The Apparent Digestibility of Corn By-products for Growing-finishing Pigs In vivo and In vitro

Two trials in vivo and in vitro were conducted, in vivo to determine the apparent digestibility of gross energy, crude protein, dry matter, acid detergent fiber, neutral detergent fiber and apparent digestible energy in 10 corn by-products. In vivo the diets included one basal corn diet, four corn gluten meal diets, four corn distillers dried grains with solubles diets and two corn distillers dried grains diets using the different methods, 12 crossbred barrows weigh 40±1.6 kg were allocated into individual metabolic crate, according to a 6×6 Latin square design. In vitro using flask technique, filter bag technique and dialysis tubing technique, the digestibilities of gross energy, crude protein and dry matter in corn gluten meal and corn distillers dried grains with solubles were investigated. Pepsin, pancreatin, intestinal fluid, rumen fluid and cellulase were used in incubation. The results showed that correlation coefficient was 0.73 in corn distillers dried grains with solubles between the digestibility of crude protein and acid detergent fiber in vivo (p<0.01); and correlation coefficient was 0.68 in corn distillers dried grains with solubles between the digestibility of gross energy and neutral detergent fiber in vivo (p<0.01). Apparent digestible energy (DE) of corn by-products in pig total tract was predicted by the percentage of crude protein (CP) and the content of gross energy (GE) in feedstuff. The equation: DE=5,601.09+26.69×CP %-0.5904×GE, (R=0.72). In vitro, filter bag technique was more convenient; furthermore, the digestibility for the treatments (pepsin+pancreatin+rumen fluid and pepsin+pancreatin+cellulase) was better. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 3 : 379-385)


INTRODUCTION
Corn by-products are valuable protein sources for animals as unconventional feedstuffs.Corn gluten meal (CGM), corn distillers dried grains (DDG) and corn distillers dried grains with solubles (DDGS) attributed to the corn by-products, are produced by different processing techniques in many countries.Corn gluten meal comes from corns by the extraction of oil and starch for food.DDGS and DDG come from corns by the extraction of oil and alcohol using different processing techniques.
Corn by-products have a good effect on animal growing performance (Brenes et al., 1985;Ham et al., 1994;Coyle et al., 1996;Koelkebeck et al., 1999).The consume for determination of animal digestibility in vivo was greater.Therefore, there is a need to found a quick and reliable method.Some methods have been attempted for relationship between in vitro and in vivo (Furuga et al., 1979;Graham et al., 1989;Cone and Vander poel, 1993).The prediction of digestible nutrients of diets in vitro has been proposed (Boisen andFernandez, 1995 and1997).However, an in vitro method with a general validity for all kinds of feedstuffs should be established to measure the digestibility of nutrient.
Two trials were conducted to investigate correlations of the nutrient digestibility in vivo and in vitro in this study.
Growing-finishing pigs were employed to estimate the apparent digestibility of crude protein (CP), gross energy (GE), dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF) and digestible energy (DE) of corn by-products for trial 1. Trial 2 was designed to apply multienzyme systems for investigating the in vitro digestibility of CP, DM and GE of corn by-products.Data from both trials were collected and used to find the correlations among digestibilities of CP, GE, ADF, NDF, DM and DE in corn by-products.
groups.The trials were simultaneously conducted under the same condition.Each experimental group had six pigs and were fed diets according to a 6×6 Latin Square.The first group was fed diets containing one corn basal diet, four CGM diets and one DDG diet; while the second group was fed diets containing one corn basal diet, four DDGS diets and one DDG diet.Each experimental diet of Chinese corn by-products had six replicates.Each pig was fed diet for a 7 day period, which consisted of 4 days adaptation followed by 3 days collection of feces.The diets (Table 1) were based on nutrient requirements of pigs between 50 and 80 kg (NRC, 1998).Throughout the experiment, the barrows were individually housed in 0.5×1.5 m cast-iron metabolism crates equipped with a 0.25 m 3 round bottom feeder located at the front of the crate.The crates were located in an environmentally controlled barn with the temperature set at 18°C.The barrows were fed at 08:00 and 17:00 h each day.Feed intake was maintained at a constant level (3-4% body weight) for all pigs during each period of fecal collecting.The amount fed was the amount consumed by the pig eating the least during the trial adaptation phase.Water was added to the diets at feeding to form a moist mash.The barrows typically consumed their rations within 40 minutes of feeding.Collection of feces was started after the morning feeding, and the feces were collected for 9 h during each day of collection.Each collection was placed in freezer and stored at -20°C.At the completion of the third day collection, the two frozen feces samples were thawed and mixed with the third collection.About 500 g of the mixed feces were refrozen and stored at -20°C.Prior to analysis, the feces were thawed, then freeze-dried and analyzed.

Trial 2
The trials in vitro were conducted by flask technology, filter bag technology and dialysis tubing technology.Some digestive enzymes were applied to, which were pepsin, pancreatin, intestinal fluid, cellulase and rumen fluid.Corn by-products included one corn gluten meal (CP 47%) and one DDGS (CP 30%) and the feedstuffs were ground by a 1 mm screen.Four treatment groups in every technology trial, which were pepsin+pancreatin, pepsin+intestinal fluid, pepsin+pancreatin+cellulase and pepsin+pancreatin+ rumen fluid.Each treatment group had six replications.The samples were incubated by enzymes to investigate the digestibility of DE, DM and CP.The temperature, time and pH were presented in Table 2.

Flask technique
The trial procedures are as follow : Pepsin+pancreatin : Step 1.The sample of about 1 g was added to a conical flask (100 ml volume); then 20 ml HCl -NaCl (pH 2.0) buffer and 0.1 g pepsin were added to the flask, which were sealed with a parafilm under anaerobic condition, and placed in the oscillator.The temperature of shaking bath was controlled to 39°C, shaking frequency was 60 times in a minute, and incubating time was 6 h.
Step 2. To the mixture, 20 ml phosphate buffer (pH 7.0) and 2.5 ml pancreatin solution were added, then, the flask with mixture was put into the shaking bath at 39°C for 18 h after sealed with a parafilm under anaerobic condition.
Step 3. Filtered with whatman filter paper, the undigested residues were separated, and dried at 105°C.DM, GE and CP in the residues were analyzed.
Pepsin+intestinal fluid : The methods were similar to flask technique (pepsin+pancreatin treatment), except pancreatin solution was not added to but 20 ml intestinal fluid solution was added to the flask.
Pepsin+pancreatin+rumen fluid : The methods of step one and step two were similar to flask technique (pepsin+pancreatin), except that the incubated time of pepsin and pancreatin was one hour and six hours respectively.The procedure of step three was that 10 ml rumen fluid was added to the flask with sample solution (step one and step two), while pH in flask solution was 6.8.Then, shaking at 39°C for 17 h.
Step four was similar to the step three in flask technique (pepsin+pancreatin).
Pepsin+pancreatin+cellulose : The methods were similar to flask technique (pepsin+pancreatin+rumen fluid), rumen fluid was not added to the flask but 10ml cellulase solution was added to.

Filter bag technique
Pepsin + pancreatin : Step 1.The methods were similar to flask technique (pepsin+pancreatin) but the sample was added to a filter bag.The entrance of filter bag was sealed by sealing machine, and was put into the flask.The filter bag with undigested remnants was dried under 105°C for 4 h and analyzed.
Pepsin+intestinal fluid, pepsin+pancreatin+rumen fluid and pepsin+pancreatin+cellulose : The method was respectively similar to flask technique (pepsin+intestinal fluid, pepsin+pancreatin+rumen fluid and pepsin+ pancreatin+cellulase) but the sample was added to filter bag.

Dialysis tubing technique
The dialysis tubing should be prepared beforehand.Firstly, the dialysis tubing was cut 20 cm long bag.Glycerin from surface of dialysis tubing was washed out by running water for 3-4 h.Then, Dialysis tubing was soaked by 0.3% (w/v) sodium sulfide solution at 80°C for one minute to remove off sulfur compounds, and washed with hot water (60°C) for two minutes, followed by acidification with 0.2% (v/v) sulfuric acid, rinsed with hot water to remove the acid.One end of dialysis tubing would be sealed by cord before adding sample and buffer, another end of dialysis tubing would be sealed by cord after sample and buffer was added to.The trial treatments are as follow: Pepsin+pancreatin, Pepsin+intestinal fluid, pepsin+ pancreatin+rumen fluid and pepsin+pancreatin+cellulose : The trial steps were respectively similar to that of the flask technique (pepsin+pancreatin treatment, pepsin+intestinal fluid treatment, pepsin+pancreatin+rumen fluid treatment and pepsin+pancreatin+cellulase treatment), except that the sample, enzyme and buffer were added into the dialysis tubing, then, the entrances of dialysis tubing with sample, enzyme and buffer were sealed by cord, the dialysis tubing was put into a bottle (500 ml volume), which contained 40 ml phosphate buffer.The sample solution in dialysis tubing was required to soak in the bottle of phosphate buffer.Finally, the bottle with dialysis tubing was sealed with a parafilm and shaken in the oscillator bath.The end-products in dialysis tubing were filtrated, dried and analyzed.

Chemical analysis
Samples of corn by-products, trial diets and feces were analyzed for their crude protein (CP), gross energy (GE), dry matter (DM), ash, acid detergent fiber (ADF) and neutral detergent fiber (NDF) content using the methods of the AOAC (1990).Nitrogen (N) was analyzed using the Kjeldahl method (AOAC method 988.05).NDF and ADF were analyzed by Fiber Analyzer (ANKOM220); GE was analyzed by Bomb Calorimeter (PARR 1281).The apparent digestibility of CP, GE, DM, ADF and NDF in corn byproducts was determined by the equation (Sauer et al., 2000): Where D A was apparent digestibility of a nutrient in the assay feed ingredient (%); D B was apparent digestibility of a nutrient in the basal feed ingredient (%); S B was the contribution level (%) of a nutrient in the basal ingredient to the assay diet; S A was the contribution level (%) of a nutrient in the assay ingredient to the assay diet.

Statistical analysis
Both the in vivo and in vitro trials had six replicates per treatment.Analyses of variance from filter bag method, dialysis tubing method, flask method in vitro and the results in vivo were carried out by comparing means according to the One-way ANOVA of SPSS.Four corn gluten meals, 4 DDGS and 2 DDG were separately compared their correlate coefficient with correlate of Kendall's test.Prediction equation was established using the General Linear Model Procedure.

RESULTS
The pigs remained healthy throughout experiment.The chemical composition of DDGS, DDG and corn gluten meal is presented in Table 3.The digestibility of diets and feedstuffs is presented in Table 4.The correlation (CP %: the percentage of crude protein in corn byproducts; GE: the content of gross energy in corn byproducts; DE: the apparent digestible energy of corn byproducts in pig total tract).
The digestibility of GE, DM and CP in vivo and in vitro is presented in Table 5.

Digestibility of GE, CP, NDF and ADF in vivo
Corn by-products were used as protein sources for pigs.In the present study, corn gluten meal had the higher digestibility of CP and GE than that of DDGS and DDG.Thus, it is regarded that corn gluten meal can supply high quality protein and energy sources for animal.As observed in Table 4, there was significant difference (p<0.01) in the digestibility of ADF and NDF among four DDGS.
Two explanations are possible for this phenomenon.Firstly, it is likely that processing technique affected the chemical composition of corn by-products.Corn gluten meal came from corns after the extraction oil and starch; DDGS and DDG came from corns after extraction oil and alcohols, additionally, the process techniques were different between DDGS and DDG.Secondly, the chemical composition of corn by-products may have direct effect on the digestibility of nutrients.
It was shown in the present study that processing methods had some effect on the physical and chemical characteristics of feedstuffs.Similar results were also reported by Boisen and Eggum (1991) and Yang et al. (2001).Clearly, the digestibility was influenced by the characteristics of feedstuffs.
Although DDGS and DDG contained higher NDF and ADF, they had higher nutrients digestibility.This result showed that fiber did not affect the digestion of other nutrients in pig diets.Apparently, growing-finishing pigs had the potential ability of digesting fiber diet.This conclusion is in agreement with the previous study (Varel and Yen, 1997).
Furthermore, it was showed in this study that there was a significant correlation between digestibility of NDF and GE (R 2 =0.68).In spite of the higher digestibility of ADF, significant correlation between ADF and GE was not found.This result demonstrated that NDF markedly influenced GE digestion.Meanwhile, it was also found that the digestibility of CP was not affected by the digestibility of NDF.However, it was significantly affected by the digestibility of ADF (R 2 =0.73).Further studies should be carried out to establish correlation between CP and ADF in DDGS.Fortunately, for corn by-products in this study, the prediction equation of DE in vivo can be determined by its percentage of CP and GE.

Comparison of techniques in vitro
Filter bag technique was used to measure CF, NDF and ADF of food or feed with ANKTM fiber analyzer in many countries.However, there were very few reports applying this technique in determining the digestibility of nutrients in

Comparison of digestibility in vivo and in vitro
It is shown in Table 5 that the CP digestibilities of corn gluten meal in vivo were markedly higher than that of in vitro (p<0.01);but the CP digestibilities of DDGS in vivo had lower tendency than that of in vitro (p<0.01);except that the CP digestibility in filter bag (pepsin+pancreatin+ rumen fluid) had not markedly difference from in vivo.For GE digestibility, corn gluten meal in vivo was significantly lower than that of filter bag and dialysis tubing (pepsin+ pancreatin+cellulase), but was significantly higher than GE digestibility of flask method (p<0.01);DDGS in vivo was significantly higher than that of in vitro (p<0.01).For DM digestibility, corn gluten meal in vivo was significantly higher than that of flask method (p<0.01);DDGS in vivo was significantly higher than that of in vitro (p<0.01).
There were four possible explanations for difference between in vitro and in vivo.Firstly, it was likely that the difference came from different trial techniques.Secondly, digestive environment was different between in vivo and in vitro, as pH, temperature, incubating time, enzyme activity, multi-enzyme compound systems, shaking frequency and sample size.Thirdly, the characteristics of corn by-products can affect the digestibility of nutrient.Fourty, it is explained that endogenous N might have a great influence on the digestibility in vivo, thus, it is showed in this study that the some nutrient digestibility in vivo was lower than in vitro.Boisen and Fernandez (1995) also reported that in vivo digestibility was influenced by endogenous loss.

CONCLUSION
It can be concluded that the digestibility in vitro with three-step enzyme was close to that in vivo.Filter bag method to evaluate the digestibility of nutrients in feedstuff was more convenient than that of flask method and dialysis tubing method.There was significant correlation between the digestibility of GE and NDF or between the digestibility of CP and ADF in pig diet.The prediction of DE in vivo from corn by-products can be determined by using the nutrient concentrations of the feedstuffs.

Table 1 .
Ingredient and chemical composition of experimental diets (%)

Table 2 .
Multi-enzyme systems used in vitro incubation Enzyme systems procedure Time, pH and temperature in enzyme incubation (1) Pepsin+(

Table 3 .
The chemical composition of feedstuffs

Table 4 .
Digestibility of the feedstuffs and the diets

Table 5 .
Digestibility of CP, GE and DM from feedstuffs in vivo Filter bag can make liquid or gas pass through freely; but the solid contents are retained in bag.In addition, filter bag do not contain N and can not be eroded below 72% sulfuric acid solution.It was indicated in this study that filter bag technique was superior to dialysis tubing technique and flask technique in evaluating the digestibility of CP and GE, as the filter bag technique was convenient.