This work is supported by the Basic Scientific Research Business Project of Chongqing Academy of Traditional Chinese Medicine (Project Number: jbky20200013), the Performance Incentive Guidance Project of Chongqing Scientific Research Institutions (Project Number: cstc2021jxjl 130025), and the Chongqing Municipal Health Commission Key Discipline Construction Project of Chinese Materia Medica Processing.

All animal-related experiments were conducted with approval from the Experiment Ethics Committee of the Chongqing Institute of TCM (laboratory animal ethics review certificate number: ZJS2022-03).
1. Determination of the bioactive components
NOTE: The species used in this research was Epimedium sagittatum, and the samples were collected in Fengdu County, Chongqing. The sample was identified as a dry above-ground part of E. sagittatum (Sieb. et Zucc.) Maxim. by researchers of The Institute of Biological Medicine, Chongqing Institute of Traditional Chinese Medicine.
<ol>
	<li>Prepare the control product solution by accurately weighing the appropriate amount of each reference substance, namely, icariin, epimedin A (EA), epimedin B (EB), epimedin C (EC), and baohuoside I (BI), using an electronic analytical balance, and dissolve in methanol. Using these, prepare a mixed reference stock solution containing 381.61 &#181;g/mL icariin, 124.14 &#181;g/mL EA, 110.24 &#181;g/mL EB, 1091.75 &#181;g/mL EC, and 184.98 &#181;g/mL BI.</li>
	<li>Prepare the test product solution by crushing EF through a No. 3 sieve. Place approximately 0.2 g (using an electronic analytical balance) of crushed EF into a stoppered Erlenmeyer flask, add 20 mL of dilute ethanol, and then ultrasonicate at 400 W power and 50 kHz frequency for 1 h. Shake well, and pass through a 0.22 &#181;m membrane filter to obtain the test solution.</li>
	<li>Perform the chromatography as follows. Use high-performance liquid chromatography (HPLC) with a C18 column with dimensions of 4.6 mm x 250 mm and an inner diameter of 5 &#181;m. Use acetonitrile as mobile phase A and ultrapure water as mobile phase B. Use the following gradient elution parameters: 0-30 min, 24% A to 26% A; 30-31 min, 26% A to 45% A; 31-45 min, 45% A to 47% A. Use a detection wavelength of 220 nm (for the detector used, see Table of Materials). Keep the column temperature at 30 &#176;C and the current velocity at 1.0 mL/min, and use a sample size of 10 &#181;L.</li>
	<li>To investigate the linear relationship, use the mixed reference solution as in step 1.1 diluted 2 times, 4 times, 8 times, 16 times, and 32 times, for icariin, EA, EB, EC, and BI, respectively. Use acetonitrile as mobile phase A and ultrapure water as mobile phase B.</li>
	<li>Use the following gradient elution parameters: 0-30 min, 24% A to 26% A; 30-31 min, 26% A to 45% A; 31-45 min, 45% A to 47% A. Use a detection wavelength of 220 nm (for the detector used, see Table of Materials). Keep the column temperature at 30 &#176;C and the current velocity at 1.0 mL/min and use a sample size of 10 &#181;L. Finally, record the peak areas. Plot the linear regression with the reference concentration (x-axis, &#181;g/mL) as the abscissa and the peak area (y-axis) as the ordinate using professional software (see Table of Materials).</li>
	<li>Perform the precision test by measuring the mixed control solution six consecutive times by HPLC using the chromatographic conditions shown in step 1.3. Record the detection time and peak areas of each chemical composition, and calculate the relative standard deviations (RSD) of the peak areas to assess the precision (reproducibility) using the formula below: 
	RSD% = standard deviation (SD)/arithmetic mean of calculated results (X) x 100 %</li>
	<li>To perform the reproducibility test, accurately weigh the EF powder, and prepare six parts of the test product solution in parallel according to the method in step 1.2. Subject the prepared solutions to HPLC under the chromatographic conditions presented in step 1.3. Record the retention times and peak areas of each chemical composition and calculate the amounts of each compound from a standard curve (peak areas versus concentrations). Calculate the RSD% as above.</li>
	<li>To perform the stability test, store the test solutions at room temperature, and measure their contents by the HPLC method described in step 1.3 at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation to assess the stability. Record the retention times and peak areas of each chemical composition and calculate the RSD% of the peak areas as above.</li>
	<li>To perform the sample recovery test, weigh 0.2 g of EF powder into a stoppered Erlenmeyer flask for six replicates. Add an appropriate amount of the reference solution (the amount of reference substance added to the sample is equivalent to 100% of the known content of the sample) and prepare the test solution according to the method presented in step 1.2.</li>
	<li>Inject the samples into the chromatograph and analyze according to the chromatographic conditions in step 1.3. Record the peak areas, and calculate the average recovery and RSD% values as below: 
	Spiked sample recovery rate = (spiked sample content &#8722; sample content)/sample amount x 100%</li>
</ol>
2. Optimization of the EF mutton-oil processing technology using the Box-Behnken design-response surface methodology
<ol>
	<li>Select the key parameters in EF processing, such as the amount of mutton oil (A; 15%-35%), the mutton oil temperature (B; 50-120 &#176;C), and the frying temperature (C; 80-300 &#176;C), as influential factors. Use the comprehensive scores of icariin, EA, EB, EC, and BI content as the evaluation indexes. The percentage of mutton oil here is the mass percentage.</li>
	<li>Use the response surface analysis software (see Table of Materials) to design the Box-Behnken response surface experiments, explore the quadratic response surface, and construct a second-order polynomial model. Select the new Box-Behnken Design, and set the Numeric Factors option to 3; set factors A, B, and C. Click on Continue. Set the Responses option to 1 (which was the comprehensive score). Click on Continue to complete the design. A total of 17 experiments were planned (see Table 1). 
	NOTE: For the independent and dependent variables, along with their low, middle, and high levels, see Table 2.</li>
	<li>Process the EF according to the specific parameters in Table 1; for example, for order number 1, weigh refined mutton oil as 15% v/v, and then heat to 50 &#176;C to melt it. Add the crude EF to the melted mutton, stir-fry over a gentle fire (190 &#176;C) until it is evenly shiny, and then remove and cool. Performed 17 experimental operations. A total of 17 groups of EF-processed products were obtained in this work. 
	NOTE: Mutton oil is solid at room temperature (25 &#176;C) and melts into liquid when heated. Mutton oil in a liquid state can be used as the excipient.</li>
	<li>Prepare the test solutions of the processed products according to the method described in step 1.2. Then, analyze them using HPLC according to the chromatographic conditions described in step 1.3. Record the retention times and peak areas of each chemical composition, and calculate the contents of the icariin, EA, EB, EC, and BI in each test solution against an external standard curve. Use the comprehensive score calculation formula below to calculate the comprehensive scores of the 17 experimental groups: 
	Comprehensive score = Z/Zmax &#215; 0.5 + BI/BImax &#215; 0.5 
	where Z is the sum of the icariin, EA, EB, and EC contents; Zmax is the maximum value of the sum of the icariin, EA, EB, and EC contents in the 17 experimental groups; BI is the BI content; and BImax is the maximum value of the BI content in the 17 experimental groups.</li>
	<li>Import the comprehensive scoring results for the 17 groups of experiments into the data analysis software (see Table of Materials) to analyze the experimental data. Under the evaluation items, select the quadratic process order option and polynomial model type option.</li>
</ol>
3. Testing the effect of processing on zebrafish embryonic development
<ol>
	<li>Sample preparation
	<ol>
		<li>Crush the crude and processed EF through a No. 3 sieve (see Table of Materials). To 100 g of each EF sample, add 1,000 mL of ultrapure water. Soak the EF for 0.5 h, boil the water twice for 30 min each, and then filter with filter paper.</li>
		<li>Combine the filtrates and concentrate the sample by heating. Add ultrapure water to a final volume of 100 mL to obtain the processed EF (PEF, 1 g/mL) and the crude EF (CEF,1 g/mL) stock solutions. Measure the amount of raw drug in each stock solution.</li>
		<li>Place aliquots of 1 mL, 1.5 mL, 2.5 mL, 5 mL, and 7.5 mL stock solutions in 10 mL volumetric flasks, and then add ultrapure water to volume to prepare the test solutions with concentrations of 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 500 mg/mL, and 750 mg/mL for the zebrafish embryotoxicity study. 
		NOTE: The concentrations of the test solutions were prepared by referring to the relevant literature20,21 and by performing preliminary experiments to give the 10-fold concentration gradient used in normal toxicology. CEF was an unprocessed sample, and PEF was a sample prepared with the best processing technology described in section 2.</li>
	</ol>
	</li>
	<li>Zebrafish husbandry and embryo treatment21
	<ol>
		<li>Adapt wild-type zebrafish (see Table of Materials) at a controlled temperature for 2 days, keep them in a flow-through aquarium at pH 7.0-7.4 and feed them twice daily. 
		NOTE: The inhibition of melanin formation in zebrafish was achieved by adding 1-phenyl-2-thiourea in a concentration of 0.003% (mass/volume) to the culture medium, which kept their bodies transparent for morphological observation.</li>
		<li>Select adult fertile wild-type zebrafish in the evening and separate them by using baffles in mating boxes. Remove the baffles the following morning, and allow the fish to spawn for 30 min. Collected the fertilized eggs with a dropper every 15 min. In total, 520 healthy wild-type embryos were collected. Keep the zebrafish embryos in an incubator at 28.5 &#176;C for 24 h.</li>
		<li>Randomly assign the healthy embryos at 24 h post fertilization (hpf) to 13 groups, and along with one control group, separately soak in 10 mL of each of the following solutions in a culture dish: PEF: 100 &#181;g/mL, 150 &#181;g/mL, 200 &#181;g/mL, 250 &#181;g/mL, 500 &#181;g/mL, 750 &#181;g/mL; CEF: 100 &#181;g/mL, 150 &#181;g/mL, 200 &#181;g/mL, 250 &#181;g/mL, 500 &#181;g/mL, 750 &#181;g/mL . Treat the blank control group with the medium as a solution. Each group contained 40 embryos in this study. 
		NOTE: The medium composition is 0.15 M NaCl, 5 mM KCl, 0.25 mM Na2HPO4, 0.45 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4 mM NaHCO3.</li>
		<li>Culture the zebrafish in a constant temperature incubator for up to 120 hpf. Count the number of dead larvae every day, observe the main organ morphology of the larvae in each experimental group under a stereomicroscope (scale bar = 500 &#181;m, see Table of Materials), and calculate the half-death concentration (LC50) of zebrafish at 72 hpf by using data analysis software (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+modern+pharmacological+effects+of+traditional+Chinese+medicine+for+tonifying+kidney+yang.">Study on modern pharmacological effects of traditional Chinese medicine for tonifying kidney yang.</a> Journal of Hubei University of Chinese Medicine. 13 (04), 63-66 (2011).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+chemical+constituents+and+pharmacological+studies+of+Epimedium.">Advances in the chemical constituents and pharmacological studies of Epimedium.</a> Asia-Pacific Traditional Medicine. 15 (6), 190-194 (2019).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+icariin+on+ER&#946;+gene+expression+and+serum+estradiol+level+in+ovariectomized+rats.">Effects of icariin on ER&#946; gene expression and serum estradiol level in ovariectomized rats.</a> Hunan Journal of Traditional Chinese Medicine. 32 (1), 150-152 (2016).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epimedin+A+on+osteoclasts+and+osteoporotic+male+mice.">Effects of epimedin A on osteoclasts and osteoporotic male mice.</a> Chinese Journal of Veterinary Science. 41 (07), 1359-1364 (2021).</li><li>Liu, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+icariin+and+epimedium+C+on+microstructure+of+bone+tissue+in+glucocorticoid+osteoporosis+model+mice+based+on+Micro-CT+technique.">Effects of icariin and epimedium C on microstructure of bone tissue in glucocorticoid osteoporosis model mice based on Micro-CT technique.</a> Drug Evaluation Research. 43 (09), 1733-1739 (2020).</li><li>Zhan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antiosteoporotic+activity+for+micro+amount+icariin+and+epimedin+B+based+on+the+osteoporosis+model+using+zebrafish.">Evaluation of antiosteoporotic activity for micro amount icariin and epimedin B based on the osteoporosis model using zebrafish.</a> Chinese Pharmaceutical Journal. (24), 30-35 (2014).</li><li>Zhan, Y., Wei, Y. -. J., Sun, E., Xu, F. -. J., Jia, X. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+zebrafish+model+combined+with+hyphenated+chromatographic+techniques+for+evaluation+anti-osteoporosis+activity+of+epimendin+A+and+its+metabolite+baohuoside+I.">Two-dimensional zebrafish model combined with hyphenated chromatographic techniques for evaluation anti-osteoporosis activity of epimendin A and its metabolite baohuoside I.</a> Acta Pharmaceutica Sinica. 49 (06), 932-937 (2014).</li></ol>

The authors declare no conflicts of interest.

Independent variables and the determination of their levels 
The EF processing technology is only described in the 2020 edition of the Chinese Pharmacopoeia and the local Chinese medicine processing specifications published by 26 provinces, municipalities, and autonomous regions across the country1. The description involves the following steps: taking mutton oil and heating it to melt, adding EF shreds, stir-frying with a slow fire until it is uniform and shiny, taking it out, and letting it cool. Additionally, 20 kg (abbreviated as 20%) of mutton oil (refined) is used for every 100 kg of Epimedium. However, the parameters of the processing process of EF are not specified. Among the independent variables in this experiment, three key factors could be quantified in the production process: the mutton oil dosage, the mutton oil temperature, and the frying temperature. The value range should be set according to the above description. From the results of the preliminary test, it can be seen that when the amount of mutton oil is 15%, the EF leaves can be evenly coated with mutton oil. When the dosage exceeds 35%, there is too much mutton oil. Finally, the range of the mutton oil amount should be 15%-35%. When the temperature reaches 50 &#176;C, mutton oil melts. When the temperature reaches &#62;120 &#176;C, the mutton oil starts to smoke, and the temperature is too high. Therefore, the temperature range of the mutton oil should be 50 &#176;C-120 &#176;C. The Chinese Pharmacopoeia 2020 edition stipulates that EF should be stir-fried with a slow fire. The slow fire should not exceed 200 &#176;C, and the frying temperature should range from 80 &#176;C-300 &#176;C.
Comprehensive scoring 
During the processing of Epimedium, the glycosidic bonds are broken, and the glycosidic components are transformed into lower glycosidic components. The determination of the content of EF-processed products in the 2020 edition of the Chinese Pharmacopoeia is based on the determination of the total content of icariin, EA, EB, and EC in the original medicinal materials, and a monoglycoside component, BI, is listed separately as an indicator. In this experiment, the total weight of the icariin, EA, EB, and EC in the processed EF was 50%, the weight of the BI was 50%, and the comprehensive score was set based on these values.
Response surface methodology (RSM) is a statistical technique for identifying optimal process parameters and solving multivariate problems. In this technique, a reasonable experimental design is used to obtain certain data through experiments, and a multivariate quadratic regression equation is used to derive a functional relationship between the factors and responses22. Uniform design and orthogonal design process optimization are commonly used, but their test accuracy is not high, and the mathematical model is not very predictable. The mathematical model underlying RSM is highly predictable. RSM requires fewer experiments and shorter cycles, which not only can eliminate the problems associated with traditional mathematical statistics but can also clarify the relationships between factors and responses23. RSM conceptualizes the system&#39;s response as a function of one or more factors and uses graphical techniques to display this functional relationship to help the user to select the optimal conditions in the experimental design by intuitive visual observation. These advantages have led to the wide use of this method in the chemical industry24, biological engineering, the food industry25, the pharmaceutical industry, and TCM preparations.
Although RSM can identify the functional relationship between responses (indexes to be investigated) and factors (independent variables), not all experiments are suitable for response surface optimization because there is not always a strong functional relationship between the response and factors. RSM often obtains continuous function relationships, which require all the factors to be continuous variables. Nevertheless, not all factors to be investigated are continuous variables or have significant effects on the response values at the beginning of the experimental design. To reduce the number of experiments and improve the accuracy of the response surface modeling, screening is necessary to select the significant factors and determine their levels through factorial design, uniform design, or orthogonal design before conducting response surface design methodology. The biggest advantage of response surface methodology is that once the model is correctly established, the response value under any combination of conditions can be predicted, and the functional relationship can be seen more intuitively and visually through the 3D response surface. This visualization is of great help to researchers in finding the optimal processing conditions26.
This study used the Box-Behnken design principle of RSM to design 17 combined experiments using the comprehensive chemical content score of EF as the response value. Finally, the best process optimization results were obtained by regression analysis. The processing technology was optimized as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, frying it with a gentle fire (189 &#176;C &#177; 10 &#176;C) until it is evenly shiny, and then removing and cooling it. For every 100 kg of EF, 15 kg of mutton oil (refined oil) should be used. Our results showed that the EF process was stable, reliable, and repeatable. Additionally, factor interactions were analyzed, and the interaction between the mutton oil amount and frying temperature, but not the interactions between other factors, was significant. This study demonstrated that the response surface design, as a method to analyze the interactions between factors and the relationships between factors and their response surface values, enabled the optimization of the processing conditions in a short period with a minimal number of experiments. The selected factors in this study were the key factors identified in the single-factor screening experiment, and their levels were determined in a preliminary experiment. The test samples conformed to the characteristics of the response surface method, so the study was able to use the response surface methodology to establish a predictive model. The experimental results can provide a reference for improving the quality and uniformity of the processed EF.
Zebrafish embryos are used as model organisms in the field of developmental genetics because they are transparent, develop in vitro, and are easy to observe27. Commonly used zebrafish toxicity indicators in developmental toxicity studies include embryonic mortality, embryonic malformation rate, yolk-sac edema, pigment formation, egg condensation, tail extension, head morphology, and body-segment formation, among others28. Compared with mammalian toxicity evaluation techniques, the specificity of zebrafish embryos for compound toxicity detection is 70%-80%, and the sensitivity exceeds 80%18. Ton et al.29 found that the accuracy of evaluating the developmental toxicity of non-teratogenic compounds with zebrafish embryos was 75%. Teratogenic compounds were evaluated with 100% accuracy here. Although TCM has the characteristics of complex components and unclear target organs of toxicity, zebrafish embryos can still be used as an experimental animal model for the accurate and rapid evaluation of developmental toxicity. He et al.30 found that emodin affected the survival and hatching rates of zebrafish embryos, causing trunk bending and yolk-sac edema. Chen et al.31 found that muscone caused zebrafish embryo pericardial edema, spinal curvature, and yolk-sac edema. He et al.32 found that Arnebiae Radix had lethal effects on zebrafish at all developmental stages, and 1.0 mg/L Arnebiae Radix inhibited embryonic development, resulting in a reduced number of somites, tail deformities, body bending, and reduced melanin in zebrafish embryos.
To investigate the effects of crude and processed EF on zebrafish embryo development, a zebrafish embryo developmental toxicity experiment was conducted in this study. The data showed that the LC50 values were 151.3 &#181;g/mL for the crude group (S) and 219.8 &#181;g/mL for the processed group (P). Observing the zebrafish bodies in each experimental group through a microscope showed an obvious degree of zebrafish teratogenicity in the crude group. Most of the fish showed varying degrees of teratogenicity, including spinal deformity, body curvature deformity, pericardial edema, swim bladder incompleteness, or liver deformation, and these observations were rare in the processed group. These experiments showed that the toxicity of EF was significantly reduced after processing, suggesting that processing could reduce drug toxicity in humans. The experimental results provide a reference for improving the clinical medication safety of mutton oil-processed EF.
Traditional Chinese medicine suggests that the function of the kidney is closely related to the growth, development, and reproduction of the human body33. The ancient books of traditional Chinese medicine record that the kidney is the bone marrow of the body. The kidney stores essence, and the marrow resides in the bone cavity to nourish the bone. When the kidney essence is deficient, the bone marrow is reduced34. The traditional Chinese medicine of tonifying the kidney yang can treat lumbar debility, osteoporosis, impotence, premature ejaculation, and uterine cold infertility35. EF is one of the representative medicinal materials for tonifying the kidney yang. Modern pharmacological studies have shown that EF has obvious effects on the skeletal system, immune system, reproductive system, cardiovascular system, and nervous system, as well as having anti-tumor effects36. In terms of activity on the skeletal system, icariin37 can improve the level of serum E2 in ovariectomized rats and upregulate the expression of ER&#946; mRNA in the bone tissue of ovariectomized rats. The synthesis of ER&#946; is increased, thereby improving the biological effect of ER, weakening the bone resorption activity of osteoclasts, and enhancing the bone formation of osteoblasts. The changes in bone resorption are greater than the negative balance of bone metabolism. Epimedin A can improve the bone microstructure and serum bone turnover markers in osteoporosis model mice by inhibiting osteoclast formation, differentiation, and bone resorption and playing a role in bone protection38. Epimedin C has obvious anti-osteoporosis activity, mainly in terms of increasing the bone mass and improving the trabecular microstructure to ultimately increase bone strength39. Other studies have shown that epimedin B40 and baohuoside I41 have anti-osteoporosis activity.

Epimedii folium (EF) is the dried leaves of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., or Epimedium koreanum Nakai. EF can be used to treat osteoporosis, menopausal syndrome, breast lumps, hypertension, coronary heart disease, and other diseases1. As a traditional Chinese medicine (TCM), EF has a history in medicine and food of more than 2,000 years. Due to its low price and remarkable effect of tonifying the kidneys, it is widely used in medicines and health foods. EF is processed by stir-frying it with mutton oil, a process first described in Lei Gong Processing Theory written by Lei Xiao in the Liu Song period2. The efficacies of crude EF and stir-fried EF are quite different. Crude EF mainly dispels rheumatism, whereas the stir-fried EF warms the kidneys to reinforce yang3. At present, EF is widely used as a raw material in drugs and health foods; there are 399 listed Chinese patent medicines, nine imported health foods, and 455 domestic health foods with EF as a raw material4. This medicinal material has great application prospects. However, in recent years, there have been increasing reports of adverse reactions and human liver injury caused by health foods and Chinese patent medicines using EF as a raw material, and related toxicity studies5,6,7 have reported that EF as a raw material has potential safety risks.
Chinese medicinal processing refers to pharmaceutical techniques that can effectively reduce or eliminate the toxicity and improve the safety of TCMs. The traditional processing method of EF is stir-frying with mutton oil, which reduces the toxicity of EF and enhances its effect of warming the kidneys and promoting yang8. This processing method is included in the Chinese Pharmacopoeia and various processing specifications1. The process of EF is only specified as follows: for every 100 kg of EF, 20 kg amniotic oil (refined) is added, and it is mild-fired until uniform and shiny1. There are no strict EF processing method parameters in the above standards, so local processing specifications have not been unified to provide consistency. Therefore, it would be useful to conduct a systematic study of the EF process. In this paper, the Box-Behnken experimental design-response surface method was used to optimize the processing technology of EF.
The Box-Behnken experimental design is a method typically used to optimize the factors in a process. The extraction parameters can be optimized by establishing the functional relationship between multiple regression equation-fitting factors and effect values. Recently, this method has been widely used to study TCM extraction5,6,7 and processing9,10,11. Various studies have reported TCM preparation methods involving salt processing, wine processing, and stir-frying following a Box-Behnken design, such as for salt-processed Psoraleae fructus12, wine-processed Cnidii fructus13, and roasted Cinnamomi ramulus14. This method has reduced test time, high test accuracy, and is suitable for multi-factor and multi-level tests. The method is simpler than the orthogonal design test method and more comprehensive than the uniform design method15. The obtained relationships can determine the predicted value of any test point within the test range, which is a great advantage. A zebrafish model can be used to test whether EF is less toxic after processing.
In TCM toxicity studies, the zebrafish model has the dual advantages of the high throughput of cell experiments and the similarities with rodent experiments16. This model is characterized by its small size, high spawning rate, short reproduction cycle, and ease of breeding. The model can be used in large-scale synchronous experiments in cell culture plates, and the experimental drug dosage is small, the experimental cycle is short, the cost is low, and the whole experimental process is easy to observe and operate17. Zebrafish embryos are transparent and develop rapidly. Therefore, the toxicity and teratogenic effects of drugs on visceral tissues at different developmental stages can be directly observed under a microscope18. The gene homology between zebrafish and humans is as high as 85%18. The signal transduction pathway of zebrafish is similar to that of humans18. The biological structure and physiological function of zebrafish are highly similar to those of mammals18. Therefore, a zebrafish model for drug testing can provide experimental animals that are reliable and fully applicable to humans19.
In this study, we used the Box-Behnken design-response surface methodology to optimize the amount and temperature of mutton oil and the frying temperature used in the EF processing technology, with the contents of icariin, epimedin A, epimedin B, epimedin C, and baohuoside I as the evaluation indexes. The zebrafish model was used to preliminarily explore the effect of an EF water extract on zebrafish embryonic development before and after processing to evaluate the attenuation effect of processing on EF.

<table><tbody><tr><td>Acetonitrile</td><td>Fisher</td><td>197164</td><td></td></tr><tr><td>Baohuoside &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65096/65096eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; (B&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65096/65096eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; )&amp;nbsp;</td><td>Chengdu Manst Biotechnology Co., Ltd.</td><td>MUST-20042402</td><td></td></tr><tr><td>Chromatographic column</td><td>Waters Corporation</td><td>Symmetry C&lt;sub&gt;18&lt;/sub&gt;</td><td></td></tr><tr><td>Design Expert software</td><td>Stat- Ease Inc., Minneapolis, MN</td><td>Trial Version8.0.6.1</td><td></td></tr><tr><td>Detector</td><td>Waters Corporation</td><td>2998</td><td></td></tr><tr><td>Disintegrator</td><td>Hefei Rongshida Small Household Appliance Co., Ltd.</td><td>S-FS553</td><td></td></tr><tr><td>Electronic analytical balance</td><td>Mettler-Toledo International Inc.</td><td>MS205DU</td><td></td></tr><tr><td>Epimedin A (EA)</td><td>Chengdu Manst Biotechnology Co., Ltd.</td><td>MUST-21112118</td><td></td></tr><tr><td>Epimedin B (EB)</td><td>Chengdu Manst Biotechnology Co., Ltd.</td><td>MUST-20080403</td><td></td></tr><tr><td>Epimedin C (EC)</td><td>Chengdu Manst Biotechnology Co., Ltd.</td><td>MUST-20080310</td><td></td></tr><tr><td>Ethanol</td><td>Chongqing Chuandong Chemical ( Group ) Co., Ltd.</td><td>20180801</td><td></td></tr><tr><td>Graphpad software</td><td>GraphPad Software Inc., San Diego, CA, USA</td><td>6.02</td><td></td></tr><tr><td>High Performance Liquid Chromatography (HPLC)</td><td>Waters Corporation</td><td>2695</td><td></td></tr><tr><td>Icariin</td><td>Chengdu Glip Biotechnology Co., Ltd.</td><td>21091401</td><td></td></tr><tr><td>Methanol</td><td>Chongqing Chuandong Chemical (Group) Co., Ltd.</td><td>20171101</td><td></td></tr><tr><td>Microporous membrane</td><td>Tianjin Jinteng Experimental Equipment Co., Ltd.</td><td>0.22&amp;mu;m</td><td></td></tr><tr><td>Mutton oil</td><td>Kuoshan Zhiniu Fresh Food Store</td><td>20211106</td><td></td></tr><tr><td>Office Excel office software</td><td>Microsoft</td><td>Office Excel 2021</td><td></td></tr><tr><td>Pharmacopoeia sieve</td><td>Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.</td><td>R40/3</td><td></td></tr><tr><td>Pure water machine</td><td>Chongqing Andersen Environmental Protection Equipment Co., Ltd.</td><td>AT Sro 10A</td><td></td></tr><tr><td>Qualitative filter paper</td><td>Shanghai Leigu Instrument Co., Ltd.</td><td>18cm</td><td></td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss, Oberkochen, Germany</td><td>Stemi 2000</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Branson Ultrasonics (Shanghai) Co.,Ltd.</td><td>BUG25-12</td><td></td></tr><tr><td>Zebrafish</td><td>China Zebrafish Resource Center (CZRC)</td><td>The AB strain</td><td></td></tr></tbody></table>

optimization of the epimedii folium mutton-oil processing technology and testing its effect on zebrafish embryonic development

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

Methodological investigation results 
A linear relationship between the concentration of icariin, EA, EB, EC, BI, and chromatographic peak areas was observed (see Table 3). The RSD% values (n = 6) of the chromatographic peak areas of icariin, EA, EB, EC, and BI were 0.28%, 1.22%, 0.65%, 1.67%, and 1.06%, respectively, indicating that the precision of the HPLC measurements was good. The RSD% values (n = 6) of the contents of icariin, EA, EB, EC, and BI were 1.59%, 1.46%, 1.86%, 2.29%, and 0.98%, respectively, indicating that the method had good repeatability. The RSD% values (n = 6) of the peak areas of icariin, EA, EB, EC, and BI in the samples were 1.49%, 1.96%, 1.42%, 0.96%, and 0.81%, respectively, indicating that the sample solution was stable within 24 h. The average recovery rates of icariin, EA, EB, EC, and BI were 99.98%, 100.14%, 100.09%, 100.75%, and 100.94%, respectively, and the RSD% values were 0.56%, 0.78%, 0.84%, 1.10%, and 1.47%, respectively (see Table 4). These results show that the accuracy of the method met the requirements.
The above experimental results showed that the analytical method provided results that had excellent precision, reproducibility, and accuracy and were acceptable for the quality analysis of the EF-processed products.
Optimization of the mutton-oil processing technology of EF by applying the Box-Behnken design-response surface methodology 
We performed quadratic polynomial regression fitting of the above data to obtain the following model: Y = 0.86 &#8722; 0.11 x A + 0.025 x B &#8722; 0.078 x C &#8722; 0.023 x A x B &#8722; 0.037 x A x C + 0.037 x B x C &#8722; 0.045 x A2 + 2.5 x 10-3 x B2 &#8722; 0.14 x C2. The variance analysis gave a value of P &#60; 0.01, indicating that the model was significant. The P value of the lack of fit was P &#62; 0.05, indicating that the lack of fit was not significant. The R2 value was 0.9300, indicating that the fit of the model was good, and the error was small. It was feasible to use this model to analyze and predict the effect of the chemical composition content of the EF stir-fried with mutton oil. In addition, A2 and D2 had an effect on the content of the processed products, and the difference was statistically significant (P &#60; 0.01). The effects of A and C of the one-degree term and C2 of the second-order term on the comprehensive score were significant. The one-degree term B, the second-order A2, B2, and all of the interaction items had no significant effect on the comprehensive score. The analysis of the P values showed that, of the experimental parameters, the mutton oil amount (A) had the greatest effect on the comprehensive score, followed by the frying temperature (C), and then the mutton oil temperature (B). The above results are shown in Table 5.
The software was used to set the mutton oil amount, mutton oil temperature, and frying temperature to the medians and to use the comprehensive score as the index to draw a single-factor influence diagram of one factor (Figure 1). Increasing the frying temperature first increased the comprehensive score and then decreased it (Figure 1). The mutton oil temperature had a negligible effect on the comprehensive score. The mutton oil amount was the main significant factor that affected the change in the comprehensive score, and as the amount increased, the content trended downward.
To help better understand the results, the predicted models are presented in Figure 2 as 3D response surface plots. In terms of the slope of the response surface, the greater the significance of the interaction effect between factors, the gentler the slope, and the less significant the effect. An ellipse in the shape of a contour line indicates a strong interaction between factors, whereas a circle indicates the opposite. The response surface of the mutton oil amount and the frying temperature was steeper compared to the other tested factors, and the contour lines tended to be more elliptical (see Figure 2C,D), indicating that the interaction between these two factors was more significant; in contrast, the interactions between other factors were not significant (see Figure 2A,B,E,F).
The optimal mutton oil processing technology of EF was selected as follows: a mutton oil amount of 15%; a mutton oil temperature of 120 &#176;C; and a frying temperature of 189 &#176;C. Considering that the temperature cannot be very accurately controlled in actual operation, the temperature value is specified as a variable &#177;10 &#176;C. Therefore, the final parameters were as follows: a mutton oil amount of 15%; a mutton oil temperature of 120 &#176;C &#177; 10 &#176;C; and a frying temperature of 189 &#176;C &#177; 10 &#176;C. The optimal process was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, frying it with a gentle fire (189 &#176;C &#177; 10 &#176;C) until it is evenly shiny, and removing and cooling. For every 100 kg of EF, 15 kg of mutton oil (refined oil) should be used. Using these conditions, three parallel experiments were conducted, and the scores obtained were 0.96, 0.97, and 0.94 (RSD% = 1.60%), indicating stable and feasible conditions. The typical HPLC chromatograms of the crude, processed, and mixed reference substances of EF are shown in Figure 3.
Test of the effect of processing on the embryonic development of zebrafish 
The zebrafish hatched into juveniles at 72 hpf. The development of each organ was basically complete. The fish bodies remained transparent, and it was easy to lay them on their side on the glass slide. The shapes of the organs were easy to observe and identify when viewed under a microscope. The blank control group did not experience any death or organ toxicity during the administration period. Compared with the control group, at a drug concentration of 100 &#181;g/mL, no obvious abnormalities were found in the crude EF&#160;group (S) and the processed group (P) at 72 hpf. At 96 hpf and later, swim bladder incompleteness and loss of the swim bladder were more common in the juvenile fish in the crude group but were rare in the juvenile fish in the processed group. At a drug concentration of 150 &#181;g/mL, obvious spinal deformities, body curvature deformities, pericardial edema, and liver deformation were seen in the juvenile fish in the crude group at 72 hpf, but those changes were rare in the juvenile fish in the processed group, and the degree of teratogenicity was weaker than that of the crude group. At a drug concentration of 200 &#181;g/mL, all juvenile fish in the crude group died, and obvious teratogenicity appeared in the juvenile fish in the processed group. At a drug concentration of 250 &#181;g/mL, a small number of zebrafish survived in the processed group. The microscopic examination results of the zebrafish are shown in Figure 4.
The zebrafish mortality rates in the crude and processed Epimedium herb groups depended on the concentration and time of administration. The time-dose-mortality relationship is shown in Figure 5. The zebrafish mortality results showed that 24 h after administration (48 hpf), at a drug concentration of 200 &#181;g/mL, all zebrafish in the crude drug group died, whereas the mortality in the processed group was only 6.67%. At 48 h after EF administration (72 hpf), the concentration that caused the death of all zebrafish in the crude drug group was 200 &#181;g/mL, and the concentration that caused the death of all zebrafish in the processed group was 500 &#181;g/mL. The median lethal concentration of the two experimental groups at 72 hpf was calculated. The results showed that the LC50 (see Figure 6) was 151.3 &#181;g/mL in the crude group (S) and 219.8 &#181;g/mL in the processed group (P).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65096/65096fig01.jpg" /> 
Figure 1: Univariate analysis. The figure shows the single-factor influence diagram. A is the single-factor result of the amount of mutton (suet) oil; B is the single-factor result of the temperature of the mutton (suet)oil; and C is the single-factor result of the frying temperature. With an increasing frying temperature, the comprehensive score first increases and then decreases. The mutton-oil temperature has little effect on the score. The amount of mutton oil was the main significant factor affecting the change in the comprehensive score, and the content showed a downward trend with an increasing amount of mutton oil. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65096/65096fig02.jpg" /> 
Figure 2: Response surface and contour plot of the influence of different factor interactions on the comprehensive score. (A) This figure shows a 3D response surface plot of the interaction between the mutton oil amount and temperature. (B) This figure shows a contour plot of the interaction between mutton oil amount and temperature. (C) This figure shows a 3D response surface plot of the interaction between the mutton oil amount and processing temperature. (D) This figure shows a contour plot of the interaction between mutton oil dosage and processing temperature. (E) This figure shows a 3D response surface plot of the interaction between mutton oil amount and processing temperature. (F) This figure shows a contour plot of the interaction between mutton oil amount and processing temperature. The result shows that the response surface of the mutton oil amount and frying temperature was steep, than the other tested parameters and the contour lines tended to be elliptical (see C,D), indicating that the interaction between these two factors was significant, whereas the interactions between other factors were not significant (see A,B,E,F). The suet oil term used in the figure refers to mutton oil. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65096/65096fig03.jpg" /> 
Figure 3: HPLC chromatograms of the crude, processed, and mixed reference substances of EF. (A) This figure shows the HPLC chromatogram of the mixed reference substance. (B) This figure shows the HPLC chromatogram of crude Epimedii folium. (C) This figure shows the HPLC chromatogram of Epimedii folium processed products. These three pictures demonstrate that the BI content in raw EF is low, while it increases after processing. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65096/65096fig04.jpg" /> 
Figure 4: Micrographs of zebrafish. This figure shows micrographs of the zebrafish. (A) This figure shows the results for the observation of the zebrafish under a microscope in the blank group. (B) This figure shows the results for the observation of the zebrafish under a microscope in the crude group. (C) This figure shows the results for the observation of the zebrafish under a microscope in the processed group. The blank control group did not experience any death or organ toxicity during the administration period. At an EF drug concentration of 150 &#181;g/mL, obvious spinal deformities, body curvature, pericardial edema, and liver deformation were seen in the juvenile fish in the crude group at 72 hpf, whereas those changes were rare in juvenile fish in the processed group, and the degree of teratogenicity was weaker than that in the crude group. At a drug concentration of 200 &#181;g/mL, all the juvenile fish in the crude group died, and obvious teratogenicity appeared in the processed group. At a drug concentration of 250 &#181;g/mL, only a small number of zebrafish survived in the processed group. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65096/65096fig05.jpg" /> 
Figure 5: The dosing time-dose-mortality relationship. This figure shows the dosing time-dose-mortality relationship. (A) This figure shows the dosing time-dose-mortality relationship of the crude group. (B) This figure shows the dosing time-dose-mortality relationship of the processed group. n = 40. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65096/65096fig06.jpg" /> 
Figure 6: LC50 diagram of crude and processed EF. The LC50 diagram of the crude and processed EF is shown. The median lethal concentrations of the two experimental groups at 72 hpf were calculated. The LC50 was 151.3 &#181;g/mL in the crude group (S) and 219.8 &#181;g/mL in the processing group (P). n = 40. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/65096fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Experimental design and the Box-Behnken response surface method results of the 17 groups of experiments. Table 1 shows the 17 groups of experiments designed by the Box-Behnken design-response surface method and their comprehensive score results. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/Table 1 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Variables used in the Box-Behnken design. The independent and dependent variables are listed here along with their low, middle, and high levels. The Box-Behnken design enabled the identification of the most influential factors in the EF processing, with the mutton oil amount (A) (15%-35%), mutton oil temperature (B) (50 &#176;C-120 &#176;C), and frying temperature (C) (80 &#176;C-300 &#176;C) as the influencing factors. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/Table 2 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Regression equations and linear ranges of the chemical constituents of EF. The results of the regression equation and linear range of the EF chemical composition show that there was good linearity between each of the concentrations of icariin, EA, EB, EC, and BI and their chromatographic peak areas. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/Table 3 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Sample recovery test rates. The average recovery rates of icariin, EA, EB, EC, and BI were 99.98%, 100.14%, 100.09%, 100.75%, and 100.94%, respectively, and the RSD% values were 0.56%, 0.78%, 0.84%, 1.10%, and 1.47%, respectively. The results show that the accuracy of the method was suitable. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/Table 4 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Regression coefficients of the predicted quadratic model. The P value of the model was P &#60; 0.01, indicating that the model was significant. The P value of the lack of fit was P &#62; 0.05, indicating that the lack of fit was not significant. The R2 value was 0.9300, indicating that the fit of the model was good, and the error was small, so the model was suitable for analyzing and predicting the effect of the chemical composition content of the EF stir-fried with mutton oil. In addition, A2 and D2 had significant effects on the content of processed products (P &#60; 0.01). The influences of A and C of the one-degree term and C2 of the second-order term on the comprehensive score were significant. The one-degree term B, the second-order A2, B2, and all of the interaction items had no significant effects on the comprehensive score. The analysis of the P value showed that, of the experimental parameters, the amount of mutton oil (A) had the greatest influence on the comprehensive score, followed by the frying temperature (C), and then the temperature of the mutton oil (B). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65096/Table 5 .xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development at JoVE.com

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed ...

optimization-epimedii-folium-mutton-oil-processing-technology-testing

Universidad San Sebastian

Research

JoVE Journal

Medicine

401 Views.  Chongqing Academy of Chinese Materia Medica. In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

Video: Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Developmental Biology

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

Environment

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Immunology and Infection

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Biology

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

Summary

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Chapters in this video

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Metabolic Profile Analysis of Zebrafish Embryos

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

High Throughput Danio Rerio Energy Expenditure Assay