This investigation was intended to find out the estrogenic effect of chlortetracycline (CTC) on vitellogenin induction in adult male Japanese medaka (Oryzias latipes). Vitellogenin (Vtg) produced in male fish has been used to as one of a biomarker of endocrine disrupters. The positive control was $17{\beta}-estradiol$ (E2) that induced Vtg in male fish. As a result, male and female fish were exposed to 0.1, 1, 10 and 100 ppm of CTC. Western blot results showed approximately 205 kDa, that is similar to myosin at high molecular weight range Sigma maker. Vtg band was showed fainted to 10 and 100ppm for chlortetracycline. Vtg concentration of CTC was qunatified by total protein quantification and ELISA. Exposure of the male fish to CTC of 0.1, 1, 10 and 100 ppm produced Vtg concentrations of 0.24, 0.12, 7.61 and 40.02%o, respectively, that value was elevated than control male fish (0.14%o). CTC exerted as a Vtg inducer in male fish from 10 ppm, but it was a reducer in female fish from 0.1 ppm level. The results say that vitellogenin induction patterns alter in male medaka treated with CTC, and that CTC may caused endocrine disruption in fish.
The effects on CTC (T1) and CTC, Sulfathiazole and Penicillin Combination (T2) medication in feed through one life cycle of pigs, namely, weaning, mating, farrowing, lactation, growing, finishing and slaughter, were tested under local condition. In sow phase, productivity and the number of microflora in urine before and after medication of CTC were studied and average daily gain and feed conversion rate were checked during growing and finishing period. All pigs reached at 155 days old were slaughtered for pathological examination. 1. Litter size, farrowing rate and survival rate at birth were improved by CTC medication from weaning to 21th day after mating and mortality of piglet at weaning, 25 days after farrowing, was reduced in the CTC medication group, but no siginificant. 2. The number of microflora in the sow urine was changed with the medication at 200ppm of CTC in feed. In particular, the number of E coli, Samonella and Staphylococci were reduced by CTC medication. 3. The average daily gain and feed conversion rate of grower and finisher pigs was improved significantly in both treated groups, most in the high level CTC (T1) medicated group and was lowest in the control group. 4. The number of infected lungs was reduced not significant by both treatments (as % pneumonic lesions Co 66.7%, T1 47.1%, T2 31.4%) and the severity of lung lesions was significantly reduced by both high level of CTC and CTC combination medication in feed. 5. Although there were no statistical differences in atrophic rhinitis based on turbinate scores among the 3 groups, the number of mild and moderate (Grade 2 and 3) infections was higher in the control group (9/36) than in the treated groups (T1 2/34 & T2 4/35).
Rare earth(RE) is a common name indicating 17 of specific elements including 15 of lanthanides, scandium (Sc), and yttrium(Y). This study was conducted to compare the effect of RE to growth stimulating antibiotic products in broiler diet. One of the products commonly used in broiler feeding, Chlorotetracycline(CTC) was used at a level of 200ppm for the antibiotic treatment and replaced by 100ppm RE for the RE treatment. Dietary addition of RE and CTC showed similar effects in improving broiler growth and feed conversion each other, and those were significantly better when compared to those of the control group(P<0.05). Abdominal fats of chicks fed RE were significantly lower than others(P<0.05). Intestinal E. coli and total microbial numbers were reduced by RE addition(P<0.05), but the number of Lactobacillus was not affected. The number of Salmonella was also decreased but it was not significant.
Chlortetracycline (CTC) is one of the most important compounds in antibiotic production, and its distribution has been widely investigated due to health and ecological concerns. This study presents systematic approach to optimize the high-performance liquid chromatography-tandem mass spectrometry for analyzing CTC in a multiple reaction monitoring mode ($479{\rightarrow}462m/z$). One-factor-at-a-time (OFAT) test with response surface analysis (RSA) was used as optimization strategy. In OFAT tests, the fragmentor voltage, collision energy, and ratio of acetonitrile in the mobile phase were selected as major factors for RSA. The experimental conditions were determined using a composite in cube design (CCD) to maximize the peak area. As a result, the partial cubic model precisely predicted the peak area response with high statistical significance. In the model, the (solvent composition) and (collision $energy^2$) terms were statistically significant at the 0.1 ${\alpha}$-level, while the two-way interactions of the independent variables were negligible. By analyzing the model equation, the optimum conditions were derived as 114.9 V, 15.7 eV, and 70.9% for the fragmentor voltage, collision energy, and solvent composition, respectively. The RSA, coupled with the CCD, offered a comprehensive understanding of the peak area that responds to changes in experimental conditions.
Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.
Beef, pork and chicken on the market were analyzed for determination of tetracyclines residue and decomposition of tetracyclines by heating were studied. The content of oxytetracycline was trace in chicken A, 0.09 mg/kg in C and trace in beef C, pork A and B, but in the other samples was not detected oxytetracycline. Chlortetracycline residue was 0.14, 0.02 and 0.01 mg/kg in chicken A, C and beef B respectively. In HPLC analysis, two peaks of 8.1 and 9.0 min of retention time were found from beef and pork and expected to be component of meat, and because of same retention time, the one of 9.0 min interrupted determination of tetracycline. But those were not found in chicken, therefore the residue was 0.01 mg/kg in sample A. The residue were degradated rapidly by heating of roast, but slightly by that of boiling.
BACKGROUND: Tetracyclines (TCs) are mainly regulated as parent compounds by bioactivity-based screening methods in food. Especially with respect to antimicrobial residues, their metabolites/epimers are also highly concerning chemicals and traditionally applied microbial detection methods are needed to improve with validation for regulatory control. METHODS AND RESULTS: Detection capability and biological activity of tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC) and their epimers; anhydrotetracycline (ATC), epianhydrotetracycline (EATC), epitetracycline (ETC), 4-epi-chlortetracycline (ECTC), 4-epianydrochlotetra-cycline (EACTC), 4-epioxychlortetracycline (EOTC), were measured by microbial growth inhibition screening method of Korea Food Code. CONCLUSION(S): Limited detection capabilities were found, B. megarerium and B. subtilis showed for TC and CTC, and B. subtilis for OTC. Biological potency of each epimer was also presented against various microorganisms, at the level from 50% to 96%, comparing with parent TCs. It is recommended that more advanced microbial screening methods with validation are needed, and biologically active epimers are to be considered as marker residues for MRL setting of regulatory control purpose.
Release of veterinary antibiotics (VSs) to agricultural environment through application of animal manure and/or animal manure-based composts to soils is of concern. The current study was conducted to examine decline of VAs during composting the chicken manure. For this, antibiotics free chicken manure (20 kg) and sawdust (10 kg) were added to the bench-scale composting apparatus and then the mixed material was spiked simultaneously with three VAs (chlortetracycline, CTC; sulfamethazine, SMZ; tylosin, TYL) at two different levels (10 and $20mg\;kg^{-1}$). Then the decline of VAs was determined using Charm II system during 53 composting period. For comparison, composting only chicken manure was included at VAs concentration of $10mg\;kg^{-1}$. During composting, the concentration of all three different VAs declined below the prospective guideline values ($0.8mg\;kg^{-1}$ for CTC, $0.2mg\;kg^{-1}$ for SMZ, and $1.0mg\;kg^{-1}$ for TYL) except CTC at $20mg\;kg^{-1}$ spiking when the chicken manure was composted together with sawdust. Interestingly, CTC at $10mg\;kg^{-1}$ spiking appeared to be declined under the guideline value without sawdust while SMZ was resistant to be declined without sawdust. Unlike CTC and SMZ, TYL showed immediate decline right after spiking TYL to composting materials regardless the spiking concentration and existence of sawdust. Appropriate composting procedure of chicken manure was able to decline the residual VAs in the manure below the prospective guideline value and the importance of organic substances on this decline was perceived.
Objective: Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. Methods: we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. Results: The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (${\Delta}$) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (${\Delta}$) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. Conclusion: These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.
To investigate the effects of reactive oxygen species (ROS) on capacitation, acrosome reaction in human spermatozoa. Human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O$$_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5 %) condition. Chlortetracycline (CTC) staining was conducted to assess capacitation and acrosome reaction. Analysis of lipid peroxidation was done by spectrophotometric determination of malondialdehyde (MDA) production in spermatozoa. $H_2O$$_2$, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within 1 h of incubation. There was no significant difference in capacitation between low- and high $O_2$ groups. In the presence of low concentration of $H_2O$$_2$, lipid peroxidation decreased significantly. However, under the high concentration of $H_2O$$_2$, lipid peroxidation significantly increased at the end of incubation compared to control. In the presence of high concentration of lymphocytes, lipid peroxidation significantly increased compared to control at 1hr of incubation. There was no significant difference in lipid peroxidation according to $O_2$ concentration examined. Acrosome reaction (AR) was evaluated by CTC staining after the progesterone challenge. In all ROS groups, AR increased compared to control. The X(100 $\mu$M) - XO (100mIU) system was the most potent to induce AR. Taken together, it suggested positive control of AR by ROS and the positive relationship between the lipid peroxidation and AR. The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation.
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