Long-Term Storage of a Natural-Ingredient Diet within Variable Conditions of Temperature and Humidity

Abstract

Institutions with aging structures may face difficulties maintaining consistent temperature and humidity for feed storage due to continued daily use of older heating, ventilation, and air conditioning systems and maintenance of structural integrity since the environmental efficiency of buildings often reduces as structures age. Consequently, institutions can face difficult financial decisions regarding whether repairs or new structures should be considered when compliance with standards is inconsistent. Replacing running or currently functioning heating, ventilation, and air conditioning systems that lack efficiency in comparison with newer systems is often not considered wise use of institutional resources. As this concern was faced and discussed during a recent campus AAALAC site visit to our institution, our IACUC requested additional information related to safe storage of feed in our current older buildings. Three test group environments of control/Guide for the Care and Use of Laboratory Animals (Guide)-recommended temperature/humidity, variable temperature/humidity, and high temperature/Guide-recommended humidity were used to store feed for a total of 6 mo. Full feed analysis, retinol levels, thiamine levels, and mold/yeast levels were evaluated at 0, 3, and 6 mo following storage. Study findings demonstrated that the nutritional content for feed remained relatively equivalent across the timespan as well as across the different storage conditions. More importantly, all storage conditions showed no increases in yeast/mold growth and acceptable levels of both thiamine and retinol at 6 mo. Our findings suggest that the diet tested was still usable for feeding animals after storage in the tested conditions that fall outside of Guide parameters and that other institutions may consider feed stability evaluation when addressing challenges with maintaining Guide parameters in feed storage spaces.

Introduction

Nutritional guidelines for laboratory rodents were most recently updated by the National Research Council (NRC) in 1995.¹ The modifications of the AIN-73 diet to the AIN-93 diet included mineral and vitamin mixtures as well as establishing that different life stages require additional nutritional supplementation, thereby affirming the need for maintenance, growth and breeding diets.² The nutritional guidelines created in 1995 acknowledge that there are far fewer nutritional studies related to the management of mice, yet studies^3,4 have assumed that nutritional needs of maintenance are met through growth diets. Many studies^3–6 throughout the last few decades of the 20th century focused on improving research rodent diets and improving feed stability to exclude diet as a potential variable in studies.

Though it is often assumed that standard rodent diets adequately meet the nutrtional needs of the animals, genetically unique strains can pose challenge with respect to Diet and nutrition are often overlooked variables in research involving rodents. Consistency in diet formulation was considered “imperative” enough that appropriate nutrients were provided while also providing minimal exposure to contaminants.⁸ The Guide states, “Storage of natural-ingredient diets at less than 21 °C (70 °F) and below 50% relative humidity is recommended.”¹ Storage conditions that deviate from these guidelines may impair food quality.^1,9 Surprisingly, there are only a handful of other resources that discuss proper storage conditions for rodent feed. For example, previously published storage guidelines state, “Diets stored at high temperatures and humidity may deteriorate within several weeks.”⁴ Food stored within a “warm, unventilated room” would spoil or lack nutritional adequacy faster than a diet stored at 15.5 °C.⁸ These narratives prompted additional studies that evaluated feed storage length and nutritional adequacy. In the past, it was determined that the ability to extend the shelf-life of rodent diet past 30 d could allow for reproducible experiments of longer duration in addition to reducing operational costs and labor for an animal facility.¹⁰ Feed stability evaluation showed that the rodent diet storage could extend to 130 d postautoclaving if stored at 18.5 °C and 50% humidity.¹⁰ Refrigeration significantly extended the shelf-life of food when stored at 4 °C.¹¹ Appropriate testing including feed stability, ingredient potency, and bioavailability/concentration of ingredients would be needed if bags of feed undergo extended storage.^10,11 As new means to extend shelf-life developed, such as fortifying vitamin content of diets and formulation of vitamins that slow the degradation process, research on rodent diet nutritional stability slowed considerably until a recent publication in 2023.¹² Further, the “recommended” set of the ARRIVE Guidelines 2.0 suggests including in manuscripts the descriptions of extrinsic factors, such as diet, that may affect study outcomes and which could also be interpreted to include feed storage conditions.¹³ Research institutions that may not have been facing issues with adequate macroenvironmental control for food storage during earlier years could face such issues infrastructure ages and wears. Temperature can significantly impact the stability and bioavailability of some rodent diet nutrients and remains a very important factor in ensuring that rodent diets do not become a confounding variable within research.

Two of the most labile ingredients within a rodent diet are vitamin A (retinol) and vitamin B₁ (thiamine).^4,11,14,15 Deficiencies of these 2 vitamins can result in growth deficiencies, reproductive dysfunction, immunosuppression, musculoskeletal abnormalities, and neurologic dysfunction.^4,7 Exposure to increased temperatures can decrease thiamine levels when feed is stored at temperatures greater than 20 °C (68 °F).¹¹ Thiamine levels were shown to decrease in concentration by 50% when stored above 20 °C at 45 d and by 50% when feed was stored at 20 °C at 80 d.¹¹ Retinol levels were shown to steadily decrease in concentration by 41.3% following 168 d in storage.¹¹ Refrigeration of rodent feed at 4 °C preserves and extends the shelf life of both thiamine and retinol concentrations in rodent feed.¹¹ Recent concerns with macroenvironmental stability during a site visit by AAALAC International prompted us to explore extended storage of feed under conditions that are beyond the recommendations of the Guide. The Teklad 2018SC diet is used across our campus and is composed of both cereal grains and nationally sourced ingredients to compose a nutritionally complete diet according to the NRC guidelines.^16,17 This diet is one of the most commonly used research diets for laboratory rodents, and the manufacturer currently advises the shelf-life of the diet to be 9 mo, which may include minor deviations from appropriate storage conditions, as they are likely not to affect diet quality due to manufactured excess of nutrients.^16,17 However, the minor deviations from appropriate storage are not listed in detail. This study was designed to push the boundaries of minor deviations into major deviations from Guide storage recommendations. Our hypothesis is that the natural-ingredient diet Teklad 2018SC can maintain appropriate nutrient concentration, including for the 2 most labile vitamins, retinal and thiamine, when storage conditions deviate from the Guide.

Materials and Methods

Feed source and storage sites.

This study was completed within an institution accredited by AAALAC International. Within the facilities, animals are housed within IVC or Innovive (San Diego, CA) disposable caging on a 12:12 light/dark cycle. Excluded pathogens for the rodent facilities included the following: Mycoplasma pulmonis; mouse hepatitis virus; minute virus of mice; mouse parvovirus; murine norovirus; Theiler murine encephalomyelitis virus (GDVII); epizootic diarrhea of infant mice/mouse rotavirus; mouse adenovirus 1 and 2; reovirus 1, 2, and 3; pneumonia virus of mice; Sendai virus; ectromelia; hantavirus; pinworms; polyomavirus; lymphocytic choriomeningitis virus; rat coronavirus/sialodacryoadenitis virus; rat parvovirus; Killham rat virus; Toolan H-1 virus; rat minute virus; pinworms; fur mites; cilia-associated respiratory bacillus; and Pneumocystis. Norovirus is excluded from most facilities. Helicobacter is tested for annually but not excluded as a pathogen. The 2018SC Teklad rodent diet (Inotiv, West Lafayette, IN) from lot T.2018SC.15 and mill date February 20, 2024 was autoclaved upon receipt, and 90 bags were transported and equally divided to 3 different experimental groups used on campus from April to October 2024 for measurements to occur within the hottest periods of the year. This number of bags was informed by a power analysis tuned for detecting medium-large effect sizes. The Teklad 2018S diet is packaged within a perforated 2-ply paper bag to allow steam penetration during autoclaving; this design would also render the feed susceptible to increased macroenvironmental humidity. All 3 sites were located away from normal feed storage locations to prevent usage, and bags were marked as “do not disturb.” All feed bags were stored on pallets or wire shelving in accordance with Guide recommendations. Site 1 stored 20 bags at Guide recommended temperature and humidity levels, at less than 21 °C and less than 50% relative humidity (Figure 1).¹ Site 2 stored 20 bags as the variable temperature/humidity site, with minimum temperatures slightly above 20 °C (68 °F) and maximum temperatures near 27.2 °C (82 °F) with relative humidity ranging from 22% to 93% (Figure 1). Site 3 stored 20 bags at 26.7 °C (80 °F) with relative humidity below 50% (Figure 1). Both site 1 (control) and site 3 (hot/normal humidity condition) were located as interior rooms to a building on campus away from drafts, autoclave, or delivery dock to minimize fluctuation in room settings. Site 2 was located within a large autoclave room adjacent to a loading area where temperatures and humidity were known to fluctuate greatly. All locations had daily minimum, maximum, and current temperature/humidity readings using the Avantor VWR Therm/clock/humidity monitor (VWR International, Radnor, PA).

Figure 1.

Humidity and temperature ranges for the control/Guide-suggested storage conditions (red), variable temperature and humidity (green), and hot with normal humidity (blue) conditions across the experimental time frame (April 2024 to October 2024). Ranges are represented as shaded fills where the bottom indicates the minimum value and the top indicates the maximum value recorded on the given date.

Feed sampling and measurements.

Feed bags were stored at 3 sites corresponding to the experimental conditions discussed above. Once in place, bags were left for their designated storage period. This process did not apply to bags sampled on day 0, baseline measure, which were sampled shortly after being received and autoclaved; and these bags were never stored in any of the experimental conditions (total of 30 bags, 10 per condition site). Feed bags were sampled at 3 time points: baseline (day 0; April 17, 2024), 3 mo (July 17, 2024), and 6 mo (October 16, 2024). All feed bags were transported to a central location for sampling within a Type II A biosafety cabinet on the given collection date. All bags were sprayed down using Peroxiguard (Virox Technologies, Oakville, ON). Nitrile gloves and a new boxcutter were both cleaned/saturated with Peroxiguard, a 4 × 4-inch flap was cut into the center of the bag. Teklad 2018SC diet is packaged within a 2-ply paper bag with perforations; and no plastic liner is present to affect humidity levels within the bag. A commerical plastic bag (Ziploc; S. C. Johnson & Son Co., Racine, WI) bag placed over the gloved hand was also sprayed with Peroxiguard, air-dried, and used to grab feed pellets from this core area reaching down to the other side of the feed bag (Figure 2). Each sample from the bag consisted of feed weighing 250 to 270 g, which was the minimum weight requested by Midwest Laboratories (Omaha, NE) for analysis. 10 samples were collected for each site per time point and submitted via overnight shipping to Midwest Laboratories for the F2 feed stability assessment package, which measured protein, energy, fat, ash, moisture, and metabolizable energy (Figure 3 and Table 1). In addition, mold and yeast samples were assessed from 3 bags per site per time point. Retinol and thiamine levels were evaluated from samples taken from one bag at each site at time 0, because adequate levels were expected from each site. At 3 and 6 mo, 3 bags were sampled for retinol and thiamine levels from each group. None of the feed bags were excluded from the study; no tears or breaks in the bags occurred during handling or transport. Potential confounding effects for study design were minimized by purchasing food from the same lot number, performing consistent monitoring of the hygrometers to ensure desired environmental conditions, selecting the same 2 individuals for sample harvesting, and ensuring overnight transport of the feed to the test analysis site.

Figure 2.

Depiction of our sampling method for feed bags within a biosafety cabinet. Each bag was surface-sprayed with Peroxiguard, a 4 × 4″ opening at the center of the bag was cut using a clean boxcutter, and a core feed sample was grabbed from the center of the bag to accumulate an additive 250-270g of feed for analysis.

Figure 3.

Group means plotted for each nutrient measure across each of the baseline/day 0 (blue), month 3 (orange), and month 6 (green) time points split by control conditions (Guide-suggested storage conditions), variable conditions (variable temperature and humidity), and hot conditions (80 °F with normal humidity). The standard range of nutrient value acceptability based upon NRC recommendations is displayed as dashed, red lines for the top and bottom of the range.

Table 1.

Full statistical results for all equivalence tests conducted

			Effect of condition		Effect of time
		Effect of time and condition	Month 3	Month 6	Control	Variable	Hot
Moisture	F	51.2	206	49.35	14.73	13.96	256.7
Equiv margin: ±5	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.4959	0.9974	0.0741	0*	0*	0.9994
	Eff Size	0.57	6.87	1.64	0.49	0.47	8.56
Dry matter	F	49.26	206	49.35	12.23	13.96	256.7
Equiv margin: ±5	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.4127	0.9974	0.0741	0*	0*	0.9994
	Eff Size	0.55	6.87	1.64	0.41	0.47	8.56
Crude protein	F	4.34	0.07	0.4	6.08	3.95	8.23
Equiv margin: ±5	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0*	0*	0*	0*	0*	0*
	Eff size	0.05	0	0.01	0.2	0.13	0.27
Crude fat	F	5	1.24	2.42	14.33	3.56	5.7
Equiv margin: ±3	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0*	0*	0*	0.0004*	0*	0*
	Eff Size	0.06	0.04	0.08	0.48	0.12	0.19
Fiber	F	20.86	0.11	34.13	7.3	9.77	53.84
Equiv margin: ±5	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0*	0*	0.0037*	0*	0*	0.1231
	Eff size	0.23	0	1.14	0.24	0.33	1.79
Ash	F	11	1.38	1.99	0.38	8.44	1.65
Equiv margin: ±2	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.0014*	0*	0*	0*	0.0006*	0*
	Eff Size	0.12	0.05	0.07	0.01	0.28	0.05
Digestible nutrients	F	22.26	62.17	3.45	9.08	13.58	50.25
Equiv margin: ±5	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0*	0.2445	0*	0*	0*	0.0828
	Eff Size	0.25	2.07	0.12	0.3	0.45	1.67
Net energy	F	14.5	31.25	1.72	5.86	9.31	29.2
Equiv margin: ±0.3	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.9999	0.9995	0.0909	0.5821	0.8349	0.9992
	Eff size	0.16	1.04	0.06	0.2	0.31	0.97
Digestible energy	F	16.13	44.83	3.36	4.65	9.05	42.32
Equiv margin: ±0.3	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.9999	1	0.2809	0.4455	0.8221	0.9999
	Eff dize	0.18	1.49	0.11	0.16	0.3	1.41
Metabolizable energy	F	15.12	36.98	3.88	5.46	10.06	35.71
Equiv margin: ±0.3	df	(9, 80)	(2, 27)	(2, 27)	(2, 27)	(2, 27)	(2, 27)
	P value	0.9999	0.9998	0.3479	0.5396	0.8666	0.9998
	Eff size	0.17	1.23	0.13	0.18	0.34	1.19
Retinol	F	2.9	0.55	3.81	2.31	0.06	3.45
Equiv margin: ±10,000	df	(5, 12)	(2, 6)	(2, 6)	(1.0, 4.0)	(1.0, 4.0)	(1.0, 4.0)
	P value	0*	0*	0*	0*	0*	0*
	Eff size	0.16	0.06	0.42	0.39	0.01	0.58
Thiamine	F	1.3	0.07	2.08	0.66	6.32	0.02
Equiv margin: ±50	df	(5, 12)	(2, 6)	(2, 6)	(1.0, 4.0)	(1.0, 4.0)	(1.0, 4.0)
	P value	0*	0*	0*	0*	0*	0*
	Eff size	0.07	0.01	0.23	0.11	1.05	0

Measure, statistic, and equivalence (Equiv) range are indicated on the left. Results were calculated for the pooled effect of time and condition (first column), the effect of condition for month 3 and month 6 individually (second 2 columns), and the effect of time for the control, variable, and hot environments individually (last 3 columns). Significant results are indicated by bolded text, and significant P values are annotated with an asterisk. F statistic and η² (effect [Eff] size) are rounded to 2 decimal points. P values are rounded to 4 decimal points. If rounding results in a value of 0, then 0 is displayed without decimals.

Statistical analysis.

Sample size selection was based upon power analysis for ANOVA (0.8) and testing for large effect size. Equivalence of sample means was tested via one-way equivalence tests as implemented with the statsmodels python library. Equivalence testing is a statistical analysis wherein the equivalence of samples, rather than differences, is assessed. A significant result therefore indicates significant equivalence of the sample means as determined by the distributions’ alignment within established equivalence ranges. The equivalence range is a set of arbitrary upper and lower bounds applied to the mean difference between samples. These bounds represent the smallest effect size that would be interesting to the researchers and are selected on that basis.⁵ Equivalence ranges were set individually for each of the nutrient measures using veterinary experience and existing nutritional guidelines, with the goal of selecting equivalence ranges that would reasonably represent values that would not prompt concern if evaluated by a veterinary professional.^6,12 These equivalence ranges are detailed in Table 1.

Results

We evaluated 12 measures of food nutrition: moisture, dry matter, crude protein, crude fat, fiber, ash, total digestible nutrients, net energy, digestible energy, metabolizable energy, retinol, and thiamine. For each measure, we evaluated equivalence across each time point/location condition, for example, month 3/control location. In addition, we also conducted equivalence testing within each level of both time point and location. That is, we evaluated each measure across each location within the 3-mo and 6-mo time points individually, as well as across each time point within each of the control, variable, and hot conditions. Because this analysis produced a high number of results, we focused primarily on the pooled effect of both time point and location because if that result is significant, it indicates that a measure can be considered equivalent across all time points and storage conditions. In cases where the pooled time point/location effect was significant, we do not report further statistical results in the text; however, full statistical results are available in Table 1. Significant equivalence was detected across all time point/location conditions for crude protein (F_[9,80] = 4.34, P = 0.0), crude fat (F_{[9, 80]} = 5.0, P = 0.0), fiber (F_{[9, 80]} = 20.86, P = 0.0), ash (F_{[9, 80]} = 11.0, P = 0.0014), total digestible nutrients (F_{[9, 80]} = 22.26, P = 0.0), retinol (F_{[5, 12]} = 2.9, P = 0.0), and thiamine (F_{[5, 12]} = 1.3, P = 0.0), indicating that, for these measures, values can be considered equivalent across all locations and time points. We failed to find equivalence across all time points for moisture (F_{(9, 80)} = 51.2, P = 0.4959), dry matter (F_{[9, 80]} = 49.26, P = 0.4127), net energy (F_{[9, 80]} = 14.5, P = 0.9999), digestible energy (F_{[9, 80]} = 16.13, P = 0.9999), and metabolizable energy (F_{[9, 80]} = 15.12, P = 0.9999). However, we did find significant equivalence across time points specifically within the control and variable locations for both moisture (control: F_{[2, 27]} = 14.73, P = 0.0; variable: F_{[2, 27]} = 13.96, P = 0.0) and dry matter (F_{[2, 27]} = 12.23, P = 0.0; variable: F_{[2, 27]} = 13.96, P = 0.0).

In addition to equivalence testing, we also wanted to evaluate nutrition values against standards of acceptability. This investigation was conducted by simply applying a range of acceptable values from the 1995 NRC guidelines to the chart of nutrition measurements (Figure 3). Group means were observed to fall within the acceptable range for each time point/location condition for each measurement except retinol, which was found to exceed the maximum in the month 6 control location.

Discussion

Overall, our data show that nutrient concentration within the diets did not differ significantly over time, even with storage under elevated temperature and humidity conditions. Increases in protein concentration and energy density were expected in cases where a corresponding decrease in moisture was observed, because the reduction in moisture leads to increased concentration of other values in the feed.¹² All nutritional parameters evaluated by the F2 feed stability analysis produced levels above the NRC guidelines.^4,6,18 Protein requirements by the NRC are recommended within a range of 14% to 25%, and dietary fiber should not exceed 20%, as this can affect the growth of rats (Figure 2).^6,18 Protein loss from the autoclaving process is usually minimal, and good bioavailability was expected after extended storage periods.¹³ Appropriate fat concentration can range anywhere from 5% to 15%, which also can affect retinol absorption within the GI tract.⁶ Diets that have lower concentrations than the NRC recommendations can alter absorption of both carotene and retinol.⁶ Natural ingredient diets will typically have anywhere from 4% to 11% of fat.⁶ Recommended ash concentration is from 5% to 7%, and various vitamin concentrations are also discussed.⁶ Consequently, concern was presented for vitamin levels, specifically retinol and thiamine, after studies have shown that autoclaving can reliably reduce starting retinol levels by 40% to 50% and thiamine levels by 67% to 83% without even considering the effects of extended storage conditions that deviate from the Guide.^4,7

It has been recommended that thiamine levels be evaluated postautoclaving or postexposure to high temperatures, as this is the most heat-labile vitamin.^4,6 Thus, thiamine levels can be used as a general tool for assessment of the overall quality of a diet if additional resources are lacking to evaluate stability of the vitamin content of feed.⁶ In addition, vitamin requirements between rats and mice are thought to be very similar.³ Recommendations for both retinol and thiamine are 30,000 IU/g diet and 7 to 8 mg/kg diet for adult rats.^6,18 Carotenoids have poor bioavailability within diets, so only retinol levels were evaluated for functionality.⁶ Retinyl acetate is usually stabilized within a gelatin/carbohydrate matrix within feed that can retain 90% of its activity over a 6- mo shelf-life; and this is the vitamin A source that is added to Teklad 2018SC diet.¹⁹ Minimum retinol recommendations from the NRC are 2,300 IU/kg diet.¹⁸ Currently, our findings suggest that with diet fortification of these vitamins preautoclaving, and despite storage conditions that deviate from the Guide suggestions, additional mild heat exposure does not lead to sufficient degradation of retinol or thiamine within the diets to merit concern for nutritional deficiencies, though retinol levels did decrease over time as expected.¹¹ Vitamin concentrations, namely retinol, may vary within feed and appear to have higher concentrations at later time points, a finding that was mirrored in our study.¹⁷

Nonautoclaved natural ingredient diets can increase in mold and yeast counts when stored at higher temperatures over time.¹¹ Autoclaving of feed will drastically reduce the colony-forming units of mold and yeast; however, it is not established whether these numbers could change if diets are stored in alternative conditions.^9,13 Mold usually will not grow when the water activity level or humidity is below 0.7/70%, though 65% is usually recommended as a precautionary humidity level.⁶ Increased levels of mycotoxins can either stimulate or suppress the immune system, cause mutations, or lead to allergenic reactions.²⁰ Despite storage for up to 6 mo with humidity levels over 50% and temperatures generally over 20 °C (70 °F), both mold and yeast counts remained less than 10 colony-forming units per gram, which is the lowest reading that the feed evaluation laboratory we used (Midwest Laboratories, Omaha, NE) reports. Our high-temperature room that was maintained at 80 °F had humidity that was maintained at less than 50%, thus increased mold or yeast counts were not expected in these samples over time.

Limitations of this study include a lack of evaluation of peroxide values or rancidity of the fat due to increased temperatures and duration of storage. Feed did not exhibit traces of rancidity, and concurrent measurements of fat remained within acceptable ranges. Rancidity was not considered a high risk since high-fat diets were not evaluated. Additional considerations could include the evaluation of irradiated diets, as irradiation will significantly reduce retinol levels within a diet whereas thiamine levels tend to be minimally affected.⁷ Future applications of our study could include evaluating the shelf-life of irradiated and even purified diets at variable room temperature and humidity levels. Additional vitamins such as E, B₁₂, pantothenic acid and pyroxidine were not selected for evaluation due to decreased susceptibility to heat.^4,6,9,21,22 Newer formulations of vitamin E are very stable with heat exposure and have good bioavailability within rodent diets.⁶ Vitamin K levels were not evaluated; however, Vitamin K in the form of menadione is recommended to not be stored longer than 6 mo.²³ This is the most stable and common form of vitamin K in rodent diets.²³ Vitamin K can also be produced within the gastrointestinal tract of rodents for appropriate nutritional levels, thus this factor was not explored experimentally.⁴ Influences of diet on a study are minimized by thorough evaluation through peer review to ensure all data and details are supplied about diet and drinking water; and providing such details supports replicability of the experiment.^12,24 Commonly, however, storage conditions that could impact the feed quality are not mentioned or discussed. Moreover, guidelines and regulations in Europe and Asia allow storage of rodent feed for 9 to 12 mo without an expectation for noticeable adverse effects on the animals.⁶ Consequently, the findings from this study enabled our IACUC to confidently allow excursions of feed storage conditions that exceed the Guide’s recommendations. Our results may support the utility of a similar approach, using nutritional quality evaluation of feed stored under conditions that lie outside of current standards, for instutions that have difficulty maintaining consistent conditions for feed storage. Making a subjective decision on diet stability and quality requires consideration of feed formulation, sterilization technique, planned storage duration, and storage conditions.⁶ For storage concerns in conditions that may not comply with Guide recommendations, performing feed stability and labile vitamin analysis is recommended.

Author Contributions

Kristine Towns assisted with study design, preparation and storage of feed, sample collection, and construction of manuscript. Aidan Horvath assisted with sample collection and statistical analysis. Amanda Darbyshire assisted with study design and revision of manuscript.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This study was internally funded.