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@#Objective To investigate the effect of different diluents on the stability of the mixed enzyme-labeled antibody,and screen a suitable diluent for the mixed enzyme-labeled antibody,which can protect the stability of the cocktail mixture of horseradish peroxidase(HRP) conjugated secondary antibody and alkaline phosphatase(AP) conjugated secondary antibody.Methods Using Tris-HCl buffer as the base solution,four different diluent formulations(A,B,C,D) were prepared with different kinds of stabilizers contained in each formula,such as protein,metalion,surfactant and bacteriostatic agent.Mixed enzyme-labeled antibodies were prepared with different diluents and stored at 2～8 ℃ and 37 ℃,which were detected for the stability by ELISA,enzyme activity assay and immunohistochemistry(IHC) staining.Results D solution [Tris(50 mmol/L)+BSA(1.5%,g/100 mL)+Tween-20(0.3%)+Proclin-950(0.1%,g/100 mL)+sodium chloride(150 mmol/L)+L-Ascorbic Acid(1 mmol/L)+L-methionine(5 mmol/L)+L-histidine(5 mmol/L)+sodium caseinate(1%,g/100 mL)+zinc chloride(0.1 mmol/L)+trehalose(8%,g/100 mL)] was of the optimal protective effect.When stored at 37 ℃ for 10 weeks,the working solution of HRP conjugated secondary antibodies and AP conjugated secondary antibodies showed the highest titer,the activity residual ratios of HRP and AP were 93.46% and96.07%,respectively,and there was no significant difference in IHC results.Conclusion This formula diluent can be used for the preparation of mixed enzyme-labeled antibody working solution.
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Abstract The present study evaluated the effect of cryoprotectants, semen diluents and chicken lines during pellet method of semen cryopreservation. Three different experiments were conducted; Experiment 1 - semen was cryopreserved using dimethylformamide (DMF) at 6% and 9% concentrations in two semen diluents (Lake and Ravie diluent and TES/NaCl diluent), Experiment 2 - semen was cryopreserved using dimethylacetamide (DMA) at 6% and 9% with or without sucrose (100mM), Experiment 3- semen from two chicken lines (PD1 and PD6) was cryopreserved using DMA (6% and 9%). Semen was evaluated pre and post cryopreservation for progressive motility, live and abnormal sperm. Semen pellets were stored in cryovials for at least seven days before examination and insemination. Thawed semen was inseminated intravaginaly to study fertility. All the parameters studied were significantly lower (p<0.05) in cryopreserved semen. DMF in Lake and Ravie diluent gave very low fertility and TES/NaCl diluent no fertile eggs. DMA as cryoprotectant gave fertility up to 9.22 %. Addition of sucrose along with DMA produced fertility similar to other cryopreservation treatment groups. No difference in in vitro semen parameters between chicken lines was observed. There is difference in cryopreservation outcome due to semen diluent and type of cryoprotectant.
Subject(s)
Animals , Semen , Cryopreservation/methods , Fertility , Fertilization in Vitro/methods , ChickensABSTRACT
Objective To screen a suitable reagent system for measuring iodine in serum by direct dilution sampling-inductively coupled plasma mass spectrometry (ICP-MS).Methods Experiments were on sample dilution method for standard solutions and serum samples with six different diluents.After dilution,the concentration of iodine in serum was determined by ICP-MS method.The rhenium (Re) and tellurium (Te) was used as internal standard element,respectively.The effects of the two internal standard elements on determination results were compared.And the results were compared with the results determined by the current serum iodine arsenic cerium catalytic spectrophotometry standard method (WS/T 572-2017),hereinafter referred to as the standard method.The methodological evaluation of this new method was done through standard curve linearity,sample detection limit,precision and accuracy in determining serum iodine in the range of 0-300 μg/L.Results The direct dilution sampling-ICP-MS method screened for measuring iodine in serum was preparing 1 L of 2.0 g/L ascorbic acid,1.0 g/L ammonium chloride,0.10％ ethanolamine,and 1.0％ ethanol as diluent;the standard solutions and the serum samples were all diluted in a ratio of 1:19 (sample:diluent) before testing with Re as internal standard element.The linear range of the calibration curve was 0-300 μg/L and the linear correlative coefficient was 0.999 9.The detection limit for serum iodine was 1.2 μg/L (0.20 ml of serum was tested).Precision:the range of the intra assay coefficient of variation (CV) was 0.2％-1.2％ (n =6) and the range of the inter assay CV was 0.4％-1.9％ (n =3) when measuring 5 serum samples.Accuracy:The average recovery was 97.9％ with a range of 93.9％-103.8％ when measuring 8 serum samples.No significant difference was found between the results of the 35 serum samples determined by the standard method and the new method (t =0.178,P ＞ 0.05).Conclusions Re performs better than Te in determination of iodine as an internal standard element.The reagent system screened for determination of iodine in serum does not require matrix matching.This method has good standard curve linearity,high sensitivity,good precision,accuracy and anti-interference ability,and is easy to be used and quickly to be analyzed of the test results,which is suitable for widely application in determining serum iodine.
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To establish appropriate conditions for a disinfectant efficacy test at subzero temperatures, this study examined mixtures of frozen foot-and-mouth disease virus or avian influenza virus solutions and disinfectant diluents at −5℃ and monitored temperature and freezing status of an anti-freezing diluent (AFD, 15% ethanol + 30% propylene glycol + 55% distilled water) over time at various subzero temperatures. Viral solutions and disinfectant diluents froze before the mixtures reached −5℃, whereas the AFD was not frozen at −30℃. The times taken for the AFD to reach −10, −20, −30, and −40℃ from room temperature were 36, 39, 45, and 48 min, respectively.
Subject(s)
Animals , Ethanol , Foot-and-Mouth Disease Virus , Freezing , Influenza in Birds , Propylene GlycolABSTRACT
O objetivo do estudo foi comparar o efeito de três diluidores comerciais (Tryladil®, Botu-Bov® e OptiXcell®) na qualidade do espermatozoide bovino após o processo de criogenia. Para tal, foram utilizados oito touros da raça Nelore (2 ejaculados/touro). As amostras de sêmen fresco, diluído e pós-descongelamento foram avaliadas, comparando os parâmetros de motilidade total, vigor, funcionalidade da membrana (HOST) e integridade da membrana (eosina). Os dados foram expressos em média e desvio padrão. As variáveis foram submetidas às análises de ANOVA e Tukey ou teste de Friedman e Dunn's, dependendo da normalidade (p< 0,05). Os achados mostram que no momento da diluição não houve diferença (pË0,005) entre os diluidores comerciais nos parâmetros avaliados (exceto integridade da membrana plasmática). No entanto, no momento do pós-descongelamento os espermatozoides criopreservados utilizando-se o diluidor Tryladil® apresentaram maiores valores (pË0,005) referentes a integridade e funcionalidade da membrana plasmática comparado aos diluídos em Botu-Bov® e OptIXcell®. Os parâmetros relacionados a cinética espermática (motilidade e vigor) não se diferiram (pË0,005) entre os diluidores comerciais utilizados. Em conclusão, no momento pós-descongelamento o diluidor Tryladil® apresentou os melhores resultados nos parâmetros de integridade e funcionalidade da membrana plasmática. Sendo assim, recomenda-se o diluidor Tryladil® para criopreservação de sêmen de bovinos da raça Nelore.
The main of the study was to compare the quality of frozen bull semen processed with three different commercially extenders (Tryladil®, Botu-Bov® and OptiXcell®). For this, eight Nelore bulls (two ejaculate per bull). Sperm samples were analyzed fresh, diluted and frozen-thawed. The parameters analyzed were total motility, sperm vigor, functional integrity of sperm plasma membrane (HOST) and plasma membrane integrity (eosin). Date were expressed as mean and standard deviation. The variables were subjected to ANOVA (Tukey test) or Friedman (Dunn's test) test according to normality (p< 0,05). The results indicate that there was no difference (pË0,005) among all treatments in the parameters evaluated (except plasma membrane integrity) at dilution moment. However, Tryladil extender promoted an increase (pË0,005) in functional and integrity of sperm plasma membrane compared with others extenders at the post-thawing analyze. After thawing, there was no difference (pË0,005) among all treatments in the kinetic parameters. In conclusion, the Tryladil® extender promoted an increase in functional and integrity of frozen-thawed sperm plasma membrane. Therefore, the Tryladil® extender is recommended to be use as an extender for Nelore bull sperm cryopreservation.
Subject(s)
Cattle , Semen , CattleABSTRACT
Objective To establish a method for determination of iodine in urine with no need for base urine match by inductively coupled plasma mass spectrometry (ICP-MS).Methods The diluent which contains 2.5 g/L N2H4·2HCl-1.0 g/L NH4C1-0.50％HC1-2.0％C2H5OH were used to eliminate the matrix interference in determination of iodine in urine by ICP-MS.The standard solutions and the urine samples were all diluted in a ratio of 19:1 (diluents:sample) before testing.The methodological evaluation of this new method was done through standard curve linearity,sample detection limit,precision and accuracy in determining urinary iodine.And the determine results were compared with the current urinary iodine arsenic cerium catalytic spectrophotometry standard method (WS/T 107.1-2016,hereinafter referred to as the standard method).Results The linear range of the calibration curve was 0-1 000 μ.g/L and the linear correlative coefficient was 0.999 9.The detection limit for urinary iodine was 0.4 μg/L (0.25 ml of urine was tested).Precision:The average coefficient of variation (CV)was 0.8％ with a range of 0.2％-1.7％ (n =6) when measuring 33 urine samples with iodine concentration of 26.5-854.4 μμg/L.Accuracy:The iodine standard of 40-400 μg/L was added for recovery test.The average recovery was 99.6％ with a range of 94.3％-103.4％ when measuring 24 urine samples with iodine concentration of 26.5-858.3 μg/L.The test results of 8 urinary iodine national standard materials with iodine concentration ranged from 64.5 to 883.0 μg/L were all within the given value range and the relative deviations were all below 3.0％ (n =12).No significant difference was found between the results of the 51 urine samples determined by the standard method (WS/T 107.1-2016) and the new method (t =0.836,P ＞ 0.05).Conclusions The new method to determine iodine in urine with no need for base urine match is successful established.This method has wide linear range,high sensitivity,good precision,accuracy and anti-interference ability,and is easy to be used and quickly to be analyzed of the test results,and is suitable for widely application in determining urinary iodine.
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Starches from four new sweet potato genotypes were evaluate for use as tablet diluents, binders and disintegrants; using a commercially available maize starch as reference. The pre-formulation studies established low pH (5.1 - 5.9) and moisture content (10.0 - 13.1%), but high bulk density (0.50 - 0.58), tapped density (0.75 - 0.82) and true density (1.15 - 1.18) for the sweet potato starches. Hardness and friability of tablets formulated with sweet potato starches as binder were significantly better (p = 0.001) than similar compacts containing maize starch. The sweet potato starches also caused significantly faster tablet disintegration and release of paracetamol (p = 0.005). The results established the sweet potato starches as stronger pharmaceutical diluents, binders and disintegrants, compared to the commercially available maize starch.
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Objective To investigate the influence of different diluents(physiologic saline, dis-tilled water and human inactivated serum) on measurement of 15 items of biochemical parameters.Methods Fifteen items of biochemical parameters [alanine transaminase(ALT), aspartic transaminase (AST), alkaline phosphatase( ALP), gamma glutamyl transpeptidase ( GGT), creatine kinase ( CK),lactate dehydrogenase(LD), hydroxybutyrate dehydrogenase(HBDH), total bilirubin(TBIL), direct bilirubin(DBIL), total bile acid(TBA), ereatinine(Cr), uric acid(UA), eholesterol(CHO), glucose (GLU), and blood urea nitrogen(BUN)] were chosen. For each parameter, 45 serum samples with different eoncentrations of the parameter were collected. After diluted with different diluents(physio-logic saline, distilled water or human inactivated serum), the serum samples were detected by applying the fully automated biochemical analyzer. The mean value was calculated and statistical analysis was performed. Results There were some differences of detection results when the specimens were diluted with different diluents. ALT, AST, GGT, DBIL, and HBDH serum samples could be diluted by 10 times with physiologic saline, distilled water or human inactivated serums ALP and TBA serum sam-ples could only be diluted with inactivated serum, otherwise its result would be lower; GLU, TBIL samples could be diluted with distilled water and inactivated serums for BUN, CR, UA, CK, LDH,and CHO samples, physiologic saline or human inactivated serum might be optimal; if distilled water was chosen, the results of other parameters tented to decline except UA. It was BUN was improper to dilute the BUN samples with distilled water. In addition, there was no significant difference between the items diluted by 5 times and 10 times with physiologic saline. All the 15 items could be diluted with inactivated serum. Conclusion The inactivated serum should be the first choice of diluents to e-nure the accurate results of biochemical parameters. If the prepared inactivated serum is absent, we may choose other diluents according to the above-mentioned results.