Accurately assessing the nutritional value of various feeds is the key to formulating diets. As we all know, the methods for assessing the nutritional value of feed mainly include chemical analysis, in vivo method and in vitro method. Chemical analysis is a common method for assessing the nutritional value of feed, such as total energy, total phosphorus, crude protein, crude fiber, and crude fat. However, there is a big difference between the feed nutrient measured by the nutrient analysis and the digestion and absorption of nutrients in the animal, which is not enough to accurately reflect the actual nutritional value of the feed. The use of animal experiments can accurately evaluate the nutritional value of feed, but these methods are often time consuming and laborious. It is difficult to evaluate a large number of feed samples in a short period of time, and there are many biological factors, resulting in a large variation in results. Poor repeatability limits its usefulness. Therefore, people established in vitro methods to study the nutritional value of feed. With advances in technology and advances in animal digestion and physiology, it has become possible to use animal in vitro digestion simulation techniques to study the nutritional value of feed. The in vitro method is simple, fast, and reproducible, and plays an important role in the nutritional value evaluation of feeds. This article reviews the application status, application problems and application prospects of in vitro methods in different animals to raise people's awareness and attention to in vitro methods.

In vitro digestion in pigs

Due to the large individual pigs, some of the digestive enzymes needed for in vitro digestion, such as pepsin and trypsin, as well as small intestinal fluids, are relatively easy to obtain. And the one-time acquisition quantity is also relatively large. Therefore, the in vitro digestion method in the pig research and application more.

In vitro digestion of pigs includes pH method, single enzyme (such as pepsin) method, two-step method (pepsin-pancreatin method), three-step method (pepsin-pancreatin-carbohydrate method), etc. Predict the in vivo digestion of pig feed.

The NaOH was used to continuously neutralize the H+ produced during the proteolysis, keeping the pH constant and recording the amount of NaOH used. The in vitro digestibility of 31 plant protein feeds and 11 animal protein feeds measured by this method was significantly correlated with pig in vivo digestibility (r=0.85 and 0.92). In the evaluation of 89 dietary samples, it was found that when the feed sample was treated with pepsin at pH 1.5, the correlation coefficient between the in vitro digestibility of organic matter and in vivo digestibility was high (r=0.92), and it could be used to predict the feed. Digestibility in pigs. The researchers improved the pepsin-pancreatin method. In the separation of digested nutrients and residues, soluble peptides and proteins were first precipitated with trichloroacetic acid, and the digested chyme was filtered through a standard filter for measuring fibers, and the digested nutrients were separated. Afterwards, further improvements and improvements were made on the basis of the former, and the ileal digestibility of protein and amino acids in pigs was successfully predicted using improved in vitro digestion simulation methods. There was a linear correlation between the in vitro organic matter digestibility of the 90 feed samples and 30 compound feeds measured using the pepsin-pancreatin-carbohydrase method and the digestive energy in pigs (R2=0.94). This shows that the three-step method using pepsin-pancreatin-carbohydrase can predict the digestive energy of pig feed.

Application of in vitro digestion on ruminants

The application of in vitro digestion method to ruminants mainly includes in vitro fermentation method and nylon bag method. Compared with in vivo method for assessing nutritive value of feed, in vitro methods have the following advantages: consistent fermentation conditions, consistent fermentation time, and accurate determination of fermentation products. The yield of the test is low, so the application of in vitro digestion to ruminants is relatively wide.

Using sheep manure and rumen fluid as the source of bacteria, the in vitro digestibility of 9 kinds of roughage and 3 mixed-coarse neutral detergent fibers in four categories (legume, grass, straw and silage) was determined. The fiber index of these roughages was evaluated by the Fermentation Index. In vitro tests were conducted to study the degradation of soybean peptides, corn peptides, and rumen fluid peptides in rumen microbial culture fluid at 36, 72, 144, 216, 324, and 450 mg/L. Using in vitro nylon bag fermentation technology, the effects of different sources of nitrogen and carbohydrates in dairy cows and their ratios on the in vitro degradation rate of dry matter were studied. The results showed that the excess urea supplementation and the increase of the proportion of starch and sucrose in the diet decreased the in vitro degradation rate of dry matter. The effects of microbial additives and urea on the release of fatty acids from silage corn were studied by in vitro digestion. The results showed that both the microbial silage additive and urea can increase the content of volatile fatty acids in the silage after digestion, and the combined use of the two is better. The in vitro method was used to compare the effects of different drying methods on the in vitro digestibility of Medicago sativa, Cyclobacilla argenteus, and Ralstonia arborescens. The results showed that the spray desiccant had a significant effect on in vitro digestibility.

Problems in in vitro digestion applications

The digestive process in animals is a complex, highly sophisticated and complete system that is a dynamic process that responds to various stimuli through the regulation of nerves and hormones. Accurately simulating the complex digestion and absorption process of the in vivo digestive system in vitro is very difficult or even impossible. The main reason is that many in vivo chemical reactions or physical processes cannot be simulated in vitro, for example, dietary fiber for digestive tracts Effects of tract rate; effects of anti-nutritional factors on nutrient digestion and absorption in the body; endogenous loss of proteins and amino acids on the apparent digestibility of proteins and amino acids; and complex interactions between microorganisms in the digestive tract Simulation in the test. In vitro assays also make it difficult to fully account for the variability that influences in vivo digestibility.

In practical applications, it is difficult to directly compare in vitro digestibility estimates with in vivo digestibility measurements because in vitro methods for determining digestible protein and amino acid content of feeds are mostly based on the difference method, ie, the total protein and amino acid content is subtracted The protein and amino acid content of the residue after digestion (excluding protein and amino acid content in the blank residue) is the difference between digestible protein and amino acid content. This method considers soluble proteins as proteins that have been digested. This is not the case. Some soluble proteins are not absorbed by animals and thus result in in vitro digestibility estimates that are higher than in vivo measurements. In addition, in vivo digestibility is usually referred to as total digestibility. In vitro digestion generally simulates the digestive process of the digestive tract, whereas microorganisms in the digestive tract of the posterior segment have an effect on proteins that have not been digested by the anterior digestive tract. This results in in vitro digestibility estimates. The value is lower; Secondly, in the in vivo digestion, amino acids in excrement are mainly from digestive chyme, and some are from intestinal metabolites, including intestinal secretions, intestinal cell shedding, intestinal mucosal proteins, etc., among these metabolites. The amino acid content will affect the determination of feed amino acids, which will also result in a low estimate of in vitro digestibility.

Therefore, in vitro methods need to pay attention to the following points in the application: 1 in vitro digestion method used in the type of enzyme preparation and combination of methods should be consistent with the in vivo digestion reaction in order to more accurately simulate the in vivo digestion process, such as the use of multiple enzymes Formulations (including proteases, lipases, peptidases, carbohydrate enzymes, etc.) can take full account of the effects of other nutrient metabolisms on protein and amino acid digestibility. 2 The enzyme-substrate concentration ratio, enzyme specificity, and enzyme activity should also be consistent with the body. If the pepsin concentration used in in vitro digestion is too high, the inferior protein can be completely digested, instead the pepsin concentration is too high. Low, the protein is not completely digested. 3 Incubation temperature and pH need to be adjusted to the same level as in vivo. 4 The enzyme concentration and incubation time should be adjusted to the simulated in vivo conditions to make it as close as possible to the in vivo conditions. 5 To avoid possible inhibition of enzymatic reactions by digestive products, the final digested product should be separated from undigested material as soon as possible. 6 Pay attention to the effect of sample weight and particle size on digestibility. 7 Consider the effects of hormones on digestion of nutrients.

Application prospect of in vitro digestion assay

From the above, there are many limitations in practical applications of in vitro digestion assays, but this does not mean that there is no significance for the in vitro assays. On the contrary, in vitro digestion assays have great potential in assessing feed value.

In vitro digestion assay technology has been greatly developed after decades of history. The future development of in vitro digestion assay technology is from simple to complex, from single enzyme to multiple enzymes, from closed static incubation system to dynamic dialysis incubation system, by animal Digestion fluids (small intestine fluid and rumen fluid) to commercial enzyme preparations (pepsin preparations, pancreatic enzyme preparations, and microbial fiber degrading enzymes), can only estimate the nutritive value of dry matter and protein to also estimate calcium phosphate, crude fiber, etc. The development trend of the nutritional value of the material. The purpose of the continuous development of in vitro digestion assays is to make it easier, faster, and more accurate to estimate in vivo digestibility and to more scientifically assess feed nutritional value.

In summary, the in vitro digestion assay is a highly potential method for assessing the nutritional value of feeds. It can better compare the pros and cons of feed digestibility, and can study nutrient intake of dietary supplements against endogenous nutrients through the difference in digestibility. The relative impact of excretion, as long as the practical application of factors that interfere with the body digestibility results as much as possible, and quantify the impact to the established regression equation, you can get more accurate estimation of the in vivo digestibility value. (Author: Feed Research Institute, Chinese Academy of Agricultural Sciences Ye Hongtao; Liu Guohua)

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