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In vitro efficacy in feed additives — How to test for it

To register a feed additive in the European Union, its efficacy should be demonstrated in microbial testing trials that follow common feed manufacturing, animal husbandry and farming practices in the EU.


In vitro vs in vivo efficacy assessment

Efficacy can be assessed by means of in vitro studies for technological feed additives i.e. those additives, which are intended to affect only the characteristics of feed. For zootechnical feed additives, which are intended to have an effect in animals, in vivo efficacy studies are required. In specific circumstances, like in the case of coccidiostat compatibility of probiotic feed additive strains, both in vitro and in vivo trials are used.

The basic requirements for an in vitro efficacy study

An in vitro efficacy study should outline a range of materials (feed materials, complete or complementary feed or water) to which the additive will be applied, compared to untreated control feed. The study should follow the criteria of recognized quality assurance schemes like GLP and ISO.


Update your knowledge on Transparency Regulation and In vitro efficacy studies

Efficacy criteria depend on the additive

Technological additives are divided into 12 different functional groups, all of which have different criteria for demonstrating efficacy.

For example, for acidity regulators, you evaluate the pH and/or buffering capacity of the additive in the feedingstuffs and/or water. Testing the capacity of the acidity regulator is a simple procedure. You measure the pH of the feed matrix in both additive-supplemented samples and non-supplemented control samples at several time points. However, the setting up the study is not as straightforward, since several factors must be taken into account:

  • What are suitable feed matrices for my target species?
  • How many replicates per treatment should I have, to expect statistically significant differences?
  • What is a sufficient inclusion level of the additive?
  • What is the duration of the trial and at which time points should I measure the pH?
  • How do I mix the additive homogeneously with the matrix?

Do your preliminary trials

Proper planning and use of relevant preliminary trials are the key to success. Every variable should be analyzed in preliminary trials to make the actual efficacy trial go as smoothly as possible. It is good to remember that feed matrices are usually not sterile, and background contamination may cause unexpected surprises by altering the pH of the feed. While the trials should be performed under the intended practical conditions, it is essential to select feed matrices, which won’t interfere with the assay.

Selecting feed matrices

Most likely, the effect of your additive strain will be different in liquid and dry matrices. Measuring the effect in nutrient-rich calf milk replacers (CMR) results commonly in a rapid pH decrease, especially with lactic acid bacteria, while in fibre-rich ruminant total mixed rations (TMR) the decrease may be just marginal. It is also possible, that an additive inclusion level that was efficacious with CMR will not be efficacious with TMR, so you may have to raise the inclusion level for it.

Variation means more work

Having an understanding of the basics of how the statistics work is also worth an evening crunching numbers. The more statistical variation you have in your results, the more replicates you will need in your study to achieve statistical significance. This shows the significance of homogeneous samples.

Our recommendation to save you some grey hairs

For your feed additive to succeed in an actual in vitro efficacy trial, our strong suggestion is to invest in preliminary trials to close the knowledge of how your additive will perform in different feed matrices. Once you have exhausted what different outcomes might come out of the study, you will be a lot wiser. And in the end, you’ll have saved time and money not going through the same trials again.


If you are looking to test the efficacy of microbial feed additives, download our brochure on in vitro efficacy.
Download our brochure on in vitro efficacy


Published: 23.03.2021

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