We would like to acknowledge research support from the Department of Defense Prostate Cancer Research Program Award W81XWH-20-1-0274 and Prostate Cancer Foundation Challenge Award 16CHAL13. We would like to thank and acknowledge Dr. Michelle Rudek, Dr. Noushin Rastkari, Dr. Nicole Anders, and Linping Xu of the Analytical Pharmacology Shared Resource at Johns Hopkins for assistance with equol LC/MS/MS.

All animal studies were performed following institutional guidelines and under Johns Hopkins University Institutional Animal Care and Use Committee (IACUC) approved protocols M021M197 and M023M195. Mouse strains used (male mice, 7 weeks old) are described in the Table of Materials. The use of male mice was due to their use in the ongoing studies of prostate cancer. The MDA method has previously been shown to be effective in female mice as well7,17. Mice were randomized to their respective cages/treatment groups. The mice were treated in cages and in no particular order. The experimenter administering the treatments was blinded to the treatment group. The number assigned to a mouse referred to their ID and not the order in which they were treated. The data was collected throughout the timeline of the study and analyzed at the end to avoid any bias. Experimental assays were blinded to the treatment group until all data was collected.
NOTE: This protocol has been modified from Scarborough et al.7.
1. Preparation of treatments
<ol>
	<li>Prepare a solution of sweetened condensed milk (ingredients: milk and sugar) and molecular biology grade water using a 3:10 ratio of milk to water. Measure the sweetened condensed milk using a 15 mL conical tube due to the viscosity of the milk.</li>
	<li>Using a 50 mL conical tube, mix molecular biology-grade water with the sweetened condensed milk by slowly mixing 5 mL of water at a time with the milk. 
	NOTE: This step is performed under sterile conditions using a biosafety cabinet.</li>
	<li>Prepare aliquots of the milk solution (stored as 500 &#181;L aliquots) and store the aliquots at 4 &#176;C until combined with the treatment of interest.</li>
	<li>Resuspend treatments in their respective vehicles at the correct concentration based on the animal&#39;s body weight before combining them with the sweetened condensed milk/water solution.</li>
</ol>
2.&#160;MDA training session
NOTE: This section describes mouse models; however, the MDA technique could be scaled up as needed with larger volumes/pipette tips for larger rodent models.
<ol>
	<li>OPTIONAL: Gently restrain the study animal by holding the scruff of the neck with one hand.</li>
	<li>Offer the study animal 100 &#181;L of sweetened condensed milk/water solution only (without the addition of the experimental treatment) using a single channel pipette and a 200 &#181;L pipette tip which is guided to the mouth.</li>
	<li>Dispense the milk/water solution slowly to avoid missing the animal&#39;s mouth. Ensure the animal readily consumes the milk solution between 5-15 s. 
	NOTE: This training occurs for a minimum of 2 consecutive days (one training session per day), with no changes to the volume administered. A gentle restraint by scruff was needed for the MDA training sessions. Scarborough et al.7 recommend full restraint of the animal on the first day of MDA training and then restraint by the tail on the second day. The method of restraint may differ for larger rodent models. In contrast, Vanhecke et al.17 only held the mice slightly by the tail during training.</li>
</ol>
3. Mouse treatments using the MDA method
<ol>
	<li>Combine the study treatment with the sweetened condensed milk/water solution (see examples with validation experiments [section 4 and section 5] below).</li>
	<li>OPTIONAL: Gently restrain the study animal by holding the scruff of the neck with one hand. 
	NOTE: Scarborough et al.7 report that mice no longer require restraint beyond the training period and will readily drink from the pipette tip. In contrast, Vanhecke et al.17 report that for some treatments (e.g., tamoxifen) that might have mild taste aversion, MDA is best performed by gently restraining the mice via scruff during all treatments. This study also determined that positioning the animal on their back with gentle scruff improves the time and efficacy of treatment consumption.</li>
	<li>Administer 100 &#181;L of treatment using a single channel pipette and a 200 &#181;L pipette tip guided to the animal&#39;s mouth, as performed during the training sessions.</li>
	<li>Treat a single animal at a time.</li>
	<li>Place each animal back into their respective cage after treatment. 
	NOTE: Treatments using the MDA method can be given daily or in a repeated fashion7,17.</li>
</ol>
4. Validation experiment #1 - oral delivery of gut bacteria to mice using MDA
NOTE: This section describes the use of the MDA method to orally deliver live bacteria for gut colonization studies in mice. In this representative pilot experiment, the gram-positive bacterium Clostridium scindens is delivered to C57BL/6J mice. The mice were treated with antibiotics (cefoxitin) for two consecutive days in the drinking water prior to bacterial inoculation with MDA.
<ol>
	<li>Bacterial culture
	<ol>
		<li>Culture C. scindens strain ATCC 35704 in reinforced clostridial broth (RCB) using a frozen glycerol stock to inoculate 500 &#181;L of bacteria into a 7 mL RCB tube. Grow anaerobically overnight at 37 &#176;C.</li>
		<li>Use 500 &#181;L of the overnight bacteria culture to inoculate a new RCB tube and incubate anaerobically for 24 h. Remove this tube from the anaerobic chamber and use it to prepare the bacterial treatment for MDA. Use this same tube to calculate the colony-forming units per milliliter (CFU/mL).</li>
	</ol>
	</li>
	<li>Calculating C. scindens CFU/mL
	<ol>
		<li>Create a 1:3 dilution of the C. scindens culture using 3.5 mL of the culture inoculated into 7 mL of RCB media. Use a sample of this dilution to make a 20% 1 mL glycerol stock (1:1) that is stored at -80 &#176;C.</li>
		<li>Plate 100 &#181;L of the 1:3 dilution onto reinforced clostridial agar (RCA) plates. Use the first dilution to then make subsequent serial dilutions and plate for a total of three more times.</li>
		<li>After incubation of the RCA plates, count the colonies and calculate the CFU. Use the following equation to calculate the concentration of bacterial cells in the diluted and original samples: 
		CFU in diluted sample (cells/mL) = (number of colonies counted on the RCA plate)/(amount of diluted sample added to the RCA plate in mL) 
		&#8203;CFU in original sample (cells/mL) = (CFU in diluted sample)/(dilution of the RCA plate)</li>
	</ol>
	</li>
	<li>Preparing the C. scindens treatment for MDA
	<ol>
		<li>Combine a solution of 50 &#181;L (1.265 x 106 CFU/mL in this example) of C. scindens + 50 &#181;L of the sweetened condensed milk/water solution. 
		NOTE: If a specific CFU is needed, an alternative method of bacterial cell quantification may be used, such as creating a standard curve with optical density (OD) measurements at 600 nm.</li>
	</ol>
	</li>
	<li>Oral treatment of mice with C. scindens using MDA
	<ol>
		<li>On days 1-3, familiarize the mice with the milk solution as described in section 2 (MDA training session).</li>
		<li>On day 4, administer 100 &#181;L of the C. scindens + milk solution or PBS (control) + milk solution via MDA to C57BL/6J mice. Treat mice once for the duration of the study.</li>
	</ol>
	</li>
	<li>Fecal sample collection
	<ol>
		<li>Hold the mouse by gentle scruff and wait for them to discharge a fecal pellet. Collect fecal pellets directly from the rectum into a sterile 1.5 mL microcentrifuge tube. Store the samples at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>C. scindens quantitative PCR (qPCR)
	<ol>
		<li>Perform fecal DNA extraction using a previously published protocol19,20.</li>
		<li>Determine DNA concentration using a DNA fluorometer for high-sensitivity detection. Normalize DNA samples to 10 ng/&#181;L and store at -20 &#176;C until use.</li>
		<li>Use qPCR to investigate the abundance of: (i) C. scindens bacteria using conserved primers against the desA gene of C. scindens strain ATCC 35704, (ii) total bacteria using primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene as previously described20,21. 
		NOTE: The qPCR conditions and primers used are listed in Table 1. A standard curve using C. scindens strain ATCC 35704 DNA was used to estimate the copies of desA+ bacteria.</li>
	</ol>
	</li>
</ol>
5. Validation experiment #2 - experimental drug delivery to mice using MDA
NOTE: This section describes the use of the MDA method to deliver an experimental compound to mice. In this representative pilot experiment, the soy metabolite equol (S-equol) is delivered to NSG and FVB/NJ mice.
<ol>
	<li>Preparation of S-equol treatment 
	NOTE: Both S-equol and dimethyl sulfoxide (DMSO) are aliquoted and stored at -20 &#176;C until use.
	<ol>
		<li>Reconstitute S-equol in DMSO combined with corn oil (90%). Mix aliquoted S-equol in corn oil (220 &#181;L) with 480 &#181;L of the sweetened condensed milk/water solution. Combine and mix this solution in the animal room to avoid the separation of the oil from the milk. 
		NOTE: If separation of the solution is observed, mix the solution before treatment. The dose of S-equol administered is 25 mg/kg/day. The mice used in this study were all similar in weight, so they received the same dose. The dose of the treatment may need to be adjusted and made into multiple preparations based on the study animal weights.</li>
		<li>Combine DMSO vehicle with corn oil (90%). Mix aliquoted DMSO in corn oil (220 &#181;L) with 480 &#181;L of the sweetened condensed milk/water solution. Combine and mix this solution in the animal room to avoid the separation of the oil from the milk. 
		NOTE: If separation of the solution is observed, mix the solution before treatment.</li>
	</ol>
	</li>
	<li>Oral treatment of mice with S-equol using MDA
	<ol>
		<li>On days 1 and 2, familiarize the mice with the milk solution as described in section 2 (MDA training session). 
		NOTE: For validation experiment #1, the training time described in Scarborough et al. was followed, which suggested a 3-day acclimation time. In this experiment, a shorter training time (2 days), was tested and found that it did not alter the administration of the treatments of interest.</li>
		<li>On days 3-43, treat mice with (i) PBS control, (ii) DMSO vehicle, or (iii) S-equol, all combined with the sweetened condensed milk/water solution. Make a master mix of the solution to deliver a homogenous solution of the treatments to all study animals. Administer a total volume of 100 &#181;L to each mouse once daily, 5 days a week, for a total of 43 days.</li>
	</ol>
	</li>
	<li>Blood collection
	<ol>
		<li>Collect blood after euthanasia (carbon dioxide overdose) via heart puncture. Transfer blood to a microtainer serum separator tube, mix, and store at room temperature (RT) until further processing.</li>
		<li>Centrifuge blood sample to complete separation at 254 x g for 5 min. Collect serum (top layer of separation) and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>S-equol liquid chromatography with tandem mass spectrometry (LC/MS/MS) assay
	<ol>
		<li>Refer to the Table of Materials file for the chemicals and reagents used for this experiment.</li>
		<li>Calibration standards and quality control: Prepare stock solutions of S-equol and internal standard (racemic equol-d4) in DMSO at concentrations of 1 mg/mL and store at -20 &#176;C.</li>
		<li>Dilute the stock standard solutions with acetonitrile:water (50:50) to prepare a mixed working solution with different concentrations. Prepare internal standard in ethyl acetate as extraction solution at a concentration of 100 ng/mL. Store stock and working solutions at 4 &#176;C and use daily, diluted, or directly.</li>
		<li>Sample extraction
		<ol>
			<li>Prepare the sample using protein precipitation by extraction solution (ethyl acetate plus internal standard, see section 5.4.3), followed by centrifugation and evaporation of supernatant under dry nitrogen (50 &#176;C).</li>
			<li>Reconstitute the sample in 100 &#181;L of 50% acetonitrile and transfer to autosampler vials for LC/MS/MS analysis.</li>
		</ol>
		</li>
		<li>Sample separation
		<ol>
			<li>Achieve separation with a C18 column, 2.1x50 mm, 1.7 &#181;m. Use 0.1% formic acid as mobile phase A and 0.1% formic acid in acetonitrile mobile phase B.</li>
			<li>Use a gradient and hold mobile phase B at 40% with a flow rate of 0.3 mL/min for 0.5 min. Then, increase the flow rate to 100% over 1 min, hold there for 2 min, and then return to 40% B for 1 min.</li>
			<li>Monitor the column effluent using a mass-spectrometric detector using electrospray ionization, which is operating in negative mode. Program the spectrometer to monitor the following multiple reaction monitoring (MRM) transitions: 241 &#8594; 119.1 for S-equol and 245 &#8594; 123 for the internal standard, equol-d4.</li>
			<li>Compute the calibration curve for S-equol using the area ratio peak of the analysis to the internal standard by using a quadratic equation with a 1/x2 weighting function using the calibration ranges of 5-500 ng/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Measuring the effect of MDA treatment on core gut microbiota
NOTE: This section describes using qPCR to measure levels of core gut microbiota after MDA treatment.
<ol>
	<li>Perform fecal sample collection and DNA extraction as described in steps 4.5.1 and 4.6.1.</li>
	<li>Determine DNA concentration using a DNA fluorometer for high-sensitivity detection. Normalize DNA samples to 10 ng/&#181;L and store at -20 &#176;C until use.</li>
	<li>Use qPCR to investigate the abundance of members of the gut microbiota: (i) Akkermansia muciniphila, (ii) Bifidobacterium spp., (iii) Streptococcus salivarius, (iv) Lactobacillus spp., and (v) total bacteria using primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene as previously described20,21,22. 
	NOTE: The qPCR conditions and primers used are listed in Table 2.</li>
</ol>

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

<ol>
	<li>Vandenberg, L. N., Welshons, W. V., Vom Saal, F. S., Toutain, P. -. L., Myers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+oral+gavage+be+abandoned+in+toxicity+testing+of+endocrine+disruptors.">Should oral gavage be abandoned in toxicity testing of endocrine disruptors.</a> Environ Health. 13 (1), 46 (2014).</li><li>Craig, M. A., Elliott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+fed+radiolabeled+protein+by+gavage+show+sporadic+passage+of+large+quantities+of+intact+material+into+the+blood,+an+artifact+not+associated+with+voluntary+feeding.">Mice fed radiolabeled protein by gavage show sporadic passage of large quantities of intact material into the blood, an artifact not associated with voluntary feeding.</a> Contemp Top Lab Anim Sci. 38 (5), 18-23 (1999).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Gulin, J. E. N., Bisio, M., Garc&#237;a-Bournissen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+drug+administration+in+a+murine+model+of+acute+infection+with+Trypanosoma+cruzi.">Refining drug administration in a murine model of acute infection with Trypanosoma cruzi.</a> Lab Anim Res. 36, 37 (2020).</li><li>Bonnichsen, M., Dragsted, N., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+welfare+impact+of+gavaging+laboratory+rats.">The welfare impact of gavaging laboratory rats.</a> Anim Welf. 14 (3), 223-227 (2005).</li><li>Brown, A. P., Dinger, N., Levine, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+produced+by+gavage+administration+in+the+rat.">Stress produced by gavage administration in the rat.</a> Contemp Top Lab Anim Sci. 39 (1), 17-21 (2000).</li><li>Scarborough, J., 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=Preclinical+validation+of+the+micropipette-guided+drug+administration+(MDA)+method+in+the+maternal+immune+activation+model+of+neurodevelopmental+disorders.">Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders.</a> Brain Behav Immun. 88, 461-470 (2020).</li><li>Germann, P. G., Ockert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomatous+inflammation+of+the+oropharyngeal+cavity+as+a+possible+cause+for+unexpected+high+mortality+in+a+Fischer+344+rat+carcinogenicity+study.">Granulomatous inflammation of the oropharyngeal cavity as a possible cause for unexpected high mortality in a Fischer 344 rat carcinogenicity study.</a> Lab Anim Sci. 44 (4), 338-343 (1994).</li><li>Martins, T., 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=Comparison+of+gelatin+flavors+for+oral+dosing+of+C57BL/6J+and+FVB/N+mice.">Comparison of gelatin flavors for oral dosing of C57BL/6J and FVB/N mice.</a> J Am Assoc Lab Anim Sci. 61 (1), 89-95 (2022).</li><li>Huynh, N., Arabian, N., Lieu, D., Asatryan, L., Davies, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+an+orally+dissolving+strip+for+pharmacological+and+toxicological+studies:+A+simple+and+humane+alternative+to+oral+gavage+for+animals.">Utilizing an orally dissolving strip for pharmacological and toxicological studies: A simple and humane alternative to oral gavage for animals.</a> J Vis Exp. (109), e53770 (2016).</li><li>Hoggatt, A. F., Hoggatt, J., Honerlaw, M., Pelus, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spoonful+of+sugar+helps+the+medicine+go+down:+a+novel+technique+to+improve+oral+gavage+in+mice.">A spoonful of sugar helps the medicine go down: a novel technique to improve oral gavage in mice.</a> J Am Assoc Lab Anim Sci. 49 (3), 329-334 (2010).</li><li>Neto, T., Faustino-Rocha, A. I., Gilda Costa, R. M., Medeiros, R., Oliveira, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+low-intensity+method+for+oral+administration+to+large+numbers+of+mice:+A+possible+alternative+to+oral+gavage.">A quick and low-intensity method for oral administration to large numbers of mice: A possible alternative to oral gavage.</a> Lab Anim. 56 (2), 185-190 (2022).</li><li>K&#252;ster, T., 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=Voluntary+ingestion+of+antiparasitic+drugs+emulsified+in+honey+represents+an+alternative+to+gavage+in+mice.">Voluntary ingestion of antiparasitic drugs emulsified in honey represents an alternative to gavage in mice.</a> J Am Assoc Lab Anim Sci. 51 (2), 219-223 (2012).</li><li>Gonzales, C., 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=Alternative+method+of+oral+administration+by+peanut+butter+pellet+formulation+results+in+target+engagement+of+BACE1+and+attenuation+of+gavage-induced+stress+responses+in+mice.">Alternative method of oral administration by peanut butter pellet formulation results in target engagement of BACE1 and attenuation of gavage-induced stress responses in mice.</a> Pharmacol Biochem Behav. 126, 28-35 (2014).</li><li>Teixeira-Santos, L., Albino-Teixeira, A., Pinho, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+oral+drug+administration+by+voluntary+intake+in+male+and+female+mice.">An alternative method for oral drug administration by voluntary intake in male and female mice.</a> Lab Anim. 55 (1), 76-80 (2021).</li><li>Schalbetter, S. M., 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=Oral+application+of+clozapine-N-oxide+using+the+micropipette-guided+drug+administration+(MDA)+method+in+mouse+DREADD+systems.">Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems.</a> Lab Anim (NY). 50 (3), 69-75 (2021).</li><li>Vanhecke, D., Bugada, V., Buch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+tamoxifen+administration+in+mice+by+encouraging+voluntary+consumption+of+palatable+formulations).">Refined tamoxifen administration in mice by encouraging voluntary consumption of palatable formulations).</a> bioRxiv. , (2023).</li><li>Heraudeau, M., 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=Micropipette-guided+drug+administration+(MDA)+as+a+non-invasive+chronic+oral+administration+method+in+male+rats.">Micropipette-guided drug administration (MDA) as a non-invasive chronic oral administration method in male rats.</a> J Neurosci Methods. 398, 109951 (2023).</li><li>Sfanos, K. 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=Compositional+differences+in+gastrointestinal+microbiota+in+prostate+cancer+patients+treated+with+androgen+axis-targeted+therapies.">Compositional differences in gastrointestinal microbiota in prostate cancer patients treated with androgen axis-targeted therapies.</a> Prostate Cancer Prostatic Dis. 21 (4), 539-548 (2018).</li><li>Shrestha, E., 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=Profiling+the+urinary+microbiome+in+men+with+positive+versus+negative+biopsies+for+prostate+cancer.">Profiling the urinary microbiome in men with positive versus negative biopsies for prostate cancer.</a> J Urol. 199 (1), 161-171 (2018).</li><li>Peiffer, L. B., 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=Composition+of+gastrointestinal+microbiota+in+association+with+treatment+response+in+individuals+with+metastatic+castrate+resistant+prostate+cancer+progressing+on+enzalutamide+and+initiating+treatment+with+anti-PD-1+(pembrolizumab).">Composition of gastrointestinal microbiota in association with treatment response in individuals with metastatic castrate resistant prostate cancer progressing on enzalutamide and initiating treatment with anti-PD-1 (pembrolizumab).</a> Neoplasia. 32, 100822 (2022).</li><li>Jones, C. B., Peiffer, L. B., Davis, C. M., Sfanos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+effects+of+4He+exposure+on+the+gut-brain+axis.">Examining the effects of 4He exposure on the gut-brain axis.</a> Radiat Res. 197 (3), 242-252 (2022).</li><li>Walker, M. K., 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=A+less+stressful+alternative+to+oral+gavage+for+pharmacological+and+toxicological+studies+in+mice.">A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice.</a> Toxicol Appl Pharmacol. 260 (1), 65-69 (2012).</li><li>Miguelena Chamorro, B., Swaminathan, G., Mundt, E., Paul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+more+translatable+research:+Exploring+alternatives+to+gavage+as+the+oral+administration+route+of+vaccines+in+rodents+for+improved+animal+welfare+and+human+relevance.">Towards more translatable research: Exploring alternatives to gavage as the oral administration route of vaccines in rodents for improved animal welfare and human relevance.</a> Lab Anim (NY). 52 (9), 195-197 (2023).</li></ol>

OG can be a significant source of stress in research animals that may create a confounding variable as previously assessed in multiple studies7,9,11,12,13,14,15,23. Due to the invasiveness of OG, alternate techniques have been employed to minimize the chall...

Drug administration to rodent models is commonly achieved via oral gavage (OG), which consists of administering a liquid preparation directly to the stomach using a cannula attached to a syringe containing the solution. This technique results in a consistent and precise dosage of the treatment to the animal, but also carries multiple disadvantages. OG has been scrutinized for not adequately modeling human dietary exposures1,2. Furthermore, OG increases the risk of unintentional injuries to the upper digestive system (perforation of the esophagus and stomach), aspiration of the administered treatment, and respiratory tract lesions3. OG is also associated with discomfort4, increased blood pressure and heart rate5, as well as stress6,7, and sometimes death8 due to decreased tolerance to gavage by the animal. These physiologic changes might interfere with or confound experimental results; thus, new procedures have been explored to avoid these side effects. Studies have utilized alternative procedures to OG, such as the use of gelatin as a drug vehicle9, orally dissolving strips (ODS)10, sucrose-coated gavage needles11, flexible feeding tubes, wheat cookies12, honey13, and peanut butter pellets14. Unfortunately, there are limitations with these modifications to the OG technique, including incompatibility with water-insoluble drugs, longer preparation time for the treatment15, drug palatability, and stability15, and familiarization of the animal with the food. Furthermore, there is potential for less precise dosing when animals feed ad lib.
Scarborough et al.7 previously developed an alternative oral treatment method in mice, which they termed micropipette-guided drug administration (MDA). This method of administration is based on a sweetened condensed milk solution as a vehicle for pharmacological substances, motivating the study animals to consume the prepared vehicle and/or drug solutions readily via dispensing the solution with a single channel pipette and pipette tip. To introduce this technique, rodents undergo a training session (minimum 2 days) to shorten handling times and to allow the study animal to become familiar with drinking from the pipette tip4. Initial validation studies by Scarborough et al.7 and Schalbetter et al.16 suggest that the MDA procedure is easy to implement, cost-effective, minimally invasive, and less stressful for the animals than conventional oral gavage methods. Scarborough et al. introduced the use of the MDA technique in a mouse model of maternal immune activation (MIA) of neurodevelopmental disorders7. This study demonstrated that the pharmacokinetic profiles of mice treated with the antipsychotic drug risperidone using MDA were comparable to the use of OG. Furthermore, MDA did not induce an increase in corticosterone (a stress hormone) levels in the mice, and chronic treatment with risperidone using the MDA technique led to a dose-dependent decrease in MIA-induced social interaction deficits and amphetamine hypersensitivity7. Additional studies have explored the efficacy of MDA versus OG in both mouse17 and rat18 models. MDA has also been compared to intraperitoneal injection and was shown to be as effective in delivering clozapine-N-oxide to mice16. Due to MDA&#39;s reported success in reducing animal stress and therapeutic efficacy, we now aim to further explore the MDA technique as an effective method of drug delivery using additional murine models. Here, we describe the implementation of the MDA method to treat different mouse strains, including the immune-competent FVB/NJ and C57BL/6J strains and the immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) strain with different oral treatments, including live bacteria, experimental compounds delivered in water-insoluble solutions (corn oil), and vehicle controls. We assessed the activity and presence of the different treatments in serum and feces, and we evaluated alterations to core gut microbiota in relation to the MDA method.

<table><tbody><tr><td>200 &amp;micro;L pipette tips</td><td>Mettler Toledo</td><td>17005860</td><td></td></tr><tr><td>AB SCIEX Triple QTRAP 5500 mass-spectrometric detector</td><td>Sciex</td><td>N/A</td><td></td></tr><tr><td>Akkermansia muciniphila strain muc genomic DNA</td><td>American Type Culture Collection</td><td>BAA-835D-5</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma&amp;ndash;Aldrich</td><td>5.43834</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Jackson Laboratories</td><td>Strain# 000664</td><td></td></tr><tr><td>&lt;em&gt;C. scindens&lt;/em&gt; strain 35704</td><td>American Type Culture Collection</td><td>35704</td><td></td></tr><tr><td>Cefoxitin</td><td>Sagent&amp;nbsp;</td><td>NDC25021-109-10</td><td></td></tr><tr><td>Corn oil</td><td>MedChemExpress</td><td>HY-Y1888</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>ethanol</td><td>Fisher Scientific</td><td>AC611050040</td><td></td></tr><tr><td>Formic acid</td><td>Sigma&amp;ndash;Aldrich</td><td>5.33002</td><td></td></tr><tr><td>FVB/NJ mice</td><td>Jackson Laboratories</td><td>Strain# 001800</td><td></td></tr><tr><td>Glycerol</td><td>Sigma&amp;ndash;Aldrich</td><td>G5516</td><td></td></tr><tr><td>Hexane</td><td>Fisher Scientific</td><td>02-002-996</td><td></td></tr><tr><td>LC-MS grade water</td><td>Fisher Scientific</td><td>14-650-357</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>02-003-340</td><td></td></tr><tr><td>Microtainer serum separator tube</td><td>Becton Dickinson</td><td>02-675-185</td><td></td></tr><tr><td>Molecular biology grade water</td><td>Corning</td><td>46-000-CI</td><td></td></tr><tr><td>NSG mice</td><td>Jackson Laboratories</td><td>Strain# 005557</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Qubit DNA HS kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>Racemic&amp;nbsp;equol-d4</td><td>Santa Cruz Biotechnology</td><td>sc-219827</td><td></td></tr><tr><td>Reinforced Clostridial agar</td><td>Anaerobe Systems</td><td>AS-6061</td><td></td></tr><tr><td>Reinforced Clostridial broth</td><td>Anaerobe Systems</td><td>AS-606</td><td></td></tr><tr><td>S-equol</td><td>MedChemExpress</td><td>HY-100583</td><td></td></tr><tr><td>S-equol reference standard for LC-MS</td><td>Cayman Chemical</td><td>10010173</td><td></td></tr><tr><td>Single channel pipette</td><td>Rainin</td><td>17008652</td><td></td></tr><tr><td>Streptococcus salivarius genomic DNA</td><td>American Type Culture Collection</td><td>BAA-1024D-5</td><td></td></tr><tr><td>Sweetened condensed milk</td><td>California Farms</td><td>B09TGQ7WV8</td><td></td></tr><tr><td>VSL#3</td><td>VSL#3</td><td>B07WX1LVHL</td><td></td></tr><tr><td>&amp;beta;-glucuronidase from Helix pomatia</td><td>Sigma&amp;ndash;Aldrich</td><td>G7017</td><td></td></tr></tbody></table>

use of micropipette-guided drug administration as an alternative method to oral gavage in rodent models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

MDA can be used in the oral delivery of bacterial strains in mouse models. C57BL/6J mice were treated with antibiotics (cefoxitin in the drinking water) for 2 days to clear commensal microbial communities before starting the MDA training session. The sweetened condensed milk/water solution was administered once daily consecutively for 3 days prior to treatment administration. Mice were briefly restrained by gentle scruff during MDA treatment administration. On day 4, mice were treated once with PBS (nega...

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

use-micropipette-guided-drug-administration-as-an-alternative-method

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

869 Views.  Johns Hopkins University School of Medicine. Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort. 

Video: Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Neuroscience

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Behavior

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Biology

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

Medicine

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

Compound Administration I

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
As many research protocols require that a substance be injected into an animal, the route of delivery and the amount of the substance must be accurately determined. There are several routes of administration available in the mouse and rat. Which route to use is determined by several factors of the substance to be injected: the pH of the solution, the volume required for the desired dosage, and the viscosity of the solution. Severe tissue damage can occur if a substance is administered incorrectly. This video looks at the various restraint methods and technical details for the most commonly used injection routes.

Compound Administration in Rodents- Injection Techniques

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
As many research protocols require that a substance ...

Education

JoVE Science Education

Basic Biology

Lab Animal Research

Compound Administration II

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN

Compound administration is often an integral component of an animal study. Many factors need to be evaluated to ensure that the compound is delivered correctly. The route of administration affects the mechanisms of absorption. The characteristics of the substance to be introduced (the pH, viscosity, and concentration) may dictate which route of administration is selected.1,2,3

Compound Administration in Rodents- Oral and Topical Routes

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN

Compound administration is often an integral ...