Storage conditions in the UK and Western Europe are less challenging than those that you face in India.

As a standard, we usually work on an ideal temperature and humidity of about 20C and 65% RH. It is important to understand that these are average values. The diet will be largely unaffected if the temperature goes several degrees above this for a few hours per day since the diet is usually in one tonne lots on a pallet and has a large thermal capacity i.e. it takes a long time for its temperature to change despite fluctuations in environmental temperature. While if the temperature increases by 10C continuously, shelf life will be halved (Q10 effect see below), it that were to occur only for a few hours per day, you can appreciate that the overall effect would be quite small. We do run samples periodically for vitamin levels after storage. It is a very expensive exercise so we don't have a huge amount of data, and there are so much published data that it provides little in terms of new knowledge. We have sent off several batches of diet covering several product types (Rodent pelleted, RGP [rabbit/guinea pig] pelleted, extruded (2027 Dog diet, 2021, 2055 Primate diet) once they had reached their expiry date (6 months). I have expressed the values as a percentage remaining of the amount added or measured after manufacture. The stability of the vitamins is shown for each group and combined. The absolute levels of the vitamins all exceeded NRC requirements for the appropriate species at the end of the six-month period. Some vitamins such as B6 are at low levels and difficult to measure: this typically gives very variable recovery results. Interestingly the data show that storage losses of vitamins are generally lower in extruded diets than in pelleted diets, which is consistent with earlier reports. Note that the vitamin C is the stabile form and as you can see shows minimal losses.

I.

Storage of Diet

The main risks for stored diet are degradation of nutrients (particularly fats and vitamins), microbiological spoilage and attack and contamination by rodents, birds, insects and other pests (vermin). All of these risks can be managed by appropriate practices. Suppliers usually provide the manufacture date and its shelf life or expiration date. The expiration date is typically based on moderate storage conditions. Many users may provide very high quality storage with full environmental control. Users should be able to extend diet expiration dates when evidence (for example, vitamin levels and measures of fatty acid oxidation) can be provided demonstrating that their storage conditions reduce the rate of decline in nutritional quality. Spencer (1985) showed that irradiated natural ingredient rodent diet was useable up to 22 months with no problems when sealed and stored at 22-23C and 3565% humidity. However there were significant changes in vitamin A levels. It is important to appreciate that although diets may be used over a wide period of storage, the variation in nutrient levels might affect experimental studies, despite the diets meeting nutritional requirements. Purified diets present special problems, particularly since a large proportion contain high fat levels. Fullerton et al. (1982) reported that vitamin losses and rancidity could be substantially decreased by reducing storage temperature from ambient to 4C, and in the case of AIN76A (which would be typical of many purified diets) expiration could be extended to 6 months. Vitamin losses and rancidity could also be minimised by storage under argon. It seems prudent that high fats should be vacuum packed and stored at a maximum of 4C; for practical purposes the diet should be packed in small (e.g. 2 kg packs) that can be kept in a freezer or refrigerator allowing the diet to be removed and used in small amounts as needed. Recently oxygen scavengers have been introduced: these small packs can be placed in small diet packs to mop up any oxygen that might contribute to vitamin destruction and rancidity.

A.

Microbiological Spoilage of Diet

Microbiological spoilage occurs when the moisture content of the diet is high. This may occur because: the moisture content of the product per se is higher than recommended; condensation has taken place (perhaps because the product has been packed while still warm, or because of a sudden large drop in temperature, for example in the transportation of diet surrounded by plastic pallet wrap); the diet has become wet because of poor storage or transportation. Moisture in mouse diets will be usually determined gravimetrically. However part of the total water in the feed will be tightly bound to chemicals or be acting as a solvent for water-soluble components. The balance, termed available water, is in practice the important component since it represents the water that is available to take part in chemical reactions, and thus food spoilage processes. It is defined as the ratio of vapour pressure of water in the diet (p) to the vapour pressure of pure water (po) at the same temperature, and is measured as the relative humidity in the headspace above the diet in a sealed container, at equilibrium.

At this equilibrium relative humidity, the diet will neither gain nor lose water. A product with an Aw value of 0.65 will be in equilibrium with the moisture in the air at 65%. For each ingredient and product, there will be a relationship between total moisture content (determined gravimetrically) and Aw, and this is known as the moisture absorption isotherm. The point at which the total moisture content of the diet is in equilibrium with the atmosphere is called the equilibrium moisture content (EMC). Typically, microorganisms require a minimum Aw to grow: typical values are 0.91 for bacteria, 0.88 for yeasts, and 0.80 for molds. This means that molds will start to grow first but as they do so they generate more moisture that allows bacteria to develop. In a diet that has been dried below an Aw of 0.80, our experience is that the numbers of vegetative organisms decline after manufacture. Eva and Rickett (1983) also showed that total viable count declined in dried diet up to 9-12 months and then very slowly increased. Fullerton et al. (1982) showed no consistent change in bacteria and mold growth in NIH-07 diet stored at 4C and 20C over 168 days, but the values were already high by current standards and moisture content was not reported.

B.

Storage Temperature and Humidity

There are two major physical factors affecting quality of diet in storage: temperature and humidity. 1. Temperature

For every 10C increase in temperature, metabolic processes double in rate (Q10 effect). If it is assumed that these processes lead to a proportional change in vitamin levels then increasing the storage temperature from 20C to 30C would double the rate of vitamin destruction and the oxidation of fats, and would reduce shelf life by half. 2. Humidity

The relationship between Aw and food spoilage allows some estimates of appropriate storage humidity. Since the lowest Aw at which most molds will grow is 0.80, one would predict that the relative humidity to which diet is exposed in storage or transport should not exceed 80% for any sustained period of time. To allow a margin of safety an average of about 65%RH at 20C is preferable. The equilibrium between the product and the environment may be delayed by the packaging, which may provide a barrier to moisture. In addition, the palletizing of diet means that penetration of the load by moisture may be slow. Consequently diet may be little affected by transient fluctuations in humidity. Aw is affected by temperature, and in the case of feed it decreases with decreasing temperature, which means that as temperature decreases, relative humidity must also be decreased to maintain product moisture constant. This is important in climate-controlled stores. However at lower temperatures the risk from food spoilage agents by the increased moisture will be offset to some extent by the Q10 effect. A series of curves describes the relationship between temperature, relative humidity and equilibrium moisture content. Different ingredients have different curves: at 60%RH and 20C, the EMC will be 13.6% for wheat; 11.8% for oats; 10.7% for soybeans and 12.8% for corn. The moisture content of diet should be no more than 13% and ideally less than 12.5% to prevent spoilage. Some vitamins will also be sensitive to high moisture content. 3. Monitoring

Measurements of temperature and humidity on either a continuous basis or as daily minimum and maximum values should be made in diet storage areas. The data should be recorded and maintained on file. Relatively inexpensive freestanding battery-operated temperature and humidity recording units, placed at appropriate points in storage areas, are capable of transmitting continual temperature and humidity data by wireless communication to recording computers.