Effects of melatonin on sperm quality, enzyme activity, antioxidant gene expression and fertility of cryopreserved bovine semen.

Melatonin (MLT) has strong antioxidant capacity and can reduce the damage caused by oxidative stress in sperm, but there is still little content in the field we have studied. In this study, we are committed to scientific research on adding melatonin to Belgian blue bull semen diluent for cryopreservation. Different concentrations (0, 0.1, 0.3, 0.5 or 0.7 mg/mL) of MLT were added diluent. Sperm kinetic parameters, enzyme activity, antioxidant gene expression and fertility were analyzed after thawing. The results showed that MLT concentration of 0.3 mg/mL exerted positive effects on post-thaw kinetic parameters. Compared with other groups, 0.3 mg/mL MLT treated sperm acrosome and plasma membrane integrity, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels significantly increased. Meanwhile, the mRNA expression of antioxidant genes SOD2, CAT and GPx increased in the 0.3 mg/mL MLT treatment group, and the mRNA expression of apoptosis genes Caspase-3 and Bax were significantly reduced. In addition, in vitro fertilization (IVF) embryo cleavage, blastocyst rate and artificial insemination (AI) pregnancy rate were higher in 0.3 mg/mL MLT. Therefore, MLT showed cryoprotective capacity to the freezing diluent used for Belgian blue bull sperm during the process of freezing-thawing, and the optimal concentration of MLT for the frozen diluent was 0.3 mg/mL.

Preservation of Simmental bull sperm at 0°C in Tris dilution: effect of dilution ratio and long-distance transport.

OBJECTIVE: This study aimed to assess the impact of the dilution ratio of Tris diluent, storage at 0°C, and long-distance transportation on the spermatozoa of Simmental cattle. It also validated the feasibility of the regional distribution of fresh semen. METHODS: In experiment 1, semen was diluted at four dilution ratios (1:6, 1:9, 1:12, and 1:15) to determine the optimal dilution ratio of Tris diluent. In experiment 2, we assessed sperm viability, progressive motility (objectively assessed by computer-assisted sperm analyzer), and acrosome intactness in Tris dilutions kept at constant 0°C for 1, 3, 6, 9, and 12 days. We compared them to Tianshan livestock dilutions (Commercial diluent). In experiment 3, semen was diluted using Tris diluent, and sperm quality was measured before and after long-distance transport. Artificial insemination of 177 Simmental heifers compared to 156 using Tianshan Livestock dilution. RESULTS: The outcomes demonstrated that 1:9 was the ideal Tris diluent dilution ratio. The sperm viability, Progressive Motility, and acrosome integrity of both Tris and Tianshan dilutions preserved at 0°C gradually decreased over time. sperm viability was above 50% for both dilutions on d 9, with a flat rate of decline. The decrease in acrosome integrity rate was faster for Tianshan livestock dilutions than for Tris dilutions when stored at 0°C for 1 to 6 days. There was no significant difference (p>0.05) in sperm viability between semen preserved in Tris diluent after long-distance transportation and semen preserved in resting condition. The conception rates for Tris dilution and Tianshan livestock dilution were 49.15% and 46.15% respectively, with no significant difference (p>0.05). CONCLUSION: This shows that Tris diluent is a good long-term protectant. It has been observed that fresh semen can be successfully preserved for long-distance transport when stored under 0°C conditions. Additionally, it is feasible to distribute semen regionally.

HGF/c-Met signaling promotes the migration and proliferation of deer antler MSCs.

The complete regeneration of deer antlers is based on the proliferation and differentiation of stem cells. Mesenchymal stem cells (MSCs) of antlers have an important role in antler regeneration and rapid growth and development. HGF is mainly synthesized and secreted by mesenchymal cells. After binding to its receptor c-Met, which transduces signals into cells to stimulate cell proliferation and migration in various organs to promote tissue morphogenesis and angiogenesis. However, the role and mechanism of the HGF/c-Met signaling pathway on antler MSCs are still unclear. In this study, we established antler MSCs with overexpression and interference of HGF gene by lentivirus and small interference RNA, observed the effect of HGF/c-Met signal pathway on the proliferation and migration of antler MSCs, and detected the expression of downstream related signal pathway genes, to explore the mechanism of HGF/c-MET signal pathway on the proliferation and migration of antler MSCs. The results showed that the HGF/c-Met signal affects the expression of RAS, ERK and MEK genes, regulates the proliferation of pilose antler MSCs through Ras/Raf, MEK/ERK pathway, affects the expression of Gab1, Grb2, AKT and PI3K genes, and regulates the migration of MSCs of pilose antler through Gab1/Grb2 and PI3K/AKT pathway.

Simulation study of oxytetracycline contamination remediation in groundwater circulation wells enhanced by nano-calcium peroxide and ozone.

The widespread use of antibiotics in recent years has led to increasing antibiotic contamination of shallow groundwater. As the most widely used tetracycline antibiotic, oxytetracycline has received a lot of attention from researchers due to its stable molecular structure and difficulty in degradation. Aiming at remediation of oxytetracycline pollution in shallow groundwater, nano-calcium peroxide (nCaO2) and ozone (O3) are used to enhance the degradation of oxytetracycline in groundwater circulation well (GCW). A three-dimensional sand box test device for circulation wells is designed to explore the repair efficiency of circulation wells strengthened by different oxidants. The results show that after nCaO2 and O3 enhancing circulation wells operate for 10 h, the average removal rate of OTC reaches 83%, and the highest removal rate is 88.13%, which is 79.23% and 13.96% respectively higher than that of nCaO2 and O3 enhanced circulation wells alone, and there is no rebound phenomenon after aeration stops. The in-situ treatment of enhanced GCW by nCaO2 and O3 has potential applications for the removal of OTC in groundwater environments.

Ram semen preserved at 0°C with soybean lecithin Tris-based extender substituted for egg yolk.

OBJECTIVE: The present study evaluated the preservation of ram semen at 0°C using soybean lecithin with a Tris-fructose extender. METHODS: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0°C in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination. RESULTS: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05). CONCLUSION: These results suggest that ram sperm is capable of fertilization after preservation at 0°C with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

A rapid reporter assay for recombinant human brain natriuretic peptide (rhBNP) by GloSensor technology.

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor™ 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p = 0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.

[Characterization and comparison of interferon reference standards using UPLC-MS].

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.

Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.

Mouse NGF (mNGF) extracted from mouse submaxillary gland has been approved on the market in China for treating nerve damage caused by N-hexane poisoning for over a decade, and many researches showed the clinical effectiveness of mNGF for the treatment of other nerve system diseases. The extracted mNGF have risks of potential viral contamination due to the animal origin. Here, we report the successful expression, purification, and characterization of recombinant mNGF (rmNGF). An expression plasmid of mouse nerve growth factor (mNGF) was constructed and transfected into CHO-S cells. Stable transfectants were obtained using a two-phase selection scheme with the addition of different concentrations of methotrexate and puromycin. Recombinant mNGF (rmNGF) was purified from cell culture medium by a two-step procedure: cation exchange followed by size-exclusion chromatography. The purity of rmNGF was 98.6% determined by size exclusion high performance liquid chromatography (SEC-HPLC). The molecular weight, isoelectric point and N-terminal sequence of rmNGF were identical to the theoretical values entirely. In TF-1/MTS, the specific activity of the protein was approximately 1.7×10(6)U/mg against rhNGF (the reference standard). In DRGs, the specific activity was approximately 7.3×10(5)AU/mg against mNGF (the reference standard). Our results showed that a high quality of rmNGF with marked biological activity comparable with mNGF was produced, and laid the basis for further research and development of rmNGF.

[Factors affecting benzene diffusion from contaminated soils to the atmosphere and flux characteristics].

The influencing factors of benzene diffusion fluxes from sand and black soil to atmosphere were investigated using a flux chamber (30.0 cm x 17.5 cm x 29.0 cm). In this study, the benzene diffusion fluxes were estimated by measuring the benzene concentrations both in the headspace of the chamber and in the soils of different layers. The results indicated that the soil water content played an important role in benzene diffusion fluxes. The diffusion flux showed positive correlation with the initial benzene concentration and the benzene dissolution concentration for both soil types. The changes of air flow rate from 300 to 900 mL x min(-1) and temperature from 20 degrees C to 40 degrees C resulted in increases of the benzene diffusion flux. Our study of benzene diffusion fluxes from contaminated soils will be beneficial for the predicting model, and emergency management and precautions.

[Research on the characteristic of toluene migration and distribution in fluvoaquic soil and its simulation using STOMP model].

Frequently accidental spill of hazard materials into soil environment posed significant threats to human health and natural environment in China. In this paper, simulated nonaqueous phase liquid (NAPLs) toluene sudden spill in fluvo-aquic soil was performed in a two-dimensional tank to investigate the migration and distribution characteristics of toluene as well as its simulation through STOMP model. Visual observation showed that the horizontal expansion of toluene with concentration > 1 g x kg(-1) and > 20 g x kg(-1) were approximate 2.3 and 3 times the length of the corresponding vertical transportation, respectively. The result revealed that toluene exerted a preferential tendency to lateral spread compared to the vertical migration trends, which may contribute to the impeding effect as a result of the low permeable capacity of soil (permeability coefficient was 0.12 cm x h(-1)). The behavior and fate of toluene in heterogeneous soil layers (combined fluvo-aquic-sand and sand-fluvo-aquic soil layers, respectively) after spill were simulated using STOMP model and the results indicated that the significant difference in relative permeability of interface layer, due to the much higher value of permeability coefficient of sand (29.70 cm x h(-1)) than that of fluvo-aquic soil, played an important role on the redistribution of toluene after its spill into the heterogeneous soil layers. For practical purposes, the results of this study may be beneficial to identify the distribution property of NAPLs in time after its release into the soil environment.

Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products.

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 µg protein per ampoule (95% C.I: 212.407-218.486 µg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.

[Quality control of recombinant oncolytic adenovirus/p53].

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

[Health risk analysis of VOC/SVOC contaminated soil in an abandoned chemical plant].

Environmental health risk of contaminated soil in a typical abandoned industry was analyzed based on the full field investigation according to the site assessment procedure of American Society for Testing and Material (ASTM). Parameters were modified with the combination of Chinese crowd character and site specifics. Results indicated that the site was mainly contaminated with volatile and semi-volatile organic compounds in soil profiles. And the contents of carbon tetrachloride, tetrachloroethylene, pentachloroethane, hexachlorobutadiene, hexachloroethane and hexachlorobenzene in soil samples were exceeded the national environmental standard. These contaminants ranked the carcinogenic risks and hazard quotients more than 10(-2) and 1 in some locations with the exposure by oral ingestion, dermal contact and inhalation. Contaminants in this site had resulted in the high health risks to the residents and surrounding communities. The risk should be reduced to the health acceptable level by the treatment and remediation before further development for residential and commercial utilization.

[Comparative study on eight trace elements in twelve flower medicines].

Eight trace elements such as Ca, Cu, Fe, Mn, Zn, K, Mg and Na in twelve kinds of flower medicines were determined by flame-atomic absorption spectrometry with air-acetylene flame. The flower medicines include Pueraria lobata Ohwi., Gomphrena globosa L., Chrysanthemum morifolium Ramat., Prunus persica (L.) Batsch., Canna indica L., Pyrus bretschneideri Rehd P. spp, Rosa chinensis Jacq., Celosia cristata L., Sophora japonica L., Saussurea medusa Maxim. , Iris lactea var. chinensis (Fisch.) koidz. and Gentiana straminea Maxim.. All of the flowers were commonly used in Tibetan medicines. Three kinds of the flowers were bought in the market and the others were picked in Qinghai province. These flower medicines were selected, dried and powdered, 4.000 g was weighed accurately with analytical balance, and five portions were used for each kind of sample. The content of eight trace elements in these flower medicines was determined and the difference in the content was observed. The recovery rate obtained by the standard addition method was between 96.76% and 102.93%, and the RSD was between 1.13% and 3.46%, so the accuracy of the method was better and the precision of the method was good. The results of the experiment indicated that the contents of the eight trace elements were rich in the twelve kinds of flower medicines, and the content of three trace elements including K, Mg, Na were more than other trace elements in the twelve flower medicines. There were considerable differences in the content of the eight trace elements in different flower medicines and there were more trace elements in Saussure medusa Maxim., Iris lactea var. chinensis (Fisch.) koidz. Canna indica L. and Celosia cristata L. and less trace elements in Sophora japonica L. and Gentiana straminea Maxim.. The data of the experiment could provide an accurate and credible evidence for the reasonable medicinal use and deeper exploitation of these flower medicines.

[Quality control methods and requirements for recombinant human lymphocyte function associated antigen 3 IgG1 fusion protein (rhLFA3-IgG1)].

To establish methods and requirements for quality control of rhLFA3-IgG1, biological potency of rhLFA3-IgG1 was determined by CD2 molecule competitive binding assay on Jurkat cell surface. Purity of rhLFA3-IgG1 was analyzed by SEC-HPLC and IEC-HPLC. Peptide mapping was preformed by tryptic digestion and RP-HPLC after sample reduced and carboxymethylation by DTT and indoacetic acid, respectively. CHO host cell protein and Protein A residual were detected by ELISA separately. The quality control methods and requirements, such as biological potency, the physical-chemical characteristic of rhLFA3-IgG1 had been established. The methods and requirements for quality control of rhLFA3-IgG1 showed advantages of assuring the products safety and efficacy, which can be used for routine quality control of rhLFA3-IgG1.

[Peptide mapping analysis of recombinant human interleukin-11 with HPLC-ESI-Q-TOF/MS spectrometry].

AIM: To analyze the peptide mapping of recombinant human interleukin-11 (rhIL-11) by HPLC-ESI-Q-TOF/MS spectrometry. METHODS: The trypsin digested rhIL-11 at 37 degrees C over night, and the peptide mapping was performed by HPLC. The relative molecular weight of the peptides fragments was measured by ESI-Q-TOF/MS, and amino acid sequence was analyzed by MS/MS. RESULTS: The peptide fragments of rhIL-11 in the peptide mapping were assigned by analyzing the retain time, relative molecular weight and amino acid sequence. And 97% of the expected peptides were detected in this way. CONCLUSION: The study proves that HPLC-ESI-Q-TOF/MS is a good method to analyze peptide mapping of protein with the advantage of sensitivity, high speed and accuracy.

[Study on parentage testing in JIRONG rabbit by microsatellite markers].

Thirteen microsatellite markers, including Sat2,Sat3,Sat4,Sat5,Sat7,Sat8,Sat12, Sat13, Sat16,Sol08,Sol28,Sol30 and Sol03, were studied for their parentage-testing application in a group of 30 JIRONG Rabbits in the present report. The 13 microsatellite loci were successfully amplified with specific primers designed according to known sequences. The PCR products amplified from the microsatellite loci were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The results demonstrated that the average alleles and the mean heterozygosity (Hs) and the polymorphism information content (PIC) and the combined exclusion probability (PE2) of the 13 microsatellite loci were 3.46, 0.578, 0.531, and 0.999329, respectively. The exclusion probability (PE1) of the 13 loci was 0.935226, and the confidence of the parentage testing was less than 80% when data of both parents were unknown, while the exclusion probability (PE1) and the confidence were 0.999329 and 95% respectively with known data of a single parent. Since the data of the rabbit maternal lines studied were known, the paternal lines of the group were successfully identified using the 13 microsatellite loci with high confidence.

[Study on methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein].

AIM: To establish methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein (rhTNFR-Fc). METHODS: Biological potency of rhTNFR-Fc was determined by neutralizing the bioactivity of TNF-alpha. rhTNFR-Fc samples were reduced by beta-mercaptoethanol and the peptide map was performed by tryptic digestion. Residual protein A and the host cell protein content were detected by ELISA. Anti-TNFR and anti-Fc antibodies were used in ELISA for detection of the rhTNFR-Fc content. RESULTS: The quality control methods, such as bioassay, peptide map, residual protein A detection, were established and used for quality control of rhTNFR-Fc. The unit of rhTNFR-Fc (AU) was defined according to the international unit of TNF-alpha. The specific activity was up to 8 x 10(4) AU.mg-1. The requirements for quality control of rhTNFR-Fc were established. CONCLUSION: The methods and requirement were used for quality control of rhTNFR-Fc products.

[Quality control methods for recombinant human endostatin].

AIM: To establish the quality control methods for recombinant human endostatin. METHODS: Biological activity was determined by endothelial cell migration assays. Peptide mapping was tested by trypsin digestion and RP-HPLC. Purity was determined by non-reduced SDS-PAGE and RP-HPLC. Other tests including molecular weight, isoelectrical point, etc. were done according to the National Requirements for Biological Products (2000). RESULTS: The method of bioassay was established and used for determining activity of endostatin. Specific activity of the three batchs of drug substance was 1.45 x 10(6), 1.57 x 10(6) and 2.73 x 10(6) u.mg-1 proteins. Peptide mappings of the three batches of drug substance were completely identified. Both purity results of the products tested by SDS-PAGE and RP-HPLC were more than 99%. CONCLUSION: The established methods can effectively control the quality of recombinant human endostatin.