Effect of Se and VE Supplement on Semen Quality,Antioxidant Enzyme Activities and Heat Shock Protein Expression of Goat in High Temperature Season

This experiment was conducted to study the effect of selenium and vitamin E supplement on semen quality, antioxidant enzyme activities and heat shock protein expression of goat in Hainan high temperature season.16 adult Hainan Black goat with good health and approximate weight were randomly divided into 4 groups, fed with basal diet(control group), basal diet+0.5 mg/kg Se(Se group), basal diet+100 mg/kg VE(VE group), and basal diet+0.5 mg/kg Se+100 mg/kg VE(Se+VE group), respectively.The experimental period was 93 d.Semen samples were collected in the last week of the experiment on two consecutive days.The semen quality, antioxidant enzyme activities and heat shock protein expression were analyzed.The results showed that compared with control group, the ejaculate volume was not significantly affected by Se or VE supplement(P>0.05).Sperm density and sperm motility were increased significantly by Se and VE supplement(P<0.05), and the abnormal rate was decreased extremely significantly(P<0.01).The goats fed with Se and VE also had higher activities of GSH-Px(P<0.01), SOD(P<0.05), CAT(P<0.05) and T-AOC(P<0.01), and lower MDA concentration in seminal plasma(P<0.05).The relative expression levels of HSP70 and HSP90 mRNA in supplement groups were decreased extremely significantly(P<0.01).However, there were some certain differences between the Se and VE supplement groups on semen quality and heat shock protein expression.In conclusion, the supplementation of Se and VE could help to improve goat semen quality by increasing the sperm density, sperm motility, the antioxidant enzyme activities, and decreasing the abnormal rate in hot season of Hainan.Finally, Se and VE supplement had good effects on relieving the environment heat stress.     

Construction and Identification of Yeast Surface Display Libraries of Ovis aries DRA Gene Exon 2

The specific primers were designed according to Ovis aries DRA gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1,which was a vector used for protein surface display on Saccharomyces cerevisiae.The gene encoding DRA was amplified by PCR using the genomic RNA of Ovis aries.The 762 bp fragment was cloned and released in GenBank and registration number was KR422362.The PCR product was inserted into the yeast surface display plasmid vector pYD1 by double enzyme digestion.It was indicated that DRA gene was successfully integrated into the genome.Dot mutation was made at both ends of exon 2 in DRA gene for making restriction enzyme cutting site and design the exon 2 specific primers according to mutated Ovis aries DRA gene sequence.Sequenced exon 2 amplification products based on DNA pooling of sheep large sample template was analyzed the polymorphic loci.The polymorphic exon 2 246 bp fragment was obtained by double enzyme digestion and connected to surface display restructuring mutation carriers pYD1-DRA by the same double enzyme digestion,and then we successfully constructed yeast surface display libraries.We transformed it into Saccharomyces cerevisiae EBY100 cell.Yeast monoclone was identified by PCR amplification and sequencing,and we confirmed that DRA gene had been integrated into Saccharomyces cerevisiae genome.After galactose induced,it was detected that DRA gene library had been successfully demonstrated on the yeast cell surface under the fluorescence microscope by immunofluorescence method.     

倍他米松ELISA检测方法的建立

为建立倍他米松(BET)ELISA检测方法,先使BET与琥珀酸酐反应,再采用活化酯法与牛血清白蛋白(BSA)偶联制备免疫原;其次,利用免疫动物获得多克隆抗体,经Sephrose 4B-proteinA方法对抗体进行纯化;最后,建立倍他米松ELISA检测方法。结果表明,免疫抗原偶联成功,获得了亲和力较高的多克隆抗血清,间接竞争ELISA测定其滴度为102 400、IC15为6.60×10-5 ng/mL和IC50为0.97ng/mL。该方法适用于残留在动物性食品中的BET现场大批量检测。     

Effects of dietary L-threonine levels on antioxidant capacity,digestive enzyme activities,and antibody production of Xinyang green-shell laying hens

Xinyang green-shell laying hens (n = 720), 32 wk of age were allocated to 5 dietary treatments groups, each of which included 6 replicates of 24 hens. The control group was given a basal corn-peanut-soybean meal diet containing 0.47% L-threonine (L-Thr). L-Thr levels of the experimental groups were 0.57, 0.67, 0.77, and 0.87%, respectively. The trial lasted 10 wk. Serum concentrations of total superoxide dismutases (T-SOD) and superoxide dismutases (SOD) were maximized at 0.67% L-Thr group. The 0.57% L-Thr group had the highest serum total antioxidative capacity (T-AOC), while the 0.67% L-Thr group had the lowest serum malondialdehyde (MDA) concentration. Hens fed 0.67% L-Thr of the diet resulted in the highest SOD and T-AOC levels in the liver. The activity of amylase and lipase in the jejunum was maximized at 0.77% L-Thr level. Hens fed 0.77% L-Thr in the diet resulted in the highest IgM and IgG levels in the jejunum. In conclusion, we suggest that a L-Thr level above 0.67% may have had an antioxidant capacity and positive effect on digestive enzyme activities and antibody production in the jejunum of Xinyang green-shell laying hens.     

Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.     

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.     

Effects of thermo‐resistant non‐starch polysaccharide degrading multi‐enzyme on growth performance,meat quality,relative weights of body organs and blood profile in broiler chickens

This research was conducted to study the performance and carcass parameters of broiler chickens fed diets supplemented with heat‐treated non‐starch polysaccharide degrading enzyme. A total of 432 one‐day old Ross 308 broiler chickens were allocated to five treatments: (i) CON (basal diet), (ii) E1: CON + 0.05% multi‐enzyme, (iii) E2: CON + 0.1% multi‐enzyme, (iv) E3: CON + 0.05% thermo‐resistant multi‐enzyme and (v) E4: CON + 0.1% thermo‐resistant multi‐enzyme, each treatment consisted of six replications and 12 chickens in each replication. The chickens were housed in three floor battery cages during 28‐day experimental period. On days 1–7, gain in body weight (BWG) improved by feeding the diets supplemented with thermo‐resistant multi‐enzyme. On days 7–21 and 1–28, chickens fed the diets containing thermo‐resistant multi‐enzyme showed improved (p < 0.05) BWG and feed conversion ratio (FCR) compared to CON group. Supplementing the diets with multi‐enzyme or thermo‐resistant multi‐enzyme affected the percentage of drip loss on d 1 (p < 0.05). Drip loss percentage on days 3 and 5 and also meat colour were not affected significantly. Supplementing the diets with multi‐enzyme or thermo‐resistant multi‐enzyme did not affect the relative weights of organs but compared to CON group, relative weight of breast muscle increased and abdominal fat decreased (p < 0.05). Among measured blood constituents, chickens fed supplemented diets with thermo‐resistant multi‐enzyme showed higher (p < 0.05) IgG. Counts of red and white blood cells and lymphocyte percentage were not affected. In conclusion, the results demonstrated that supplementing pelleted diets with thermo‐resistant multi‐enzyme improved performance of broiler chickens.     

管理措施对三江源区"黑土滩"土壤肥力及土壤酶活性的影响

对三江源区典型退化草地“黑土滩”采取施肥及种植垂穗披碱草的恢复措施,结果表明,土壤中的全氮与有机质的相关性显著,全磷与有机质的相关性较明显,变化趋势相一致。短期的恢复措施造成了恢复样地20~30 cm土壤层中有机质、全氮和全磷含量的下降,长期的恢复措施使恢复样地中的这3种营养物质的含量得到了上升;人工种植措施使土壤中的全钾含量显著下降(P<0.05)。此外,速效养分的变化不一致,其中速效氮的含量表现为先降低后升高,速效磷和速效钾的含量变化表现出升高趋势。植被恢复对土壤脲酶活性有促进作用,但蔗糖酶活性没表现出规律性变化;这2种酶的活性与速效养分的含量表现出较强的正相关,而与有机质和全价养分的含量相关性不显著。