA treatment group than in those from the PA treatment group. Furthermore, LA supplementation also significantly increased AMP‐activated protein kinase (AMPK) mRNA levels (p < 0.05), which is an upstream regulator of PGC‐1α. Collectively, these findings demonstrated that maternal dietary LA supplementation promoted muscle fibre transformation to type I fibre and that this process may be mediated through an AMPK‐dependent pathway.     

Effect of grazing cows in an intensive silvopastoral system with Leucaena leucocephala on Panela cheese properties: yield,composition, fatty acid profile and sensory acceptability

Yield, chemical composition, fatty acid profile, and sensory acceptability of Panela cheese produced from cows grazing in an intensive silvopastoral system (ISS) with Leucaena leucocephala and Cynodon nlemfuensis were evaluated and compared with Panela cheese from cows grazing a monoculture system (MS) of C. nlemfuensis only. The experiment lasted for 9 weeks in a tropical area in Mexico using ten crossbred cows (30–90 days of milking) assigned homogenously as five cows in each experimental group. No significant differences were found between the two systems for milk and cheese gross composition. Panela cheese from ISS showed lower content of the hypercholesterolemic fatty acids, accompanied with higher content of omega‐3, omega‐6 and polyunsaturated fatty acids. In addition, Panela cheese from ISS showed higher preference for the attributes of appearance, texture, flavor, and overall acceptability. It is concluded that Panela cheese from cows grazing in ISS has better acceptability and nutritional properties than that produced from MS with grass only.