Interaction between the sequence of feeding of hay and concentrate,and boiling of barley on feed intake,the activity of hydrolytic enzymes and fermentation in the hindgut of Arabian mares

The interaction between the sequence of feeding of hay and concentrate and the hydrothermal processing of barley in alleviating concentrate effects on intake, and hindgut fermentation in horses was tested. Six Arabian mares (4–10 years of age, 410 ± 35 kg body weight) were used to evaluate the effects of feeding sequence (FS) and type of barley (TB) on intake, and faecal volatile fatty acids (VFA), activities of α‐amylase (AA: EC 3.2.1.1), carboxymethyl cellulase (CMCase: EC 3.2.1.4), microcrystalline cellulase (MCCase: EC 3.2.1.91) and general filter paper degrading activity (FPD). Mares were offered a ration of air‐dried alfalfa and concentrate (70:30 as‐fed) in four subsequent periods of 14 days including 8 days of adaptation and 6 days of sampling. In each period and each meal, mares received concentrate either 30 min after (HC) or 30 min before (CH) alfalfa hay. Barley was either milled or boiled in water. Rectal samples were grabbed directly from rectum once per period. Mares subjected to CH had higher dry matter intakes than mares under HC regime. The acetate:propionate ratio (A:P ratio) in rectal content was higher with CH than HC. The AA activity was higher under CH than under HC. Mares fed boiled barley had lower rectal concentrations of VFA and propionate and a higher A:P ratio than mares fed milled barley. Furthermore, the rectal content showed a higher MCCase activity but a lower AA activity when mares were fed boiled compared with milled barley. Interactions between FS and TB were observed with respect to CMCase activity, and concentrations of propionate and valerate. In conclusion, the present results suggest that both, feeding concentrate before hay and boiling the barley, might improve the hindgut environment in Arabian mares, and that the two measures were mostly additive and sometimes even synergistic.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

石榴转录组密码子使用偏向性

对石榴(Punica granatum)转录组数据进行从头组装,预测完整CDS序列的密码子使用偏向性;将石榴转录谱与其他8种蔷薇分支物种基因组编码序列进行同义密码子相对使用度(RSCU)比较,并对石榴WRKY和MYB家族基因进行亚家族进化分析和RSCU比较。结果表明:(1)石榴转录组主要受自然选择压影响。密码子偏向性极强的基因多为细胞相关功能基因;没有密码子使用偏向性的基因多与维持生命活动本质相关。(2)基因差异高表达影响RSCU,CpG二核苷酸富集影响密码子适应性。(3)物种间亲缘关系越近,RSCU值越相似。对蔷薇分支物种密码子进行RSCU比较,石榴与巨桉(Eucalyptus grandis)偏向性基本一致。(4)亚家族功能分化影响密码子偏向性。石榴WRKY亚家族1、4和13在翻译精氨酸、亮氨酸时分别偏向使用AGG、CUC,这与其不断进化的抗逆功能相关。石榴MYB亚家族8在翻译苏氨酸时偏向选择ACA,这与其扩张中的木质化功能相关。     

蛋鸡OC-116基因的克隆和序列分析

参考原鸡（G. gallus） OC-116编码基因序列（登录号：NM_204569.1）设计3对引物，特异性地从仙居鸡子宫组织中克隆OC-116编码基因的N端、中段和C端，测序鉴定后通过酶切连接获得家鸡OC-116的全长编码基因。根据本研究的测序结果，初步发现突变概率较高的碱基有：第1233位（A→G）、1336位（C→G）、1358位（T→G）、1391位（T→G）、1491位（T→G）、1754位（G→T）、1841位（T→C）、1843位（C→T）、1983位（C→T）和2057位（T→C）；其中第1358,1754,1841,1983和2057位的碱基突变均为同义突变，第1233、1336、1391、1491和1843位是错义突变。而且突变碱基主要属于T：G颠换类型，其次是T：C转换类型。另外根据同源性分析，家鸡的OC-116编码基因在进化过程中很保守。总之，本研究已经从仙居鸡子宫组织中克隆到OC-116编码基因，为进一步研究该基因的功能奠定基础。     

青花菜锌指蛋白基因BoCCCH1的克隆和表达分析

CCCH锌指蛋白广泛存在于真核生物中,在植物的生长、发育和逆境胁迫响应中起着重要作用。为获知青花菜CCCH转录因子基因的序列特征和表达特性,从青花菜叶片中克隆到1个CCCH基因,命名为Bo CCCH1,同时,利用RT-PCR方法研究了其在不同器官及霜霉菌侵染叶片中的表达模式。序列分析结果表明,Bo CCCH1的基因组全长为1 568 bp,包含1个长度为599 bp的内含子,2个长度分别为627 bp、342 bp的外显子,编码322个氨基酸,蛋白质的分子量与等电点分别为35 296.42 Da和8.47;Bo CCCH1有2个锌指结构,类型均为C-X8-C-X5-C-X3-H。系统发育分析结果表明,Bo CCCH1与其它植物的21条同源序列在进化树上分为6组,来自十字花科的CCCH与Bo CCCH1处于同一分支。基因表达结果表明,Bo CCCH1在叶、花茎、嫩角果、花蕾和花中表达,其中花茎和嫩角果的表达量最高,但在根中未检测到表达;在霜霉菌侵染下,叶片Bo CCCH1的表达量升高,到24 h和36 h时达到最大,推测Bo CCCH1与霜霉病抗性相关。本研究为进一步探明Bo CCCH1在霜霉病抗性反应中的功能奠定了基础。     

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.