Please give some information on your experimental procedures. E.g. Did you feed only the ingredient to the animals or did you add them to a basal diet?

Thank you for your response C.A. Kan. I replaced 30% by weight of a basal diet with the ingredient (70% basal diet - reference diet - and 30% of the test ingredient). All diets fed to Clarias gariepinus  juvenile contained 0.5g/100g Chromic oxide as indicator. Fecal droppings collected and stored in freezer over a period of 15 days were analysed for proximate composition and quantity of Chromic oxide in test diets and fecal droppings determined.

The answer is yes to both of your questions. basically you can have a negative digestibility because the endogenous excretion of the test nutrient is large enough to elicit this mathematical situation. If the feeding period is long enough you should see the physiological effects too- for example you would observe negative biomass accretion. At least some of the animals will definitely lose weight. And this should not be a surprise if you are studying non-conventional ingredients which are high in anti-nutrients (oxalates, tannins etc). In your case I don't know the nutrient that you are looking into but this is common occurrence in amino acid nutrition when dealing with low crude protein diets. Tere is already a great deal of information on endogenous losses - especially the obligatory aspects of it at the ileal level. We recently produced a review in this regard. Yours is faecal but it is generally the same principle so you can read it up (see attachment). On the second question- yes it may happen although this is not the expectation nor is it a common occurrence. Such data suggest you might not have done a very good job at completely collecting all voided faecal materials (sorry about that). Your marker recovery is another source of error. Hope you have got the marker analytical procedure right too. What was your CV in laboratory repetitions you have got? Mind you all these errors are magnified and make one to easily overshoot 100 when dealing with assay ingredients (e.g corn) low in nitrogen- in case you are dealing with protein research (you have not disclose that yet). There are other reasons I can give you if I have better details of your protocol. Hope I have been able to help. Cheers.

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The answer by David nicely illustrates the most importants points in doing this type of work.

One more remark: in using this approach, you assume that the digestibility of the basal diet does not change regardless of the ingredient  and its amount added to that basal diet. Also you assume that digestion goes on more or less undisturbed.

 This is certainly not always true. We have seen that fat containing ingredients might easily cause problems in the digestive process making the results sometimes hard to interpret.

I am actually dealing with a non-conventional protein feed ingredient of plant source with high amount of anti-nutritional factors. The first phase of the study dealt with reduction of these anti-nutritional factors by subjecting the ingredient to different processing methods - Sprouting, soaking, toasting and cooking. I am now trying to establish the processing method that would give the best digestibility coefficient when fed to C. gariepinus before moving on to the growth performance evaluation.

I think I may have to re-run the determination  of the marker in the diet and faecal droppings. I spoke with a colleague whose opinion suggested that the amount of the marker in diet in most cases are lower than that in the faecal droppings - condition which is contrary to mine. And, one of your suggestions provided a pointer to the inaccuracy of the determination of the marker in diet and faecal droppings.

Thank you for your suggestions David O. Akande and C.A. Kan

Hi Banjo. Now it is clear that your marker recovery data is the cause of the problematic data. Of course to have a positive digestibility the marker values in the faeces must supersede  values in the diets. Based on some studies we did in the past, marker recovery rate is the most important factor for accurate and repeatable digestibility estimation. We know that Marker application in digestibility studies is supposed to be an easy way out of a tough job. But don't get carried away it depends on your ability to have very precise quantification in both feed and faeces. And that can be a tall order for anyone not running such analysis on routine basis. Don't use the lab values until your repeats are within CV of 1-2%, definitely not above 3%. All your equipments have to be well calibrated and be careful in your weighings. You also must use faecal samples that are very representative of the true excretion. Make sure the animal consume the diets for a good number of days before sampling to minimise diurnal effect on GIT clearance. And mix very very well. On your choice of Cr: yes it works well for some investigators yet it is not the best to be recommended. Titanium dioxide would do better any time and the analytical procedure is also very straight forward if you follow Brandt and Alam (1987). After doing a good work, faulty lab results can still rubbish it- so marker selection is quite important to succeed. The last and most important point I should mention to you is that- even after doing your marker analysis again and again- you may still have problems!!! This is because marker recovery rate and thus digestibility is affected by crude fibre and anti-nutritional factors. I have unpublished data on this aspect. This interaction means that your assay ingredient which you have told us is rich in anti-nutrients is part of the disturbances to your ability to get good marker recovery results; thereby making your final digestibility estimates questionable!!!! But you can defend it depending on how good is your literature base. Anyhow my point is that in your follow up studies use your cromium oxide but also undertake quantitative faecal collection- simultenously. With these two approaches you can use one to control the other, essentially meaning you will have a factorial analysis at the end of the day.

Once again David has made a lot of very to the point comments, which I fully support.

Two small additions: if the anti-nutritional factors have not been fully inactivated, they might reduce the digestibility of your basal diet. As the basal diet is a quite large portion of your diet, a small influence on the digestibility of the basic part will result in a large influence on the digestibility of your test material.

Secondly the use of both quantitative collection of feces and a marker will not always solve all your problems. On the contrary; in the study attached we faced the situation that some experimental units deviated from the rest in feed to feces ratio on the basis of total collection and others on the basis of Cr  data. Than once again you have to decide, which value is more likely to be true and how to deal with the situation!

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Exactly correct Kees. Just to speculate: your second point may arise due to variation in intestinal transit time in the animals subjected to the total collection rpotocol. Thanks for the sharing the paper.  

This is impossible if you do your experiment in laboratory since no natural feed found, if you see the formula for calculating digestibility (TADC) it depend on % of marker if it was determined correctly in diet and feces so 100% was achieved only if no feces, negative values if marker conc. in feces was less than in diet which mean wrong result. Attached good Ref.

A.Y.Al_dubakel  

I agree with Dr. Adel Al-Dubakel explanation. So, my advice is to recheck indicator concentrations in feed and fecal samples before searching for another reason justifying your observations. The links below could be helpful.

Regards

SM Najim

https://www.was.org/documents/.../AQ2013/AQ2013_0714.pdf

http://cals.arizona.edu/clubs/ansci/journal%20club/desale_fall02.pdf

www.pvj.com.pk/pdf-files/27_3/page%20121-125.pdf

www.thejaps.org.pk/docs/21-4/28.pdf

https://naldc.nal.usda.gov/download/27525/PDF

Thank you all for your comments and advice.

Based on your suggestions, I re-analyzed the content of Chromium in the diets and feces with a more experience laboratory technician. We got a different result from the previous and the apparent digestibility values were less than 100 and no negative results were obtained. The unprocessed seed meal (Raw) had a significantly lower (p < 0.05) apparent crude protein, crude fiber, ether-extract and energy digestibility when compared with processed seed meal.

I want to again thank you so much for your support. It is a privilege to be part of this research forum.

Glad that (with our help) you have managed to come the satisfactory and explainable result