Gavage-Needle Voluntary Consumption Administration of a Dose-Specific Measure to Mice (Mus musculus)
Orogastric gavage is a common technique used to administer test articles to rodents with risks ranging from increased stress to death of the animal. In this study, we propose a novel technique to administer treatments to mice for voluntary consumption through a plastic gavage needle: gavage-needle voluntary consumption (GVC) with a sweetened condensed milk carrier. GVC was successful at orally dosing mice in 95.0% (n = 459) of 483 total GVC opportunities. BALB/c mice (n = 61 [27 males/34 females] for analysis) were randomized into 8 intervention-treatment groups with 2 treatment allocations. Mice habituated to the GVC technique in less than 5 d and continued to consume the condensed milk with or without capromorelin for a 3-d treatment period that commenced after receiving their assigned interventions. The GVC technique allowed for efficient transition to orogastric gavage when the treatment was not consumed in 3.8% (n = 7) of 183 GVC attempts in the treatment period, thereby ensuring that every mouse received its treatment. The proportion of mice that consumed their treatments voluntarily was not affected by the intervention the mice received prior to the treatment period but was affected by the addition of the treatment article capromorelin. It is recommended to consider the GVC technique as a replacement and refinement to oral gavage when orogastric administration of treatments to mice is required.
Introduction
Oral dosing of medications is often used in humans because it is convenient, cost-effective, and safe. Oral administration is also a common method of administration of test articles to rodents and other animals. 32 Mimicking the route of administration in in vivo studies to the route of administration in humans increases translatability of the research. 28 Oral administration of compounds in rodents is often mimicked by orogastric administration, with a recent review showing that 89% of recent oral vaccine studies in rodents used orogastric gavage; 18 however, direct gastric administration in humans and rodents may alter pharmacokinetics 40 and fully bypass the oral cavity. Administration of medications through the oral cavity allows for direct absorption through oral mucosa, 39 stimulation of the immune system, 31 and physiologic responses to oral stimuli. 15 Sugar and fats in the mouth could be particularly important for stimuli in studies involving gustation, mastication, body weight, stimulation of gastrointestinal motility, and/or enzymatic digestion. 15,22,33,38,41
Confidence that a precise dose of test article is administered to an animal is a benefit of direct gastric administration. In addition, gavage is useful when a drug is odiferous or unpalatable and, therefore, unpleasant for voluntary consumption, or when an animal is experiencing dysphagia due to illness or disability. Gavage needles with a small smooth knob at the end come in variable lengths and gauges to accommodate different species and viscosity of solutions to be given via gavage. The disadvantages of direct gastric administration via gavage have been reviewed elsewhere. 1,3,4,9,19,34 There have been efforts to seek alternatives to or refinements for orogastric gavage in rodents, including voluntary administration through consumption of compounded water solutions, gels, and foods or treats. 5,7,8,19,21,29,35,36 Coating gavage needles with sucrose has been shown to reduce time to passage and decrease stress reactions to gavage in mice. 12 Most voluntary consumption methods rely on calculations of normal consumption amounts to estimate the consumed dose of medication added to the formulation, and consumption over a prolonged period may not reach peak concentrations that can be obtained when given as a full dose at a defined period. 25 Animals may have to be singly housed, at least temporarily, to accurately account for intake, 19 and food or water deprivation may be used to encourage consumption. 24 In both instances, animal welfare must be closely monitored to ensure health and provide adequate social and environmental enrichment. 13
The micropipette-guided drug administration (MDA) method 25 has proven an effective means of precisely orally dosing mice 6,25,26 and rats 11 with minimal stress. The MDA method involves administering a test article in a palatable solution directly into a rodent’s mouth for voluntary consumption using a micropipette for accurate, timed dosing. Syringe feeding has been proven as a means of delivering oral medications to rats in a sweetened solution while minimizing stress. 2,27,30 Both the MDA and syringe feeding allow for administration of a known dose of test article at a defined time; however, neither method can account for what to do if an animal refuses to voluntarily consume the solution.
The initial objective of this research study was to investigate the effect of capromorelin, a ghrelin receptor agonist, on body weight in mice after receiving various research-related interventions; those results have been published. 23 A secondary aim of this study, and the primary objective of this paper, was to explore voluntary consumption of a medicated solution through a gavage needle (gavage-needle voluntary consumption [GVC]). We hypothesized that mice could habituate to GVC, and they would continue to consume a condensed milk solution voluntarily after adding capromorelin to the solution. The benefit of using the gavage needle was that the medication could be given via gavage with minimal extra effort, time, or wastage if the mice failed to consume it initially. The utility of a plastic gavage needle to administer a defined dose of medication to mice for voluntary oral consumption has not been investigated prior to this study.
Materials and Methods
This research was conducted under a United States Army Medical Research Institute of Infectious Diseases (USAMRIID) IACUC-approved protocol in compliance with the Animal Welfare Act, Public Health Service Policy for the Humane Care and Use of Laboratory Animals, and other federal statutes and regulations relating to the use of animals in research. Animals were housed in an AAALAC-accredited facility that adheres to principles stated in the Guide for the Care and Use of Laboratory Animals. 13
Animals.
Seventy BALB/c mice (34 males, 36 females) were acquired from Charles River Laboratories (Wilmington, MA). The mice were aged 98 to 112 d at the start of the protocol. There was attrition throughout the study due to fighting, unexplained death, and humane euthanasia due to clinical decline of unattributable cause: 3 after arrival (1 male and 1 female with unknown cause of death; 1 male due to fighting), 3 during habituation (1 male with unknown cause of death; 2 males due to fighting), and 3 during the intervention phase (1 male and 1 female with unknown cause of death; 1 male humane euthanasia). Sixty-seven mice were used in the habituation phase, and 64 mice received interventions and treatments (29 males, 35 females). The 3 mice that were lost during the intervention phase were excluded from statistical analysis as to not confound clinical decline with a failure to voluntarily consume the treatment or control solution. Therefore, 61 mice were included in the data analysis (27 males, 34 females).
The mice arrived to USAMRIID with documentation that they originated from a colony free of murine respirovirus (Sendai), pneumonia virus of mice, mouse hepatitis virus, murine norovirus, mouse parvoviruses, reovirus, epizootic diarrhea of infant mice, mouse encephalomyelitis virus, ectromelia virus, lymphocytic choriomeningitis virus, murine adenovirus 1 and 2, murine cytomegalovirus, K virus, polyoma virus, hantavirus, lactate dehydrogenase–elevating virus, murine chapparvovirus, Bordetella bronchiseptica, Citrobacter rodentium, Corynebacterium bovis, Filobacterium rodentium, Mycoplasma pulmonis, Salmonella spp., Clostridium piliforme, Streptobacillus moniliformis, Streptococcus pneumoniae, Rodentibacter pneumotropicus, Corynebacterium kutscheri, Rodentibacter heylii, Helicobacter bilis, Helicobacter hepaticus, and pathogenic endoparasites, helminths, and protozoa. There were no known pathogens endemic in the USAMRIID mouse colony. The mice were allowed a minimum of 2 wk of acclimation. Weights after acclimation and prior to randomization ranged from 20.51 to 31.50 g (mean ± SD = 25.767 ± 3.419 g [males, 28.920 ± 1.762 g; females, 22.883 ± 1.395 g]). After randomization, the mice were housed in same-sex groups of 4 to 6 mice by intervention-treatment group, except when fighting necessitated removing males for individual housing (19 males); all intervention-treatment groups had at least 1 male removed, resulting in remaining male groups of 2 to 3 mice. The microisolators were maintained in the same position on the rack after randomization, unless mice were separated after randomization.
Mice were housed in individually ventilated microisolation caging (Tecniplast, West Chester, PA) with pressed cellulose bedding (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, MI). Their cages included an assortment of enrichment items such as crinkled paper (Enviro-dri, Shepherd Specialty Papers, Kalamazoo, MI), tissue paper (certified Rodent Nesting Sheets, Bio-Serv, Flemington, NJ), Manzanita gnaw sticks (Bio-Serv, Flemington, NJ) cotton nesting squares (Nestlets, Ancare, Bellmore, NY), and plastic igloos and forts (Bio-Serv, Flemington, NJ). Microisolator cage bottoms and disposable enrichment items were changed weekly. Microisolator lids, durable enrichment devices, and cage card holders were changed every 2 wk. The mice were fed a standard laboratory rodent diet (5001 Rodent Diet, Lab Diet, Brentwood, MO) ad libitum. They were provided filtered (5-μm) domestic water via Lixit bottles ad libitum. Water from animal rooms was tested quarterly (DoD Food Analysis and Diagnostic Laboratory, San Antonio, TX) for heavy metals, chlorinated hydrocarbons, nitrates, and microbial contamination, with no aberrant results on all reports reviewed for the year prior to this study. Mice were offered a standard volume of forage mix twice weekly; the forage mix was made in the facility from commercially available grain cereals. Housing rooms were maintained on a 12-h light/12-h dark cycle (lights on at 0600, lights off at 1800) with fluorescent lights. Humidity was controlled between 30% and 70%, and temperature was controlled between 68 and 76 °F (20 and 24.4 °C; set point 74.5 °F [23.6 °C]). The mice were checked no less than twice daily by animal care and veterinary staff to ensure adequate health and welfare.
Experimental design.
A prospective, randomized, factorial experiment was designed to study the effects of capromorelin on body weight in mice that received specific medical and/or surgical interventions. 23
One week before study activities started, the mice were weighed, ear tagged, and randomized using SAS Proc Plan (SAS 9.4, SAS, Cary, NC) into 8 intervention-treatment groups with 2 treatment allocations: capromorelin (t) or control (o), and 4 intervention allocations, based on interventions received on day 7: no intervention (group N [none]); buprenorphine extended release (XR) alone (group B [buprenorphine XR]); buprenorphine XR, meloxicam, and isoflurane anesthesia (group I [isoflurane]); or surgery under isoflurane anesthesia with buprenorphine XR, meloxicam, and bupivacaine administered (group S [surgery]). The statistician that performed randomization was blinded with regard to the intervention and treatment groups.
Intervention-treatment groups were initially balanced by sex and weight; however, more males than females were lost during the initial acclimation and habituation periods without enough unassigned males that had completed the habituation period remaining to assign to the corresponding group; to maintain statistical power between intervention groups, additional females were added to the corresponding female intervention groups from which the males were lost.
Habituation.
Daily during the 5-d habituation period (days 0 to 4), mice were individually weighed and subsequently offered 0.1 mL of condensed milk from a 20-gauge, 38-mm medical-grade polypropylene gavage needle (Instech Laboratories, Plymouth Meeting, PA) attached to a 1.0-mL Luer lock syringe (Figure 1). Sweetened condensed milk (Meadow Gold, Eagle Foods, Cleveland OH) and capromorelin solution were held at room temperature, 68 to 76 °F (20 to 24.4 °C; set point 74.5 °F [23.6 °C]), as recommended in the capromorelin formulation product insert 10 and on the condensed milk label. A new condensed milk can was opened daily. Habituation started at approximately 0900 daily. Mice were allowed to sit on the food hopper, held in the palm of the technician’s hand, or lightly held by the scruff of the neck, based on the technician’s preference and their evaluation of the mouse’s preference. The gavage needle’s elastomer tip was gently placed at the mouse’s lips while a small volume of condensed milk was expressed. If the mouse did not begin licking the needle tip within approximately 10 s, the tip would be introduced into the rostral portion of the mouse’s mouth to encourage them to consume the milk. Each mouse’s daily acceptance of condensed milk was documented. Mice that did not voluntarily consume condensed milk during the habituation period did not receive the dose via gavage. There was a scheduled 2-d period between the habituation period and when interventions and treatments started on day 7.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-139
Interventions.
Mice in intervention group N (none) received no interventions. Mice in intervention group B (buprenorphine XR) received 3.25 mg/kg buprenorphine XR injectable suspension (Ethiqa XR, 1.3 mg/mL, Fidelis Animal Health, North Brunswick, NJ) subcutaneously. Mice in intervention group I (isoflurane) received 3.25 mg/kg buprenorphine XR injectable suspension subcutaneously and 6 mg/kg meloxicam (Alloxate, 5 mg/mL, Pivetal, Liberty, MO) subcutaneously after anesthetic induction with isoflurane. Mice in intervention group S (surgery) received 3.25 mg/kg buprenorphine XR injectable suspension subcutaneously and 6 mg/kg meloxicam subcutaneously immediately after anesthetic induction, and a 3 mg/kg bupivacaine hydrochloride (2.5 mg/mL, Hospira, Lake Forest, IL) splash block after surgical incision. The detailed surgical procedure has been described. 23 Briefly, the mice in groups I and S were anesthetized with isoflurane in 100% oxygen (3% to 5% isoflurane induction; 0.5% to 3% isoflurane maintenance) with a precision vaporizer via a rodent anesthesia machine. Group S mice received a simple laparotomy procedure with no internal organ manipulation using aseptic techniques. The median surgery time for group S mice was 13 min, so each mouse in group I was also anesthetized for 13 min. All mice were monitored and provided supportive care until ambulatory, with full recovery within 3 to 6 min. The anesthetized mice were observed 4 h after anesthesia to ensure welfare and surgical site integrity (group S mice).
Treatments and observations.
Control-assigned animals received 0.1 mL of condensed milk only, and capromorelin-assigned animals received 0.1 mL of condensed milk with flavored capromorelin oral solution (Elura, 20 mg/mL, Elanco, Greenfield, IN). Capromorelin was dosed 10 mg/kg with a 100-µL variable volume, single-channel pipette, and the dose was mixed into the condensed milk in the hub-end of the syringe to ensure that the mouse received the entire dose. The needle hub residual volume was 70 µL, and the gavage needle residual volume was 7 µL, so the 0.1 mL (100 µL) of unmedicated condensed milk in the syringe flushed all medicated solution through the gavage tip to the mouse. A treatment syringe was prepared for each mouse. The gavage needle was transferred between treatment syringes for up to 6 mice; each new pan of mice received a new gavage needle to ensure that no control group mice used a gavage needle that had touched capromorelin. The gavage needle was inspected for damage between each mouse, with no damage to the needles noted. All mice were offered their treatment for GVC in a similar manner to the habituation period; however, the dose was administered via oral gavage with scruff restraint if the mouse did not consume the treatment voluntarily. Each mouse’s route of daily treatment administration, voluntary or gavage, was documented.
Mice were given their assigned treatment for 3 d (days 7 to 9), approximately 24 h apart starting around 0900. Treatment duration was based on the primary objectives of the designed study. 23 Mice were given their first treatment on day 7 after receiving their assigned interventions and fully recovering from anesthesia, as applicable. Four technicians with 4, 9, 18, and 23 y of experience, respectively, conducted the treatments. The technicians administering the treatments were blinded to treatment assignment, but not necessarily intervention due to the presence of a surgical incision on group S mice.
The mice were observed by study personnel daily during the habituation and treatment phases, and their appearance and behavior were scored using a standardized chart to ensure welfare and identify adverse outcomes during the treatment phase. Each mouse’s appearance and behavior were scored from 0 to 3, with 0 being normal and 3 being hunched with piloerection and immobile. Mice that received surgery also had their surgery sites inspected for evidence of infection or dehiscence. Measurements and treatments were conducted in the same order each day, starting with the treated group N females, and finishing with untreated control group S males.
Statistical analysis.
Voluntary consumption frequency by animal was calculated as a binary outcome variable indicating whether a mouse consumed the offered condensed milk during the period of interest. Failure-to-consume event frequency was calculated as number of times an animal failed to consume the dose over all feeding opportunities during the period of interest. Frequencies were calculated separately for the 5-d habituation period (days 0 to 4) and for the 3-d treatment period (days 7 to 9). In the treatment period, a failure-to-consume event was equivalent to a gavage event.
The study design had the following 4 factors: capromorelin treatment; buprenorphine XR; anesthesia + meloxicam; and surgery + bupivacaine. A full factorial design was considered to allow for examination of multiple 2- and 3-way interaction effects with a reduced sample size; however, many of the possible factor combinations would have been unethical to perform, as there may have been unalleviated pain or distress. The main treatment effect on body weight comparing capromorelin with condensed milk only (control) was deemed the most important factor for investigation. Four ethically permissible combinations of the other 3 factors were identified and together were defined as the intervention factor. The intervention factor had 4 levels: 1) no intervention (control); 2) buprenorphine XR alone; 3) buprenorphine XR + anesthesia + meloxicam; and 4) buprenorphine XR + anesthesia + meloxicam + surgery + bupivacaine. Interactions between the treatment effect and the ethically permissible interventions were examined using a 2 × 4 factorial design, with specific pairwise intervention comparisons of interest as follows: 1) no intervention compared with buprenorphine XR alone; 2) buprenorphine XR alone compared with buprenorphine XR + anesthesia + meloxicam; and 3) buprenorphine XR + anesthesia + meloxicam compared with buprenorphine XR + anesthesia + meloxicam + surgery + bupivacaine.
A power analysis of the primary outcome measure of weight was conducted using PASS 14 (NCSS, Kaysville, UT) prior to the start of the study for the specific pairwise comparisons of interest. A post hoc power analysis of the exploratory outcome measure of failure-to-consume frequency was also conducted. With a sample size of 8 subjects per treatment combination, and a total number of 16 subjects, this design achieved greater than or equal to 78% power to detect an effect size of 0.5 for each factor and 1.0 for the interaction effect. Group sample sizes of 29 in the capromorelin group and 32 in the control group achieved 80% power to detect a minimum difference between the group proportions of 0.324. The test statistic used is the 2-sided Fisher exact test with a significance level of 0.05.
The difference in the distribution of failure-to-consume frequency between treatments, between interventions, and between sexes was compared using either χ2 tests or, in cases where at least 50% of the cells had expected counts less than 5, Fisher exact tests. The difference in the distribution of failure-to-consume frequency between treatments controlling for the effects of either sex or intervention was compared using a Cochran–Mantel–Haenszel test for ordinal data (general association statistic). As these analyses were purely exploratory and not the main objective of the original study, corrections for multiple comparisons were not performed. SAS version 9.4 (SAS Institute, Cary, NC) was used for all analyses. Descriptive statistics are presented as number and percentage of animals that failed to consume the condensed milk during the habituation phase and number and percentage of animals receiving gavage during the treatment phase. In addition, descriptive statistics are presented as number and percentage of events in which an animal failed to consume the condensed milk during the habituation phase and number and percentage of events in which an animal received gavage during the treatment phase events. The level of significance was set at P < 0.05. All tests were 2-tailed. No adjustments for multiple comparisons were made. Missing values were treated as missing at random, and values were not estimated based on nonmissing values from the same animal. The total number of mice used for analysis was 61. The statistician performing the analysis was not blinded.
Results
Habituation period (days 0 to 4).
All (n = 61, 100%) mice consumed the unmedicated sweetened condensed milk for at least 3 out of 5 d it was offered for GVC during the 5-d habituation period. All mice voluntarily consumed the condensed milk on days 3 and 4. There were no significant differences in the proportion of the 32 control mice (n = 5, 15.6%) and the proportion of the 29 treated mice (n = 9, 31.0%) that failed to consume compared with mice that voluntarily consumed the milk (P = 0.153) over the habituation period. This lack of significant difference held true for the proportion of animals on each individual day and the proportion of opportunities in which the mice failed to consume the condensed milk over the habituation period (4.4% compared with 7.1%; P = 0.330) (Figure 2). The effect of sex was significant over the habituation period; proportionately more of the 27 males (n = 10, 37.0%) than the 34 females (n = 4, 11.8%) failed to consume the condensed milk at least once during the period (P = 0.020), and there were proportionately more failed consumption events for males (9.2%) compared with females (2.9%; P = 0.024) (Figure 2); however, the effect of sex was not significant on any individual day (P day0 = 0.123; P day1 = 0.404; P day2 = 0.275; P days3&4 values were not calculated, as all mice voluntarily consumed the milk).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-139
Treatment period (days 7 to 9).
All (n = 61, 100%) of the mice voluntarily consumed their treatment on at least 2 d during the 3-d treatment period; no mouse had to receive their treatment by gavage more than once during the treatment period. Overall, gavage events were a small proportion of the total administration events, with only 3.8% of the administration opportunities resulting in gavage (Figure 3), or 7 (11.5%) of the 61 mice in the study receiving gavage at some point during the treatment period. The effect of sex on voluntary consumption compared with gavage was not significant over the 3-d treatment period or on any individual day during the treatment period (P > 0.05).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-139
A comparison of gavage events by intervention group shows that 2 mice each in the treated intervention groups N(t) and B(t), as well as 3 mice in intervention group S (2 treated, 1 control), received their solution by gavage (Supplemental Table 1). Each mouse received solution via gavage once. There was not a significant difference in the percentage of opportunities (P = 0.525) or percentage of mice (P = 0.490) per intervention group that required gavage. No mice in intervention group I required gavage.
There was a significant difference between the proportion of 32 control animals (n = 1, 3.1%) that received gavage at least once compared with the proportion of 29 treated animals (n = 6, 20.7%) requiring gavage at least once (P = 0.042) over the 3-d treatment period (Table S1). When controlling for possible intervention effects, the proportion of gavaged animals between treatment and control was still statistically significant (P = 0.041). On each individual day in the treatment period, there were no significant differences in the proportion of animals receiving gavage between controls compared with capromorelin-treated mice (P > 0.05). The mice that had to be gavaged were only gavaged once over the 3-d treatment period, and therefore 6.9% of the capromorelin treatment opportunities resulted in gavage events, which was not significant compared with the 1.0% of the control opportunities that resulted in gavage events (P = 0.055) (Figure 3; Table S1); however, the difference between the proportion of opportunities resulting in gavage between treatment and control mice was statistically significant when controlling for possible intervention effects (P = 0.049) or sex (P = 0.045)
Discussion
This study demonstrated the utility of a plastic gavage needle to administer a defined dose of medication to mice for voluntary oral consumption such that the dose could be easily given via oral gavage if not consumed. GVC was successful at orally dosing mice in 95.0% (n = 459) of 483 GVC opportunities over the habituation and treatment periods. Voluntary consumption of oral solutions represents a significant refinement over gavage-only administration. The mice habituated to voluntary consumption of sweetened condensed milk through a gavage needle in less than 5 d and continued to consume the condensed milk when flavored capromorelin treatment was added, although more gavage events were required when capromorelin was added compared with controls (6.9% compared with 1.0%) (Figure 3). The interventions the mice received did not have an effect on the proportion of the mice that voluntarily consumed their treatment in the GVC method. Investigators should consider that sex may influence their success with the GVC method because the effect of sex on success of voluntary consumption was suggestive of increased failure rates in male mice over the habitation period (P = 0.020) (Figure 2), but a significant effect of sex was not seen in the treatment period, based on results of Cochran-Mantel–Haenszel tests.
The primary aim of the research conducted in conjunction with this GVC study was of the effect of capromorelin on body weight in mice. The positive effect of capromorelin on weight was proven, thereby demonstrating that a clinically effective dose was administered to the mice regardless of treatment administration route, GVC or gavage. 23 Specifically, when gavage was entered in the model for weight change, the method of treatment administration was not a significant factor that affected the change seen in weight (P = 0.9078).
The results of this study are consistent with research on other methods of voluntary oral administration to rodents demonstrating minimal training time and high rates of voluntary consumption of sugar-based solutions. 2,11,25,30 This study used 5 d of habituation and training, although a shorter period may have also been successful because all mice were voluntarily consuming the condensed milk by the fourth day of habituation. This present study did not explicitly evaluate the permanence of the habituation to voluntary consumption nor did the other studies cited herein. Further studies should be conducted to evaluate various intervals between habituation and implementation of the technique in a protocol to evaluate whether the learned behavior is perishable and, if so, over what period for different rodents.
Limitations of the GVC method are that the test article must be compatible with a carrier such as sweetened condensed milk, available in a solution form, and small enough to fit through the gauge of gavage needle that is also species appropriate for gavage. Furthermore, the test article must be appropriate for oral administration and not cause unexpected pharmacokinetic parameters, gastrointestinal dysbiosis, or physical harm if administered orogastrically. Pharmacodynamic or pharmacokinetic studies with the GVC method may be warranted based on research aims of investigators and their test article. Voluntary consumption may not be appropriate for all study types. For example, gavage better replicates medications given directly to the stomach through nasogastric, orogastric, or percutaneous endoscopic gastrostomy tubes. Animals may expel or chew substances administered into their mouths, and chewing may damage modified-release preparations, leading to damage of the drug at best, and severe adverse effects at worst.
The ability to seamlessly convert to gavage, if needed, ensures that investigators have confidence that their test article will be administered to the animal if they do not voluntarily consume the solution in the GVC method. A limitation of this method is that the volume must be one that could be comfortably consumed or administered via gavage, optimally 5 mL/kg or less. 32 Voluntary consumption could be altered by strain of mice due to difference in preference for sucrose and acid, 20 so a backup method of administration would be important if GVC is used for a strain with lower voluntary consumption rates or if the test article was unpalatable.
No adverse effects from administration were seen from any administration method in this study. The benefit of using a voluntary consumption technique extends beyond improved animal welfare to the welfare of the personnel conducting the interventions. Working with animals is an emotional experience for both animals and the personnel, with higher levels of compassion fatigue and lower compassion satisfaction in individuals that cause more stress to the animals in their care. 14 Gavage is a known stressor to rodents, 1,3,4 and voluntary consumption methods help avoid that stress and provide some level of choice and control to the animal.
A soft feeding tube as an alternative to gavage was demonstrated to reduce risk and discomfort in rats. 37 The present study could be repeated in mice using a flexible feeding tube first for voluntary consumption with the ability to convert to gavage if needed. The GVC technique could also be explored in other strains of mice and in other rodents as a potential refinement for oral dosing. Recent developments in oral drug delivery systems, such as orodispersible tablets and films 16 and buccal and sublingual vaccines, 17,31,38 should continue to be explored for laboratory species to refine techniques to improve animal welfare and translatability of in vivo research.
In summary, BALB/c mice habituated to GVC of sweetened condensed milk in less than 5 d. The mice continued to voluntarily consume a solution of condensed milk combined with a test article, that is, capromorelin oral solution, thereby significantly reducing the number of mice that needed to be gavaged and, similarly, the number of gavage events over a 3-d treatment period. The GVC method allowed for seamless conversion to orogastric gavage if the treatment was not consumed. The utility of a plastic gavage needle to administer a defined dose of medication to mice for voluntary oral consumption was proven with this study. The GVC technique is available as a replacement and refinement for oral gavage when studies or treatments necessitate orogastric administration to mice.
Supplementary Materials
Table S1. Number and proportion of gavaged mice and gavage events by treatment and intervention over the treatment period (days 7 to 9)
GVC dosing syringe: gavage needle attached to syringe with 0.1 mL of sweetened condensed milk. Capromorelin, when given, was pipetted directly into the hub of the syringe and gently stirred into the condensed milk in the hub prior to filling the gavage needle. The unmedicated condensed milk flushed all medicated solution through the syringe and gavage needle with a residual volume of 77 µL.
Percent of GVC events in which the mouse failed to voluntarily consume the condensed milk during the habituation period (days 0 to 4). Animals were assigned to capromorelin or control groups, but no capromorelin was given in the habituation period. The results were significant (*) between sex for capromorelin-assigned mice (P = 0.045) and for both treatment groups combined (P = 0.024).
Percent of GVC events that required gavage during the treatment period (days 7 to 9). The results were significant (*) between all capromorelin and all control mice when controlling for intervention (P = 0.049) or sex (P = 0.045).
Contributor Notes
This article contains supplemental materials online.