改良背主动脉注射法对鸡胚发育与转基因表达效率的影响

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况。本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14~16期(Hamburger-Hamilton Stage 14~16)鸡胚背主动脉中,再进行换壳培养,继续孵化至出壳,观察和分析改良背主动脉注射法与传统背主动脉注射法和胚盘下腔注射法对发育8、14、18、21 d鸡胚存活率、孵化率以及EGFP阳性检出率的影响。结果表明:改良背主动脉注射组鸡胚存活率均高于传统背主动脉注射组与胚盘下腔注射组(P<0.05或P<0.01);改良背主动脉注射组的鸡胚孵化率(37%)高于传统背主动脉注射组(16%)与胚盘下腔注射组(28%)(P<0.01);荧光蛋白手电筒检测显示,改良背主动脉注射组鸡胚EGFP阳性率(17%)明显高于传统背主动脉注射组(13%)与胚盘下腔注射组(12%)(P<0.01)。综上,背主动脉壳外注射结合换壳培养提高了转基因鸡胚胎孵化率和EGFP阳性检出率,对提高转基因鸡效率具有重要价值。     

猪流行性腹泻病毒S蛋白纳米抗体的筛选与鉴定

猪流行性腹泻病（porcine epidemic diarrhea,PED）是一种高度接触性肠道传染性疫病,主要危害1周龄以内的仔猪,仔猪感染死亡率高达100%,是目前危害世界养猪业的主要疫病之一。本研究旨在制备针对猪流行性腹泻病毒S蛋白的特异性纳米抗体并鉴定其结合活性。作者原核表达并纯化PEDV S1蛋白,将纯化后的PEDV S1重组蛋白免疫双峰驼,第4次免疫后分离其外周血淋巴细胞,提取淋巴细胞RNA,反转录得到cDNA,通过巢式PCR扩增VHH片段,并构建至pCANTAB-5E载体中,电转化至TG1感受态细胞,得到VHH噬菌体抗体展示文库;随后,对构建的噬菌体抗体展示文库进行救援和3轮富集,利用噬菌体展示技术从中筛选针对PEDV S蛋白纳米抗体,通过ELISA验证筛选的纳米抗体的特异性和结合力。通过Western blot和间接免疫荧光验证纳米抗体与PEDV的结合活性。结果显示：成功表达并纯化PEDV S1蛋白,经4次免疫后,双峰驼血清中的特异性抗体效价达到了1∶256 000。构建的噬菌体展示文库的库容量为2.1×10⁷,阳性率85%;对噬菌体展示文库3轮的淘选富集后,最终筛选出6株氨基酸序列不同的纳米抗体,ELISA结果显示,6株纳米抗体均对PEDV S1重组蛋白具有良好的结合力与特异性。随后验证了Nb3能够与PEDV结合,表明其具有良好的活性。成功筛选到针对PEDV S1蛋白的特异性纳米抗体,所筛选纳米抗体有望用于PED的诊断和治疗,同时为PEDV的致病机制研究提供抗体材料。     

骆驼乳清蛋白对热应激所致大鼠肝氧化损伤的保护作用

本试验旨在研究骆驼乳清蛋白（CWP）对热应激（HS）大鼠肝损伤、氧化应激及肝功能的保护作用。选取6周龄SD大鼠36只,适应性饲养2周后随机分为6组：正常对照组饲喂基础日粮;CWP对照组添加400 mg·kg^-1的CWP;HS组除饲喂基础日粮外,每天进行2 h HS处理,连续8 d;3个CWP干预组分别于每次HS前灌服100、200和400 mg·kg^-1的CWP。试验结束后,取大鼠肝组织,HE染色观察肝组织病理学变化,同时检测肝功能生物标志物、氧化应激标志物水平及抗氧化酶活性。结果表明：CWP降低了HS大鼠血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活性,400 mg·kg^-1的CWP干预效果最好（P<0.01）;400 mg·kg^-1CWP干预组有效降低了HS诱导的大鼠肝组织病理学改变; CWP降低了大鼠肝活性氧（ROS）及丙二醛（MDA）水平,增加了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活性及谷胱甘肽（GSH）水平,400 mg·kg^-1的CWP干预效果最好（P<0.01）。结果提示,CWP干预能够以剂量依赖性方式降低大鼠肝氧化应激,增加抗氧化系统防御能力,从而缓解HS所致大鼠肝损伤。     

Low potassium diets with different levels of calcium in comparison with different anionic diets fed to prepartum dairy cows: Effects on sorting behaviour,total tract digestibility,energy metabolism,oxidative status and hormonal response

This study investigated the effects of low potassium diets with different levels of Ca compared to two diets low in dietary cation–anion difference (DCAD) fed prepartum as a strategy to prevent hypocalcemia on sorting behaviour, total tract digestibility, oxidative status and energy and protein metabolism of transition cows. Forty-eight pregnant dairy cows were assigned to 4 treatment groups: Low Ca, low K (LCLK), High Ca, low K (HCLK), Supplementation with anionic mineral mixture (AMS) supplementation with SoyChlor (CAS). After parturition, all animals were fed a standard postpartum diet. Data were collected until 21 DIM. Prepartum urinary pH was significantly reduced by the low DCAD diets, while postpartum Ca homeostasis was affected by the HCLK ration. Feeding AMS induced sorting against particles <1.18 mm in favour of particles >19 mm prepartum. In contrast, cows fed CAS showed an increase in selective consumption of fine particles and sorted against longer particles similar to the HCLK and LCLK groups. Postpartum sorting activity was not affected by the dietary treatments. After calving, apparent digestibility of NDF was significantly reduced in the HCLK group. Prepartum, we observed effects on serum concentrations of non-esterified fatty acids were higher and insulin sensitivity was lower in the AMS group. Blood urea nitrogen (BUN) was decreased in cows fed the CAS ration. Postpartum, we found serum protein to be decreased with the low DCAD diets while BUN was decreased in the CAS group. The low DCAD rations increased prepartum serum malondialdehyde concentrations, while postpartum total antioxidant capacity was lower in the HCLK and the AMS group. From these data, we conclude that AMS decreased prepartum intake due to compromised palatability. Intermediate protein metabolism was affected by the low DCAD diets, while parameters of oxidative stress were probably affected by acid–base balance and Ca homeostasis.     

Expression of P-glycoprotein and breast cancer resistance protein in three cases of canine lymphoma showing drug resistance

In canine lymphoma, drug resistance is the major factor hindering treatment. In this study, we performed immunohistochemical examination of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which are considered as transporters related to multidrug resistance in three recurrent canine lymphomas. All cases were negative for both transporters before anticancer drug administration, but became positive after this administration. The expression was confirmed in capillary endothelial cells, such as in brain capillaries acting as the blood-brain barrier (BBB). It is suggested that both transporters expressed on capillary endothelial cells in lymphoma tissue may inhibit the spread of anticancer drugs into tumor tissues from blood, the same as the BBB. Therefore, capillary endothelial cells could act as a blood-tumor barrier, which might be involved in drug resistance in canine lymphoma.     

Selenium supplementation improves nutrient intake and digestibility,and mitigates CH4 emissions from sheep grazed on the mixed pasture of alfalfa and tall fescue

Low selenium (Se) in soil and forage can adversely affect on the quality of animal-derived foods, and hence on human health. Lambs grazed on mixed pastures of alfalfa (Medicago sativa) and tall fescue (Festuca arundinacea) were supplemented with five levels of Se [0, 3, 6, 9 and 12 µg/kg body weight (BW)]. The intake of dry matter (DM) and organic matter (OM) varied with the level of Se supplementation, with a peak at 6 µg Se per kg BW (p ≤ 0.05). Gross energy (GE) intake, digestive energy (DE) intake and metabolic energy (ME) intake were higher at 6 µg Se per kg BW than at other Se levels (p < 0.01); in addition, methane energy (CH₄-E) output was lower at 6 µg Se per kg BW. Supplementation with Se significantly increased nitrogen (N) intake, faecal N and urine N, for which the peak values were 20.2 g N/, 5.62 g N/day and 7.92 g N/day, respectively, at 6 µg Se per kg BW. Se intake, blood Se, faecal Se, urine Se and retained Se were negatively correlated with forage crude protein (CP) content (p < 0.001) but were positively correlated with the content of neutral detergent fibre (NDF) (p < 0.001) and acid detergent fibre (ADF) (p < 0.001). Thus, we recommend the addition of 6 µg Se per kg BW to sheep grazed on pastures in regions with low soil Se.     

Evaluation the effect of subchronic feeding of transgenic cotton line (CKC1) on the faecal microbiota of albino rabbits

Recent studies have demonstrated a strong relationship between the intestinal microbiota and the host health. As such, consumers are increasingly becoming more concerned about the potential effect of certain foods/feeds, particularly of transgenic origin on the gut microbiota. Although the European Food Safety Authority has recommended in their guidelines, to study the effect of transgenic food/feed on host-microbiota, yet, few studies have focused on the evaluation of such effects mainly due to culturing difficulties. Therefore, this study was intended to evaluate the potential adverse effects of transgenic diet consumption on some specific gut microflora (Lactobacillus group, Bifidobacterium genus, Escherichia coli subgroup and Enterococcus genus) of rabbits. A total of forty-eight rabbits were randomly assigned into four groups and fed a diet containing a variable proportion of transgenic cottonseeds at 0, 20, 30 and 40% inclusion level, respectively. Changes in the specific or total faecal bacterial population were monitored at five different experimental stages (i.e. 0, 45, 90, 135 and 180 days) using both the traditional plate count method (TM) and quantitative real-time PCR (qPCR). No significant differences (p > .05) were observed concerning numbers of specific bacteria or total bacteria between the control and experimental groups, though qPCR showed numerically higher values in terms of 16S rRNA gene copies as compared to the values obtained from TM. However, such numerical differences were biologically insignificant (p > .05). Similarly, no significant variations were noticed in the calculated B/E (log₁₀ copies of Bifidobacterium per g faces/log10 copies of E. coli genome per g faeces) ratios in all the groups. All the ratios were in the range of 1.24 to 1.30 throughout the experiment, indicating a good balance of intestinal microflora and greater resistance to intestinal disorders. It is therefore concluded that feeding transgenic cottonseeds could not adversely affect the gut microflora of rabbits during a long-term study.     

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

纤维和蛋白质对肠道健康和环境的影响

由于膳食纤维和蛋白质对肠道健康和环境都有潜在的有益或有害影响,因此,膳食纤维和蛋白质在猪肠道中的发酵是一个值得关注的问题。本文综述了近年来有关膳食纤维和蛋白质发酵对猪肠道环境的影响及其对猪粪和猪舍含氮气体和异味排放的影响,同时探讨了蛋白质发酵和相关代谢化合物的生产,与膳食纤维发酵、对肠道健康、细菌蛋白质合成和氮排泄的影响。生产上可以采用不同的营养策略来应对集约化养猪生产对环境的的负面影响,如降低日粮粗蛋白质水平减少氮排泄。总的来说,日粮纤维和蛋白质水平是影响猪肠道发酵、氮排泄和环境影响的一个有效策略。     

日粮蛋白质水平对云南半细毛羊空怀母羊能量代谢的影响

为研究日粮蛋白质水平对云南半细毛羊空怀母羊能量代谢的影响,试验选择25只云南半细毛羊空怀母羊,平均分为5个组,分别饲喂蛋白质水平为8.4%、10.0%、12.3%、13.6%、15.9%的日粮,用全收粪法进行消化代谢试验,试验期共19 d,预试期14 d,正试期5 d。结果表明：（1）日粮蛋白质水平对总能摄入量无显著影响（P > 0.05）。（2）从组1到组5粪能逐渐下降,且组3、组4、组5较组1分别降低了10.5%、11.4%、13.7%（P < 0.05）,组5较组2降低了9.5%（P < 0.05）。（3）从组1到组5尿能逐渐增加,除组2与组1、组3间差异不显著（P > 0.05）外,其余组间差异极显著（P < 0.01）。（4）组4和组5总能消化率较组1极显著提高13.2%和11.8%（P < 0.01）,其余各组差异不显著（P > 0.05）。组4总能代谢率较组1极显著提高9.8%（P < 0.01）,其余各组差异不显著（P > 0.05）。对于消化能代谢率,组1和组2差异不显著（P > 0.05）,组3、组4和组5依次显著降低（P < 0.05）。（5）蛋白质采食量与总能消化率、消化能代谢率间的回归方程分别为：Y=0.917X+45.872（R2=0.853）|Y=-0.370X+87.735（R2=0.899）。综上,日粮蛋白质水平与粪能呈负相关,与尿能呈正相关。日粮蛋白质水平为13.6%时,总能消化率和总能代谢率均较8.4%蛋白质水平组极显著提高。总能消化率随蛋白质采食量的提高而线性升高,消化能代谢率则随蛋白质采食量的提高而线性降低。 [关键词] 蛋白质|云南半细毛羊|空怀期|能量代谢