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turkeys)
Beni-Suef University Journal of Basic and Applied Sciences xxx (2017) xxx–xxx
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Full Length Article
Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys) Ibrahim M.I. Youssef a,b,⇑, Josef Kamphues a a b
Institute of Animal Nutrition, University of Veterinary Medicine Hannover, Germany Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Beni-Suef University, Egypt
a r t i c l e
i n f o
Article history: Received 22 April 2017 Received in revised form 14 May 2017 Accepted 14 May 2017 Available online xxxx Keywords: Digestibility Lignocellulose Fermentation Fecal liquor Swine Turkeys
a b s t r a c t Three experiments were performed to evaluate the digestibility and fermentation rates of two types of lignocellulose products that can be used in diets of pigs and turkeys. Experiment I: In vitro dry matter digestibility studies using Daisy system were conducted by using fecal or excreta fluid of swine/turkeys as a source of inoculums. The feedstuffs used as substrates were commercial swine or turkey diet, lignocellulose A and B. The dry matter digestibility was found to be about 75%, 28%, and <5% for swine/turkey diet, lignocellulose A and B, respectively. Moreover, the inoculums were incubated with the same substrates for 24 h using gas measuring technique. It was found that the product A was fermented better than B and resulted in higher amounts of acetic, propionic and lactic acids. Experiment II: Six swine were divided into 2 groups; the control group was fed on a commercial swine diet, whereas the experimental one was offered a commercial swine diet containing 2.0% lignocellulose A. The digestibility rates of dry matter, organic matter, and crude fiber of both diets were found to be nearly identical. Experiment III: At the end of study, the animals were slaughtered and the caecum contents of swine and turkeys were used as inoculums and incubated with the same tested substrates for 24 h using gas production technique. As observed with fecal/excreta inocula, lignocellulose A resulted in greater amounts of gases, volatile fatty acids and lactate compared to product B. In conclusions, lignocellulose A can be used as alternative source of fiber to maintain the health and function of digestive tract. Furthermore, the use of feces/excreta liquor provides a valid and proper estimate of digestibility or fermentation of feeds. These results provide further guide that this procedure would be an effective and practical means of approximating the digestibility of diets. Ó 2017 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Dietary fiber is major concern in nutrition of monogastric animals. It is important to maintain optimal digestion, fecal quality and health of the animals (Sarandan et al., 2008). High-starch diets support rapid fermentation, which is often complete early in the digestion process leaving little or no fermentation for the large intestine (McDonald et al., 2010). Inclusion of dietary fiber in the diet shifts the fermentation process to the large intestine and increases the growth of beneficial bacteria (Govers et al., 1999; Krieg et al., 2008, 2012). These bacteria produce short-chain fatty acids and lactic acid that improve the health and integrity of the intestinal lining as well as decrease the pathogenic bacteria in the intestine (Bach Knudsen and Jorgensen, 2001). Furthermore, ⇑ Corresponding author at: Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt. E-mail address: [email protected] (I.M.I. Youssef).
volatile fatty acids and lactic acid are essential to maintain eubiosis (stable and healthy microflora populations) in the large intestine (Johnston et al., 2003). Moreover, lactic acid is known as an essential antagonist to pathogens in the digestive tract and will therefore positively affect the gut health of the animals. Most recently, special dietary fiber products derived from lignocellulose are being used in animal nutrition with low inclusion rates. Lignocellulose is a product made from fresh wood and has been used as a high quality fiber source in animal diets (Sarandan et al., 2008). Compared to traditional fiber sources, lignocellulose is characterised by high crude fiber (>55%). Nowadays, a new eubiotic lignocellulose product has become commercially available. This product contains a blend of non-fermentable and fermentable fiber components. Thus, the digestibility of these feed materials by monogastric animals is of special interest. The digestibility value of a feed is one of the best indicators of nutritive value. Measurement of in vitro dry matter digestibility (IVDMD) has been used extensively to analyse feeds because of a
http://dx.doi.org/10.1016/j.bjbas.2017.05.006 2314-8535/Ó 2017 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
high degree of correlation to in vivo digestibility (Marten and Barnes, 1980). In vivo determinations are expensive and time consuming, rendering this technique impractical for routine analyses. Over the years, various techniques to determine IVDMD have been developed and modified. The recent development of DaisyII apparatus (ANKOM Technology Corp., Fairport, NY) allows simultaneous incubation of different feedstuffs in sealed polyester bags in the same incubation vessel. With this method, the material that disappears from the bag during the incubation is considered digestible (Mabjeesh et al., 2000). The DaisyII system is more labor efficient than traditional methods and represents a significant advantage for analysis of forage, grain and mixture of ingredients (Holden, 1999). Feeding monogastric species of crude fiber supply is on debate, especially due to its diverse effects on digestion, gut health, feces quality and animal welfare (Johnston et al., 2003). Fibrous feeds traditionally have not been used for nonruminants due to their effects on diet digestibility in pigs and poultry. However, some types of fiber and fiber sources do not exert such negative effects on nutrient digestibility in older growing pigs and sows. Dietary fiber can have positive effects on gut health, welfare, and reproductive performance. Hence, nutritionists are attempting to gain a more thorough understanding of dietary fiber in swine and poultry diets. Moreover, there is a lack of information on the fermentation process of these products by swine and poultry. Therefore, the aim of this study was to provide a deeper characterization of these new fiber ingredients (lignocellulose products) that can be used in diets of swine and turkeys by determining their in vitro dry matter digestibility in the Daisy incubator system using fresh feces (pigs) and excreta (turkeys) as sources of inocula. Furthermore, the ability of these products to ferment and produce gases or volatile fatty acids by fecal/excreta inoculums was evaluated. Afterwards, the results of the in vitro were confirmed in vivo especially in swine by testing the digestibility rate of the most digestible product of lignocellulose ingredients. In addition, the fermentation rates of the tested ingredients were evaluated using the caecum contents of swine and turkeys as inoculum precursors, and then compared with that obtained by feces or excreta inocula. 2. Materials and methods The study was carried out in vitro and in vivo. It was started in vitro to evaluate the fermentation of ingredients by Daisy system via using the fecal (swine) or excreta (turkeys) as inoculum sources. In addition, the amounts of gas, lactate and volatile fatty acids produced from the tested ingredients, by using fecal or excreta inocula, were determined. Based on the results of in vitro section, the most fermentable ingredient was tested in vivo through using swine as a model of non-ruminants. Afterwards, the swine and turkeys were slaughtered and the caecum contents were used for preparation of caecal inocula which were used in vitro to measure the gas production and fermentation of the investigated ingredients. 2.1. In vitro fermentation using fecal or excreta inocula 2.1.1. In vitro DM digestibility determination using Daisy system In vitro fermentation procedures were conducted in the Daisy II incubator (ANKOM Technology, Fairport, NY) using swine feces or turkey excreta as the source of inoculum. Three swine (50 kg BW) and three turkeys (15 kg BW) were used in this experiment as donors of feces or excreta. The animals were fed commercial diets with or without lignocellulose ingredients (product A and B). These products were added to the diets at the rate of 2.0%. The investigated substrates were commercial swine or turkey diet (P6), and lignocellulose A and B. Swine were fed individually; each animal
was fed one tested diet, while turkeys were fed together on the same diet to obtain enough amounts of excreta which were needed for an inoculum preparation. Moreover, the turkey birds were fed on the experimental diets consecutively, one after each other. The experimental diets were offered to animals for about 5 d (turkeys) to 10 d (swine) before collection of excreta or feces, to adapt the animals on these diets. Then, the feces or excreta were collected and prepared as an inoculum to be used in the Daisy incubator. The complete unit of Daisy II incubator consisted of 4 incubation vessels with a capacity of 2000 mL each. Each vessel contained 1600 mL of buffer solution, 400 mL of fecal inoculum, and 20 nylon bags. Samples of the tested ingredients were ground to pass a 0.75mm screen. Substrates (0.25 g) were weighed into nylon filter bags (Ankom F57, Ankom Technology, Fairpost, NY), three bags for each substrate, then heat-sealed. These bags were previously rinsed with acetone, air dried and then dried at 100 °C for 24 h, after which their weight was recorded. Bags containing the different ingredients/feeds were incubated in Daisy vessel with 400 ml of fecal inoculum and 1600 ml of buffer solution for 48 h at 39 °C. The microbial inoculum was prepared by collecting fresh feces or excreta. Fecal samples were placed into an air-tight freezer bag to maintain an anaerobic environment and transported to the laboratory in a cooler/thermos containing warm (39 °C) water. Once in the laboratory, a 50-g sample of feces was placed in a blender with 450 ml of warm distilled water (10:1). Samples were blended for 2 min while being gassed with CO2, then strained through a sieve (diameter: 200 lm) into the prewarmed incubation jars. Buffers consisted of 2 solutions that were combined in the incubation jars immediately before the fecal inoculum. Buffer solution A (KH2PO4, 10.0 g/L; MgSO4.7 H2O, 0.5 g/L; NaCl, 0.5 g/L; CaCl2.2 H2O, 0.1 g/L; and urea, 0.5 g/L) was added at 1330 mL to 266 mL of buffer solution B (Na2CO3, 15.0 g/L and Na2S.7 H2O, 1.0 g/L) to obtain a final pH of 6.8. The fecal inoculum was then added to each fermentation jar, after which the jars were purged with CO2 for 30 s and then sealed. The sealed jars were placed into the prewarmed Daisy II incubator. The incubator maintained a constant temperature of 39 °C throughout the incubation, and the jars were continuously agitated. The jars were removed after 48 h, and the filter bags were immediately rinsed for 30 min with cold water to stop microbial activity, then dried at 100 °C and the weight was recorded. Then, the in vitro DM digestibility of the samples was calculated. 2.1.2. Gas production and fermentation determination One hundred ml of produced fecal or excreta suspension/inoculum were mixed in a warmed glass beaker with 400 ml of warmed buffer (ratio of suspension to buffer (mixture of buffers A and B) was 1:5). The buffers applied in this trial were the same as that used before. Glass bottles (120 ml capacity) containing exactly 1 g of the tested feed/substrate (commercial swine or turkey diet, lignocellulose A and B) were prepared. Four bottles were used for each substrate. Further four glass bottles per animal were used without feed as control. The bottles were filled with 100 ml of the diluted suspension (ratio of feed to fecal suspension was 1:101) and a magnetic stirrer. Then, all bottles were sealed with rubber stoppers and lids and incubated on a magnetic stir plate in 39 °C warm water bath at about 400 rotations per minute for 24 h. After 24 h-incubation, the amount of gas produced in each bottle was measured by means of a gas pressure meter (Tracker 200 Series, DATA TRACK PROCESS INSTRUMENTS, Christchurch, Dorset) connected with syringe and cannula. Furthermore, the pH of the fermented substrate was determined using pH meter. Also, the
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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concentrations of volatile fatty acids (VFAs) and L-lactate produced in each bottle were analysed by using gas chromatography and photometer, respectively. 2.2. In vivo fermentation via using swine as a model of non-ruminants This experiment was conducted on swine only to determine the digestibility of the diet containing the product lignocellulose A as well as to measure the digestibility of this product per se. Six swine (40 kg body weight) were divided into 2 groups, 3 for each, and housed individually in metabolism pens. The control group was fed on commercial swine diet, whereas the experimental one was offered a commercial swine diet containing the recommended level of the lignocellulose product A (2.0%). The diets were chemically analysed according to AOAC (2005) and its composition is presented in Table 4. The animals were adapted on the diets for 10 days, then their fresh feces were collected and weighed daily for the next 5 days. The feces of 5 days for each animal were pooled and a representative sample was dried at 105 °C for 24 h to determine the DM content. The ash and crude fiber contents of the feces were measured. Also, the DM, ash and crude fiber contents of the diet were analysed. Accordingly, the digestibility rates of the DM, organic matter (OM) and crude fiber (CF) were calculated. Moreover, the digestibility of DM, OM and CF of lignocellulose product A per se was calculated by using the difference method between the control and the experimental diet. At the end of the digestibility trial, the animals were slaughtered 2 h postprandial and the amounts of the chymus content in the different parts of the digestive tract (stomach, small intestine, caecum and colon) were determined. Then, the pH and DM content of the digesta of each organ was measured. The caecum contents of each animal were removed and prepared as inocula to investigate its effect on fermentation of the tested ingredients, as described later in the third section. 2.3. Gas production and fermentation in vitro by using caecal inocula The caecum content of each animal was prepared as a microbial inoculum using the same procedure of fecal inoculum preparation that described previously in Daisy experiment. The same method used for gas and fermentation determination by fecal or excreta inocula was applied here. 100 ml of produced caecal suspension/ inoculum were mixed in a warmed glass beaker with 400 ml of warmed buffer. Glass bottles (120 ml capacity) containing exactly 1 g of the tested feed/substrate (commercial swine or turkey diet, lignocellulose A and B) were prepared. Four bottles were used for each substrate. Further four glass bottles per animal were used without feed as control. The bottles were filled with 100 ml of the diluted suspension and a magnetic stirrer. Then, all bottles were sealed with rubber stoppers and lids and incubated on a magnetic stir plate in 39 °C warm water bath at about 400 r.p.m. for 24 h. After 24 h-incubation, the amount of gas produced in each bottle was measured by a gas pressure meter connected with syringe and cannula. Furthermore, the pH of the fermented substrate was determined by a pH meter. Also, the concentrations of Llactate and volatile fatty acids (VFAs) produced in each bottle were analysed by using photometer and gas chromatography, respectively. The same procedure was repeated with turkeys after slaughtering them and using caecum and colon contents as an inoculum. The colon contents were used to provide sufficient amounts of chyme that needed for an inoculum preparation. 2.4. Statistical analyses
General Linear Models (GLM) procedure for analysis of variance. The results were subjected to one-way ANOVA accompanied by Tukey test to detect the differences between the treatments. Ttest was used in digestibility trial to compare between the control and experimental groups. Differences were considered to be significant when p < 0.05. Values are presented as arithmetical means with standard deviation (Mean ± SD). 3. Results 3.1. In vitro fermentation using fecal or excreta inocula 3.1.1. In vitro DM digestibility determination using Daisy system There were marked differences in the in vitro DM digestibility of the different substrates (Table 1). The same trend was noticed all over swine and turkeys. The digestibility of commercial diets was higher than that of lignocellulose products. It was also higher in lignocellulose A than B. Similar results were found among the different animals, indicating that the type of diet fed to animals had no effect on the in vitro DM digestibility. Due to this reason, the digestibility of each substrate was calculated collectively as shown in Table 2. It was found that the in vitro DM digestibility of product A was in the range of 23% (turkeys) up to 34% (pigs), whereas the product B had values of less than 5% in both species. However, the digestibility of commercial diets was higher and its value was nearly identical in swine and turkeys (about 75%). 3.1.2. Gas production and fermentation determination In swine, there was no effect of the diet type fed to animals on gas amounts, pH and fermentation products of the different substrates. Regardless the kind of diet, the amount of gas produced averaged about 26.3 and 1.20 ml/g for commercial swine diet
Table 1 In-vitro DM digestibility (%) of lignocellulose ingredients using Daisy incubator and feces/excreta of swine/turkeys as inoculums. Diet fed to animals
Commercial diet
Commercial diet + Lignocellulose A* Commercial diet + Lignocellulose B*
Substrate
In vitro DM digestibility (%) (n = 3)
Commercial diet Lignocellulose A Lignocellulose B Commercial diet Lignocellulose A Lignocellulose B Commercial diet Lignocellulose A Lignocellulose B
Swine inoculum
Turkeys inoculum
69.92 ± 8.68a 31.87 ± 4.89b 2.64 ± 3.63c 76.57 ± 1.71a 37.15 ± 5.54b 1.43 ± 1.91c 79.57 ± 2.81a 34.02 ± 2.33b 4.23 ± 4.52c
75.67 ± 1.28a 19.38 ± 1.41b 6.11 ± 1.49c 72.98 ± 1.95a 27.29 ± 3.95b 2.21 ± 2.15c 73.77 ± 1.88a 23.03 ± 2.36b 4.72 ± 3.68c
*
Lignocellulose products were added at a rate of 2.0% of the commercial diets. Means within the same column (in each consumed diet) with different superscripts are significantly different (p < 0.05). a,b,c
Table 2 In-vitro DM digestibility (%) of lignocellulose ingredients using Daisy incubator and feces/excreta of swine/turkeys as inoculums, regardless the consumed diets. Substrate Commercial diet Lignocellulose A Lignocellulose B
Swine (n = 9)1 a
75.4 ± 6.31 34.3 ± 4.51b 3.0 ± 3.01c
Turkeys (n = 9)2 74.1 ± 1.92a 23.2 ± 4.19b 4.30 ± 2.83c
1
3 animals 3 samples per substrate. 3 trials (each trial was done on 3 birds together) 3 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05). 2
The statistical analyses were performed using SAS statistical program (SAS Institute, 2002). The data were evaluated using the
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
Note: C2: acetic, C3: propionic, C4: butyric, C5: valeric. 1 n = 3 animals 4 samples per substrate. 2 3 trials (each trial was done on 3 birds together) 4 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05).
L-lactate C5 C4 C3 C2 P VFA pH Gas C2
C3
C4
C5
L-lactate
Turkeys (n = 12)2
VFA pH
As observed in the fermentation of substrates with fecal or excreta inoculums, there was no effect of the diet type on the fermentation with caecal inocula among the animals. In swine, the gas produced in the commercial diet substrate was higher (22.6 ml/g) than lignocellulose products (Table 7). However, the amount of gas in lignocellulose A was higher than B (2.50 vs. 1.0 ml/g). As a result of fermentation, the pH values were reduced in commercial diet (5.62) and lignocellulose A (6.52) in comparison to B (6.65). Moreover, the concentration of VFAs was higher in commercial diet than lignocellulose products. In addition,
Gas
3.3. Gas production and fermentation in vitro by using caecal inocula
P
The chemical analyses of diets revealed that the diets were isocaloric (13.5 MJ ME/kg) and isonitrogenous (175 g CP/kg; Table 4). However, the crude fiber was slightly higher in experimental diet than the control (46.4 vs. 39.3 g/kg). There were no differences in digestibility rates of dry matter, organic matter and crude fiber between the animals fed commercial swine diet and those fed commercial swine diet with 2.0% lignocellulose A (Table 5). The digestibility of DM, OM and CF of the tested lignocellulose product per se was nearly equal (about 50%). The amount of digesta and its DM content in the caecum and colon of the swine fed on the experimental diet was higher than the control group (Table 6). However, the amount of chymus in the stomach and small intestine was lower in the experimental group compared to the control. Moreover, the pH of chymus in the segments of gastrointestinal tract was not affected by the lignoceullose product. The fecal DM content of the experimental treatment was numerically higher than the control one.
Swine (n = 12)1
3.2. In vivo fermentation via using swine as a model of non-ruminants
Substrate
and lignocellulose A, respectively (Table 3). However, no gas was determined in case of lignocellulose B. Moreover, the pH values of the substrates after 24 h fermentation were a reflection of the gas production. Thus, the higher gas produced resulted in lower pH values. The results of volatile fatty acids (VFAs) resulting from fermentation of the substrates showed that lignocellulose product A produced considerable amounts of acetic (3.61 mmol/kg) and propionic (0.75 mmol/kg) acids in comparison to the product B which produced very low amounts of these acids (0.49 and 0.09 mmol/kg). As expected, commercial swine diet resulted in higher amounts of VFAs produced, and the values were 21.8, 9.58, 5.14 and 2.47 mmol/kg for acetic, propionic, butyric and valeric acids, respectively. Accordingly, the concentration of total volatile fatty acids resulted from the commercial diet substrate was found to be high (39.0 mmol/kg). In addition, lignocellulose A produced greater amounts of total VFAs compared to product B (4.45 vs. 0.58 mmol/kg). Moreover, the tested substrates provided low amounts of L-lactate, but the concentration in commercial diet and lignocellulose A was higher than B. In turkeys, the trend of gas amount, VFAs and L-lactate findings were similar to that found in swine. As observed in swine, there was also no effect of the diet type consumed by the birds on the fermentation process using turkey excreta as inoculum. Thus, the gas amount produced all over the birds was higher in commercial turkey diet (P6) and averaged about 29.3 ml/g. Little amount of gas was obtained in lignocellulose A (1.0 ml/g), but no gas was found in the product B. The pH values decreased with increasing the amount of gas output and the values were 5.39, 6.68 and 6.77 for commercial diet, lignocellulose A and B, respectively. The commercial diet resulted in higher amounts of total and individual fatty acids. Moreover, lignocellulose A produced greater amounts of these acids compared to the product B. L-lactate concentration was higher in commercial diet and lignocellulose A than B.
Commercial 26.3a ± 7.30 5.76a ± 0.07 39.0a ± 4.28 21.8a ± 2.89 9.58a ± 0.86 5.14a ± 1.70 2.47a ± 1.41 0.03a ± 0.02 29.3a ± 5.12 5.39a ± 0.22 40.8a ± 4.19 22.9a ± 2.33 12.2a ± 1.47 3.34a ± 2.57 2.30a ± 1.11 0.03a ± 0.02 diet Lig. A 1.20b ± 0.01 6.64b ± 0.04 4.45b ± 1.75 3.61a ± 1.52 0.75b ± 0.45 0.07b ± 0.06 0.02b ± 0.01 0.03a ± 0.02 1.0b ± 0.02 6.68b ± 0.05 3.40b ± 1.17 1.81b ± 0.85 1.28b ± 1.15 0.23b ± 0.18 0.08b ± 0.03 0.04a ± 0.02 Lig. B – 6.99c ± 0.04 0.58c ± 0.38 0.49a ± 0.39 0.09c ± 0.09 – – 0.01b ± 0.01 – 6.77c ± 0.10 1.57c ± 0.79 0.80c ± 0.50 0.64c ± 0.63 0.02c ± 0.01 0.11b ± 0.01 0.01b ± 0.01
I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 3 Gas amount (ml/g), pH of the fermented substrate, L-lactate amounts (mmol/l or kg) and volatile fatty acids (mmol/l or kg) produced after incubation of the substrates with buffer and feces/excreta of swine/turkeys for 24 h, regardless the consumed diets.
4
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 4 Chemical composition of swine diets used in digestibility trial (on fresh matter basis).
Chemical analyses (g/kg) ME (MJ/kg)* Dry matter Crude ash Crude protein Crude fat Crude fiber Nitrogen free extract Starch Sugar Macrominerals (g/kg) Calcium Magnesium Phosphorus Sodium Potassium Chlorine Sulphur Trace elements (mg/kg) Copper Zinc Iron Manganese Selenium Amino acids (g/kg) Threonine Glutamic Glycine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine
Control diet
Experimental diet
13.7 899 51.1 175.8 46.1 39.3 587 382.6 41.6
13.3 897 51.5 174.2 42.9 46.4 582 359.4 43.6
10.5 2.4 5.7 2.7 8.5 4.9 2.2
9.0 2.3 5.5 2.4 8.3 4.7 2.1
19.6 145.8 174.8 120.9 0.5
14.4 136.4 222.0 111.5 0.5
6.1 40.4 7.9 2.7 2.8 6.9 12.5 5.9 8.3 4.8 10.6
6.5 39.6 7.8 2.6 2.8 6.6 12.2 5.7 8.2 4.8 10.5
4. Discussion
* Metabolizable energy (MJ/kg) = 0.021503 CP + 0.032497 crude fat + 0.016309 starch + 0.014701 [organic matter-(CP + crude fat + starch + CF)] 0.021071 CF (Kamphues et al., 2009).
Table 5 DM, OM and CF digestibility rates (%) of swine diets as well as of lignocellulose A ingredient per se (n = 3).
Digestibility (%): Dry matter Organic matter Crude fiber
lignocellulose A resulted in greater amounts of total VFAs as well as of acetic, propionic, butyric and valeric acids when compared to the product B. Concerning the L-lactate amounts produced from the substrates, lignocellulose A was found to produce higher concentration than lignocellulose B but did not differ with the commercial diet. Using the caecum and colon contents of turkeys as inoculums, the same findings of swine were reported in turkeys. It was noticed that lignocellulose A substrate was well fermented than lignocellulose B via producing greater amounts of VFAs and gas. This was reflected by a lower pH value in lignocellulose A. However, the amounts of L-lactate produced from lignocellulose A were nearly equal to the product B. Moreover, the commercial turkey diet produced higher amounts of gas and VFAs compared to lignocellulose products, but no effect on L-lactate was observed.
Diet ( )1
Diet (+)2
Pure lign. A3
80.3 ± 0.71 83.2 ± 0.73 28.4 ± 6.63
79.6 ± 0.36 82.3 ± 0.31 30.8 ± 6.65
50.1 ± 14.7 47.0 ± 12.2 48.0 ± 23.9
1
Control: animals fed on a commercial swine diet. Experimental group: animals fed on a commercial swine diet containing 2.0% lignocellulose A. 3 Nutrient digestibility of lignocellulose A was estimated by difference between control and experimental diet. 2
The tested substrates were chemically analysed (The data are presented in another contribution, under preparation). It was found that the crude fiber content was about 38.2, 21.4, 560, and 612 g/kg for commercial swine diet, turkey P6 diet, lignocellulose A and B, respectively. Comparing lignocellulose products, lignocellulose A was noticed to contain higher CP (32.0 vs. 7.95 g/kg), ether extract (13.1 vs. 2.65 g/kg), and nitrogen free extract (267 vs. 260 g/kg) than lignocellulose B. In the present study, faces of swine or excreta of turkeys can be used as a microbial inoculum to measure the fermentation rate of feedstuffs. Successful use of a liquid suspension of feces to determine digestibility of different temperate feedstuffs has been reported (El-Shaer et al., 1987). Also, Jones and Barnes (1996) found that the use of feces may be easier rather than to collect rumen fluid as an inoculum source for in vitro dry matter digestibility. Lowman et al. (1999) noticed that equine feces were suitable inoculum sources for gas production assessment in the horse. In addition, Lattimer et al. (2007) reported that equine feces can be used successfully for in vitro experiments using the Daisy II incubator. Feces have been recognized as an inexpensive and readily available source of microorganisms that could easily be collected from several animals at a time (Earing et al., 2014). The average total gastrointestinal tract retention time in swine has been reported to be about 45 h (Wilfart et al., 2007). Therefore, the incubation period used in the Daisy system was 48 h to simulate the normal condition in vivo. Moreover, the retention time in the hindgut of swine is about 24 h, thus the incubation period included in the gas production technique was 24 h. The mean retention time in total tract or hindgut of turkeys is shorter than swine, but the incubation lengths used in this study were comparable to that of swine to facilitate the comparison between both species.
Table 6 Amount (g fresh matter/kg BW), pH values and DM (%) of the digesta in the different parts of swine digestive tract as well as fecal DM content (%) (n = 3). Amount of digesta (g fresh matter/kg BW)
Stomach Small intestine Caecum Colon Feces 1 2 a,b
Digesta pH
DM content (%)
Diet ( )1
Diet (+)2
Diet ( )
Diet (+)
Diet ( )
Diet (+)
31.2a ± 2.27 9.26a ± 0.85 2.81b ± 0.55 15.6b ± 0.65 –
26.7b ± 1.47 8.17b ± 2.91 4.70a ± 0.69 16.9a ± 1.00 –
3.89a ± 0.28 6.76a ± 0.17 6.14a ± 0.20 6.34a ± 0.31 –
3.91a ± 0.07 6.45a ± 0.16 6.18a ± 0.15 6.06a ± 0.34 –
16.5a ± 4.49 13.4a ± 2.34 11.5b ± 1.01 18.2b ± 1.75 28.99a ± 1.22
17.4a ± 1.08 12.0a ± 1.63 12.7a ± 0.21 20.1a ± 1.02 30.02a ± 3.55
Control: animals fed on a commercial swine diet. Experimental group: animals fed on a commercial swine diet containing 2.0% ligocellulose A. Means within the same row (in each parameter) with different superscripts are significantly different (p < 0.05).
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
L-lactate C5 C4 C3 C2 P VFA pH
a
pH
a
Gas
Note: C2: acetic, C3: propionic, C4: butyric, C5: valeric. 1 n = 3 animals 4 samples per substrate. 2 n = 3 birds 4 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05).
a
Gas C2 VFA
Substrate
Swine (n = 12)1
c
P
a
a
C3
a
C4
a
C5
a
L-lactate
Turkeys (n = 12)2
c
Commercial 22.6 ± 7.91 5.62 ± 0.25 41.9 ± 8.99 22.2 ± 2.01 8.48 ± 2.98 7.32 ± 3.30 3.94 ± 2.10 0.54 ± 0.71 29.8 ± 1.26 5.44 ± 0.04 30.5a ± 1.96 15.75a ± 0.98 9.49a ± 1.09 2.62a ± 0.49 2.64a ± 1.13 0.03a ± 0.01 diet Lig. A 2.50b ± 1.05 6.52b ± 0.03 4.01b ± 2.04 3.16b ± 1.63 0.55b ± 0.42 0.18b ± 0.18 0.12b ± 0.06 0.78a ± 0.61 4.0b ± 1.15 6.43b ± 0.02 3.66b ± 2.90 1.12b ± 0.74 2.26b ± 2.04 0.10b ± 0.08 0.18b ± 0.06 0.07a ± 0.03 Lig. B 1.0c ± 0.01 6.65a ± 0.03 1.46c ± 0.71 1.13c ± 0.64 0.18c ± 0.09 0.09c ± 0.08 0.06c ± 0.04 0.07b ± 0.08 1.0c ± 0.00 6.57a ± 0.01 1.49c ± 1.17 0.97c ± 0.30 0.44c ± 0.38 – 0.08c ± 0.07 0.05a ± 0.05
I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 7 Gas amount (ml/g), pH of the fermented substrate, L-lactate amounts (mmol/l or kg) and volatile fatty acids (mmol/l or kg) produced after incubation of the substrates with buffer and caecum/hind gut contents of swine/turkeys for 24 h, regardless the consumed diets.
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The type of diet fed to animals did not influence the nature of inoculum or bacteria as indicated by similar in vitro DM digestibility of ingredients or fermentation products among the animals fed different diets. The low fiber content-feedstuffs were highly digested than fiber rich ingredients, as observed in commercial diets compared to lignocellulose products. However, the in vitro DM digestibility of lignocellulose A was higher than B, indicating that the lignocellulose A contains higher amounts of fermentable fiber. The in vitro DM digestibility of swine diet and lignocellulose A was about 75.4 and 34.3%, respectively. These in vitro results are consistent with that found in vivo as the DM digestibility of swine diet and lignocellulose A per se was determined in swine to be about 80 and 50%, respectively. Moreover, the digestibility rates of organic matter and crude fiber of lignocellulose A were nearly similar to that of DM (about 48%). Thus, the findings of Daisy II technique in this study are supported with that obtained in vivo. Therefore, the Daizy II incubator is appropriate for the determination of in vitro digestibility of nutrients using fecal liquor. This finding is confirmed with the results of the previous studies (Holden, 1999; Mabjeesh et al., 2000; Laudadio et al., 2009). In vivo, no effect of diet type on fermentation was also detected as indicated by similar results in digestibility of DM, organic matter, and crude fiber between control and experimental animals. Moreover, lignocellulose A could improve the quality of feces as indicated by its role in increasing the fecal DM content by about 1% than the control (30.02 vs 28.99%). Thus, lignocelluloses can be used to provide a better consistency of feces or used to prevent loose or watery fecal matter. No effect of diet type consumed by the animals on digestibility, gas production, and fermentation products was determined. The same finding was observed by Earing et al. (2014) who found no effect of donor diet on in vitro digestibility. This assumed that the fiber source did not influence the number, nature or activity of fibrolytic bacteria in GIT or the amount added to the diet (2.0%) was not enough to encourage its multiplication. In spite of no gas production in lignocellulose B, very little fermentation occurred. This could be due to the pressure of gas formed in this substrate was lower than that of atmospheric pressure, thus it cannot be measured by the gas meter. The fermentation of substrates with fecal or excreta inocula showed higher production of gas and VFAs in lignocellulose A than B. This could be attributed to the higher contents of organic nutrients (CP, ether extract and nitrogen free extract) in lignocellulose A. Furthermore, the fiber of lignocellulose A was well fermented by caecum inocula and resulted in higher amounts of acetic, propionic, butyric and valeric acids in comparison to lignocellulose B. Thus, lignocellulose A can be used to stimulate the integrity and health of the intestinal lining. It was reported that lignocellulose had a positive effect on performance in swine (Kroismayr et al., 2008a; Bernscherer et al., 2011) and in broilers (Kroismayr et al., 2008b). Increasing the amount of digesta in caecum and colon of swine fed diets containing lignocellulose A indicating the higher fermentation of this product. Nevertheless, commercial swine or turkey diets were easily fermented by feces or caecum inoculum due to the higher starch contents (McDonald et al., 2010). In using fecal/excreta or caecal inocula in swine or turkeys, the more the gas production the lower pH values of substrates. This could be due to production of VFAs in these fermented substrates (Johnston et al., 2003). The obtained results revealed that the findings of using swine feces as an inoculum were coordinated with that resulted from using turkey excreta – inoculum. The same observations were found in caecal inocula of swine and turkeys. In addition, all over swine and turkeys, the fermentation results of the substrates with the caecum inocula were nearly parallel to that obtained by fecal or excreta inoculums.
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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5. Conclusions The present results indicate that the fecal or excreta inoculum can be used to determine the in vitro DM digestibility or fermentation of feeds. In addition, Daisy II method can be used to predict in vitro digestibility with relatively small variation. The new lignocellulose products differ markedly regarding their microbial degradability. Therefore, their effects on the hind gut flora (and its fermentation), the gut fill and the feces quality should be different. Regarding the product B mechanical effects might be dominant whereas for the product A further advantages (fermentation) could be expected. Moreover, the digestibility of DM, organic matter and crude fiber of lignocellulose A per se was estimated to be about 50%, 47.0%, and 48.0%, respectively. Therefore, lignocellulose A can be used as a good source of fiber in diets of monogastric animals in order to maintain the gut health and to improve the feces or excreta quality.
References AOAC, 2005. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists, Gaithersburg, MD. Bach Knudsen, K.E., Jorgensen, H., 2001. Intestinal degradation of dietary carbohydrates – from birth to maturity. In: Lindberg, J.E., Ogle, B., (Eds.). Digestive Physiology of Pigs. CABI Publishing, New York, p. 109. Bernscherer, M., Mair, C., Schedle, K., 2011. Einfluss eines Lignozellulosezusatzes auf die Mast- und Schlachtleistung bei Schweinen. 10. BOKU-Symposium Tierernaehrung, Wein. Earing, J.E., Cassill, B.D., Hayes, S.H., Vanzant, E.S., Lawrence, L.M., 2014. Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses. J. Anim. Sci. 88, 3954–3963. El-Shaer, H.M., Omed, H.M., Chamberlain, A.G., 1987. Use of faecal organisms from sheep for the in vitro determination of digestibility. J. Agric. Sci. Cambridge 109, 257–259. Govers, M.J., Gannon, N.J., Dunshea, F.R., Gibson, P.R., Muir, J.G., 1999. Wheat bran affects the site of fermentation of resistant starch and luminal indexes related to colon cancer risk: a study in pigs. Gut 45 (6), 840–847. Holden, L.A., 1999. Comparison of methods of in vitro dry matter digestibility for ten feeds. J. Dairy Sci. 82, 1791–1794. Johnston, L.J., Noll, S., Renteria, A., Shurson, J., 2003. Feeding by-products high in concentration of fiber to nonruminants. The Third National Symposium on Alternative Feeds for Livestock and Poultry held in Kansas City, MO on November 4.
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Jones, R.J., Barnes, P., 1996. In vitro digestibility assessment of tropical shrubs legumes using rumen fluid or faecal fluid as the inoculum source. Tropical Grasslands 30, 374–377. Kamphues, J., Kienzle, E., Coenen, M., Pallauf, J., Wanner, M., Simon, O., Zentek, J., 2009. Supplemente Zu Vorlesungen Und Übungen in Der Tierernährung. M. & H. Schaper, Hannover, Germany. Krieg, R., Schüle, S., Dohms, J., 2008. Lignocellulose als sichere Rohfaserquelle zur Leistungsstabilisierung bei Häsinnen und Jungtieren in der Kaninchenzucht. In: 7.BOKU-Symposium Tierernährung: Wien, Austria, pp. 136–145. Krieg, R., Martienssen, M., Zentek, J., 2012. Effect of the ratio lignin to cellulose (adfadl) on caecal fermentation, gut morphology and performance of rabbits around weaning. In: Proceedings 10th World Rabbit Congress – September 3–6, Sharm El- Sheikh –Egypt, 685–689. Kroismayr, A., Neufeld, K., Affentranger, P., 2008a. Einfluss einer neuartigen Lignocellulose auf Leistungsparameter in der Ferkelaufzucht. 7. BOKUSymposium Tierernaehrung, Wein. Kroismayr, A., Neufeld, N., Neufeld, K., 2008b. Einfluss einer neuen Lignocellulose Formulierung auf die Leistungsdaten beim Broiler. 7. BOKU-Symposium Tierernaehrung, Wein. Lattimer, J.M., Cooper, S.R., Freeman, D.W., Lalman, D.L., 2007. Effect of yeast culture on in vitro fermentation of a high concentrate or high-fiber diet using equine fecal inoculums in a DaisyII incubator. J. Anim. Sci. 85, 2484–2491. Laudadio, V., Lacalandra, G.M., Monaco, D., Khorchani, T., Hammadi, M., Tufarelli, V., 2009. Faecal liquor as alternative microbial inoculum source for in vitro (DaisyII) technique to estimate the digestibility of feeds for camels. J. Camelid Sci. 2, 01–07. Lowman, R.S., Theodorou, M., Hyslop, J.J., Dhanoa, M.S., Cuddeford, D., 1999. Evaluation of an in vitro batch culture technique for estimating the in vivo digestibility and digestible energy content of equine feeds using equine faeces as the source of microbial inoculum. Anim. Feed Sci. Technol. 80, 11–27. Mabjeesh, S.J., Cohen, M., Arieli, A., 2000. In vitro methods for measuring the dry matter digestibility of ruminant feedstuffs: comparison of methods and inoculum source. J. Dairy Sci. 83, 2289–2294. Marten, G.C., Barnes, R.F., 1980. Prediction of energy digestibility of forages with in vitro rumen fermentation and fungal enzyme systems. In: Pigden, W.J., Balch, C.C., Graham, M., (Eds.). Standardization of Analytical Methodology for Feeds. Int. Dev. Res. Center, Ottawa, Canada, 61–128. McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., Morgan, C.A., Sinclair, L.A., Wilkinson, R.G., 2010. Animal Nutrition, Seventh Edition, Longman Scientific and Technical. John Wiley & Sons Inc, New York, USA. Sarandan H, Nina Neufeld, Neufeld K. Effects of a new lignocellulose product for fibre supplementation on MMA symptoms, reproductive performance and faecal quality in a pig farm with evident mma problems in Romania. Lucra˘ri Stiinłifice Medicina˘ Veterinara˘ Vol. Xli, 2008, Timisoara., 851–858. SAS Institute, 2002. SASÒ User’s Guide: Statistics. Version 9.1. SAS Institute, Cary, NC. Wilfart, A., Montagne, L., Simmins, H., Noblet, J., van Milgen, J., 2007. Digesta transit in different segments of the gastrointestinal tract of pigs as affected by insoluble fibre supplied by wheat bran. Br. J. Nutr. 98, 54–62.
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Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys) Ibrahim M.I. Youssef a,b,⇑, Josef Kamphues a a b
Institute of Animal Nutrition, University of Veterinary Medicine Hannover, Germany Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Beni-Suef University, Egypt
a r t i c l e
i n f o
Article history: Received 22 April 2017 Received in revised form 14 May 2017 Accepted 14 May 2017 Available online xxxx Keywords: Digestibility Lignocellulose Fermentation Fecal liquor Swine Turkeys
a b s t r a c t Three experiments were performed to evaluate the digestibility and fermentation rates of two types of lignocellulose products that can be used in diets of pigs and turkeys. Experiment I: In vitro dry matter digestibility studies using Daisy system were conducted by using fecal or excreta fluid of swine/turkeys as a source of inoculums. The feedstuffs used as substrates were commercial swine or turkey diet, lignocellulose A and B. The dry matter digestibility was found to be about 75%, 28%, and <5% for swine/turkey diet, lignocellulose A and B, respectively. Moreover, the inoculums were incubated with the same substrates for 24 h using gas measuring technique. It was found that the product A was fermented better than B and resulted in higher amounts of acetic, propionic and lactic acids. Experiment II: Six swine were divided into 2 groups; the control group was fed on a commercial swine diet, whereas the experimental one was offered a commercial swine diet containing 2.0% lignocellulose A. The digestibility rates of dry matter, organic matter, and crude fiber of both diets were found to be nearly identical. Experiment III: At the end of study, the animals were slaughtered and the caecum contents of swine and turkeys were used as inoculums and incubated with the same tested substrates for 24 h using gas production technique. As observed with fecal/excreta inocula, lignocellulose A resulted in greater amounts of gases, volatile fatty acids and lactate compared to product B. In conclusions, lignocellulose A can be used as alternative source of fiber to maintain the health and function of digestive tract. Furthermore, the use of feces/excreta liquor provides a valid and proper estimate of digestibility or fermentation of feeds. These results provide further guide that this procedure would be an effective and practical means of approximating the digestibility of diets. Ó 2017 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Dietary fiber is major concern in nutrition of monogastric animals. It is important to maintain optimal digestion, fecal quality and health of the animals (Sarandan et al., 2008). High-starch diets support rapid fermentation, which is often complete early in the digestion process leaving little or no fermentation for the large intestine (McDonald et al., 2010). Inclusion of dietary fiber in the diet shifts the fermentation process to the large intestine and increases the growth of beneficial bacteria (Govers et al., 1999; Krieg et al., 2008, 2012). These bacteria produce short-chain fatty acids and lactic acid that improve the health and integrity of the intestinal lining as well as decrease the pathogenic bacteria in the intestine (Bach Knudsen and Jorgensen, 2001). Furthermore, ⇑ Corresponding author at: Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt. E-mail address: [email protected] (I.M.I. Youssef).
volatile fatty acids and lactic acid are essential to maintain eubiosis (stable and healthy microflora populations) in the large intestine (Johnston et al., 2003). Moreover, lactic acid is known as an essential antagonist to pathogens in the digestive tract and will therefore positively affect the gut health of the animals. Most recently, special dietary fiber products derived from lignocellulose are being used in animal nutrition with low inclusion rates. Lignocellulose is a product made from fresh wood and has been used as a high quality fiber source in animal diets (Sarandan et al., 2008). Compared to traditional fiber sources, lignocellulose is characterised by high crude fiber (>55%). Nowadays, a new eubiotic lignocellulose product has become commercially available. This product contains a blend of non-fermentable and fermentable fiber components. Thus, the digestibility of these feed materials by monogastric animals is of special interest. The digestibility value of a feed is one of the best indicators of nutritive value. Measurement of in vitro dry matter digestibility (IVDMD) has been used extensively to analyse feeds because of a
http://dx.doi.org/10.1016/j.bjbas.2017.05.006 2314-8535/Ó 2017 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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high degree of correlation to in vivo digestibility (Marten and Barnes, 1980). In vivo determinations are expensive and time consuming, rendering this technique impractical for routine analyses. Over the years, various techniques to determine IVDMD have been developed and modified. The recent development of DaisyII apparatus (ANKOM Technology Corp., Fairport, NY) allows simultaneous incubation of different feedstuffs in sealed polyester bags in the same incubation vessel. With this method, the material that disappears from the bag during the incubation is considered digestible (Mabjeesh et al., 2000). The DaisyII system is more labor efficient than traditional methods and represents a significant advantage for analysis of forage, grain and mixture of ingredients (Holden, 1999). Feeding monogastric species of crude fiber supply is on debate, especially due to its diverse effects on digestion, gut health, feces quality and animal welfare (Johnston et al., 2003). Fibrous feeds traditionally have not been used for nonruminants due to their effects on diet digestibility in pigs and poultry. However, some types of fiber and fiber sources do not exert such negative effects on nutrient digestibility in older growing pigs and sows. Dietary fiber can have positive effects on gut health, welfare, and reproductive performance. Hence, nutritionists are attempting to gain a more thorough understanding of dietary fiber in swine and poultry diets. Moreover, there is a lack of information on the fermentation process of these products by swine and poultry. Therefore, the aim of this study was to provide a deeper characterization of these new fiber ingredients (lignocellulose products) that can be used in diets of swine and turkeys by determining their in vitro dry matter digestibility in the Daisy incubator system using fresh feces (pigs) and excreta (turkeys) as sources of inocula. Furthermore, the ability of these products to ferment and produce gases or volatile fatty acids by fecal/excreta inoculums was evaluated. Afterwards, the results of the in vitro were confirmed in vivo especially in swine by testing the digestibility rate of the most digestible product of lignocellulose ingredients. In addition, the fermentation rates of the tested ingredients were evaluated using the caecum contents of swine and turkeys as inoculum precursors, and then compared with that obtained by feces or excreta inocula. 2. Materials and methods The study was carried out in vitro and in vivo. It was started in vitro to evaluate the fermentation of ingredients by Daisy system via using the fecal (swine) or excreta (turkeys) as inoculum sources. In addition, the amounts of gas, lactate and volatile fatty acids produced from the tested ingredients, by using fecal or excreta inocula, were determined. Based on the results of in vitro section, the most fermentable ingredient was tested in vivo through using swine as a model of non-ruminants. Afterwards, the swine and turkeys were slaughtered and the caecum contents were used for preparation of caecal inocula which were used in vitro to measure the gas production and fermentation of the investigated ingredients. 2.1. In vitro fermentation using fecal or excreta inocula 2.1.1. In vitro DM digestibility determination using Daisy system In vitro fermentation procedures were conducted in the Daisy II incubator (ANKOM Technology, Fairport, NY) using swine feces or turkey excreta as the source of inoculum. Three swine (50 kg BW) and three turkeys (15 kg BW) were used in this experiment as donors of feces or excreta. The animals were fed commercial diets with or without lignocellulose ingredients (product A and B). These products were added to the diets at the rate of 2.0%. The investigated substrates were commercial swine or turkey diet (P6), and lignocellulose A and B. Swine were fed individually; each animal
was fed one tested diet, while turkeys were fed together on the same diet to obtain enough amounts of excreta which were needed for an inoculum preparation. Moreover, the turkey birds were fed on the experimental diets consecutively, one after each other. The experimental diets were offered to animals for about 5 d (turkeys) to 10 d (swine) before collection of excreta or feces, to adapt the animals on these diets. Then, the feces or excreta were collected and prepared as an inoculum to be used in the Daisy incubator. The complete unit of Daisy II incubator consisted of 4 incubation vessels with a capacity of 2000 mL each. Each vessel contained 1600 mL of buffer solution, 400 mL of fecal inoculum, and 20 nylon bags. Samples of the tested ingredients were ground to pass a 0.75mm screen. Substrates (0.25 g) were weighed into nylon filter bags (Ankom F57, Ankom Technology, Fairpost, NY), three bags for each substrate, then heat-sealed. These bags were previously rinsed with acetone, air dried and then dried at 100 °C for 24 h, after which their weight was recorded. Bags containing the different ingredients/feeds were incubated in Daisy vessel with 400 ml of fecal inoculum and 1600 ml of buffer solution for 48 h at 39 °C. The microbial inoculum was prepared by collecting fresh feces or excreta. Fecal samples were placed into an air-tight freezer bag to maintain an anaerobic environment and transported to the laboratory in a cooler/thermos containing warm (39 °C) water. Once in the laboratory, a 50-g sample of feces was placed in a blender with 450 ml of warm distilled water (10:1). Samples were blended for 2 min while being gassed with CO2, then strained through a sieve (diameter: 200 lm) into the prewarmed incubation jars. Buffers consisted of 2 solutions that were combined in the incubation jars immediately before the fecal inoculum. Buffer solution A (KH2PO4, 10.0 g/L; MgSO4.7 H2O, 0.5 g/L; NaCl, 0.5 g/L; CaCl2.2 H2O, 0.1 g/L; and urea, 0.5 g/L) was added at 1330 mL to 266 mL of buffer solution B (Na2CO3, 15.0 g/L and Na2S.7 H2O, 1.0 g/L) to obtain a final pH of 6.8. The fecal inoculum was then added to each fermentation jar, after which the jars were purged with CO2 for 30 s and then sealed. The sealed jars were placed into the prewarmed Daisy II incubator. The incubator maintained a constant temperature of 39 °C throughout the incubation, and the jars were continuously agitated. The jars were removed after 48 h, and the filter bags were immediately rinsed for 30 min with cold water to stop microbial activity, then dried at 100 °C and the weight was recorded. Then, the in vitro DM digestibility of the samples was calculated. 2.1.2. Gas production and fermentation determination One hundred ml of produced fecal or excreta suspension/inoculum were mixed in a warmed glass beaker with 400 ml of warmed buffer (ratio of suspension to buffer (mixture of buffers A and B) was 1:5). The buffers applied in this trial were the same as that used before. Glass bottles (120 ml capacity) containing exactly 1 g of the tested feed/substrate (commercial swine or turkey diet, lignocellulose A and B) were prepared. Four bottles were used for each substrate. Further four glass bottles per animal were used without feed as control. The bottles were filled with 100 ml of the diluted suspension (ratio of feed to fecal suspension was 1:101) and a magnetic stirrer. Then, all bottles were sealed with rubber stoppers and lids and incubated on a magnetic stir plate in 39 °C warm water bath at about 400 rotations per minute for 24 h. After 24 h-incubation, the amount of gas produced in each bottle was measured by means of a gas pressure meter (Tracker 200 Series, DATA TRACK PROCESS INSTRUMENTS, Christchurch, Dorset) connected with syringe and cannula. Furthermore, the pH of the fermented substrate was determined using pH meter. Also, the
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
concentrations of volatile fatty acids (VFAs) and L-lactate produced in each bottle were analysed by using gas chromatography and photometer, respectively. 2.2. In vivo fermentation via using swine as a model of non-ruminants This experiment was conducted on swine only to determine the digestibility of the diet containing the product lignocellulose A as well as to measure the digestibility of this product per se. Six swine (40 kg body weight) were divided into 2 groups, 3 for each, and housed individually in metabolism pens. The control group was fed on commercial swine diet, whereas the experimental one was offered a commercial swine diet containing the recommended level of the lignocellulose product A (2.0%). The diets were chemically analysed according to AOAC (2005) and its composition is presented in Table 4. The animals were adapted on the diets for 10 days, then their fresh feces were collected and weighed daily for the next 5 days. The feces of 5 days for each animal were pooled and a representative sample was dried at 105 °C for 24 h to determine the DM content. The ash and crude fiber contents of the feces were measured. Also, the DM, ash and crude fiber contents of the diet were analysed. Accordingly, the digestibility rates of the DM, organic matter (OM) and crude fiber (CF) were calculated. Moreover, the digestibility of DM, OM and CF of lignocellulose product A per se was calculated by using the difference method between the control and the experimental diet. At the end of the digestibility trial, the animals were slaughtered 2 h postprandial and the amounts of the chymus content in the different parts of the digestive tract (stomach, small intestine, caecum and colon) were determined. Then, the pH and DM content of the digesta of each organ was measured. The caecum contents of each animal were removed and prepared as inocula to investigate its effect on fermentation of the tested ingredients, as described later in the third section. 2.3. Gas production and fermentation in vitro by using caecal inocula The caecum content of each animal was prepared as a microbial inoculum using the same procedure of fecal inoculum preparation that described previously in Daisy experiment. The same method used for gas and fermentation determination by fecal or excreta inocula was applied here. 100 ml of produced caecal suspension/ inoculum were mixed in a warmed glass beaker with 400 ml of warmed buffer. Glass bottles (120 ml capacity) containing exactly 1 g of the tested feed/substrate (commercial swine or turkey diet, lignocellulose A and B) were prepared. Four bottles were used for each substrate. Further four glass bottles per animal were used without feed as control. The bottles were filled with 100 ml of the diluted suspension and a magnetic stirrer. Then, all bottles were sealed with rubber stoppers and lids and incubated on a magnetic stir plate in 39 °C warm water bath at about 400 r.p.m. for 24 h. After 24 h-incubation, the amount of gas produced in each bottle was measured by a gas pressure meter connected with syringe and cannula. Furthermore, the pH of the fermented substrate was determined by a pH meter. Also, the concentrations of Llactate and volatile fatty acids (VFAs) produced in each bottle were analysed by using photometer and gas chromatography, respectively. The same procedure was repeated with turkeys after slaughtering them and using caecum and colon contents as an inoculum. The colon contents were used to provide sufficient amounts of chyme that needed for an inoculum preparation. 2.4. Statistical analyses
General Linear Models (GLM) procedure for analysis of variance. The results were subjected to one-way ANOVA accompanied by Tukey test to detect the differences between the treatments. Ttest was used in digestibility trial to compare between the control and experimental groups. Differences were considered to be significant when p < 0.05. Values are presented as arithmetical means with standard deviation (Mean ± SD). 3. Results 3.1. In vitro fermentation using fecal or excreta inocula 3.1.1. In vitro DM digestibility determination using Daisy system There were marked differences in the in vitro DM digestibility of the different substrates (Table 1). The same trend was noticed all over swine and turkeys. The digestibility of commercial diets was higher than that of lignocellulose products. It was also higher in lignocellulose A than B. Similar results were found among the different animals, indicating that the type of diet fed to animals had no effect on the in vitro DM digestibility. Due to this reason, the digestibility of each substrate was calculated collectively as shown in Table 2. It was found that the in vitro DM digestibility of product A was in the range of 23% (turkeys) up to 34% (pigs), whereas the product B had values of less than 5% in both species. However, the digestibility of commercial diets was higher and its value was nearly identical in swine and turkeys (about 75%). 3.1.2. Gas production and fermentation determination In swine, there was no effect of the diet type fed to animals on gas amounts, pH and fermentation products of the different substrates. Regardless the kind of diet, the amount of gas produced averaged about 26.3 and 1.20 ml/g for commercial swine diet
Table 1 In-vitro DM digestibility (%) of lignocellulose ingredients using Daisy incubator and feces/excreta of swine/turkeys as inoculums. Diet fed to animals
Commercial diet
Commercial diet + Lignocellulose A* Commercial diet + Lignocellulose B*
Substrate
In vitro DM digestibility (%) (n = 3)
Commercial diet Lignocellulose A Lignocellulose B Commercial diet Lignocellulose A Lignocellulose B Commercial diet Lignocellulose A Lignocellulose B
Swine inoculum
Turkeys inoculum
69.92 ± 8.68a 31.87 ± 4.89b 2.64 ± 3.63c 76.57 ± 1.71a 37.15 ± 5.54b 1.43 ± 1.91c 79.57 ± 2.81a 34.02 ± 2.33b 4.23 ± 4.52c
75.67 ± 1.28a 19.38 ± 1.41b 6.11 ± 1.49c 72.98 ± 1.95a 27.29 ± 3.95b 2.21 ± 2.15c 73.77 ± 1.88a 23.03 ± 2.36b 4.72 ± 3.68c
*
Lignocellulose products were added at a rate of 2.0% of the commercial diets. Means within the same column (in each consumed diet) with different superscripts are significantly different (p < 0.05). a,b,c
Table 2 In-vitro DM digestibility (%) of lignocellulose ingredients using Daisy incubator and feces/excreta of swine/turkeys as inoculums, regardless the consumed diets. Substrate Commercial diet Lignocellulose A Lignocellulose B
Swine (n = 9)1 a
75.4 ± 6.31 34.3 ± 4.51b 3.0 ± 3.01c
Turkeys (n = 9)2 74.1 ± 1.92a 23.2 ± 4.19b 4.30 ± 2.83c
1
3 animals 3 samples per substrate. 3 trials (each trial was done on 3 birds together) 3 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05). 2
The statistical analyses were performed using SAS statistical program (SAS Institute, 2002). The data were evaluated using the
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
Note: C2: acetic, C3: propionic, C4: butyric, C5: valeric. 1 n = 3 animals 4 samples per substrate. 2 3 trials (each trial was done on 3 birds together) 4 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05).
L-lactate C5 C4 C3 C2 P VFA pH Gas C2
C3
C4
C5
L-lactate
Turkeys (n = 12)2
VFA pH
As observed in the fermentation of substrates with fecal or excreta inoculums, there was no effect of the diet type on the fermentation with caecal inocula among the animals. In swine, the gas produced in the commercial diet substrate was higher (22.6 ml/g) than lignocellulose products (Table 7). However, the amount of gas in lignocellulose A was higher than B (2.50 vs. 1.0 ml/g). As a result of fermentation, the pH values were reduced in commercial diet (5.62) and lignocellulose A (6.52) in comparison to B (6.65). Moreover, the concentration of VFAs was higher in commercial diet than lignocellulose products. In addition,
Gas
3.3. Gas production and fermentation in vitro by using caecal inocula
P
The chemical analyses of diets revealed that the diets were isocaloric (13.5 MJ ME/kg) and isonitrogenous (175 g CP/kg; Table 4). However, the crude fiber was slightly higher in experimental diet than the control (46.4 vs. 39.3 g/kg). There were no differences in digestibility rates of dry matter, organic matter and crude fiber between the animals fed commercial swine diet and those fed commercial swine diet with 2.0% lignocellulose A (Table 5). The digestibility of DM, OM and CF of the tested lignocellulose product per se was nearly equal (about 50%). The amount of digesta and its DM content in the caecum and colon of the swine fed on the experimental diet was higher than the control group (Table 6). However, the amount of chymus in the stomach and small intestine was lower in the experimental group compared to the control. Moreover, the pH of chymus in the segments of gastrointestinal tract was not affected by the lignoceullose product. The fecal DM content of the experimental treatment was numerically higher than the control one.
Swine (n = 12)1
3.2. In vivo fermentation via using swine as a model of non-ruminants
Substrate
and lignocellulose A, respectively (Table 3). However, no gas was determined in case of lignocellulose B. Moreover, the pH values of the substrates after 24 h fermentation were a reflection of the gas production. Thus, the higher gas produced resulted in lower pH values. The results of volatile fatty acids (VFAs) resulting from fermentation of the substrates showed that lignocellulose product A produced considerable amounts of acetic (3.61 mmol/kg) and propionic (0.75 mmol/kg) acids in comparison to the product B which produced very low amounts of these acids (0.49 and 0.09 mmol/kg). As expected, commercial swine diet resulted in higher amounts of VFAs produced, and the values were 21.8, 9.58, 5.14 and 2.47 mmol/kg for acetic, propionic, butyric and valeric acids, respectively. Accordingly, the concentration of total volatile fatty acids resulted from the commercial diet substrate was found to be high (39.0 mmol/kg). In addition, lignocellulose A produced greater amounts of total VFAs compared to product B (4.45 vs. 0.58 mmol/kg). Moreover, the tested substrates provided low amounts of L-lactate, but the concentration in commercial diet and lignocellulose A was higher than B. In turkeys, the trend of gas amount, VFAs and L-lactate findings were similar to that found in swine. As observed in swine, there was also no effect of the diet type consumed by the birds on the fermentation process using turkey excreta as inoculum. Thus, the gas amount produced all over the birds was higher in commercial turkey diet (P6) and averaged about 29.3 ml/g. Little amount of gas was obtained in lignocellulose A (1.0 ml/g), but no gas was found in the product B. The pH values decreased with increasing the amount of gas output and the values were 5.39, 6.68 and 6.77 for commercial diet, lignocellulose A and B, respectively. The commercial diet resulted in higher amounts of total and individual fatty acids. Moreover, lignocellulose A produced greater amounts of these acids compared to the product B. L-lactate concentration was higher in commercial diet and lignocellulose A than B.
Commercial 26.3a ± 7.30 5.76a ± 0.07 39.0a ± 4.28 21.8a ± 2.89 9.58a ± 0.86 5.14a ± 1.70 2.47a ± 1.41 0.03a ± 0.02 29.3a ± 5.12 5.39a ± 0.22 40.8a ± 4.19 22.9a ± 2.33 12.2a ± 1.47 3.34a ± 2.57 2.30a ± 1.11 0.03a ± 0.02 diet Lig. A 1.20b ± 0.01 6.64b ± 0.04 4.45b ± 1.75 3.61a ± 1.52 0.75b ± 0.45 0.07b ± 0.06 0.02b ± 0.01 0.03a ± 0.02 1.0b ± 0.02 6.68b ± 0.05 3.40b ± 1.17 1.81b ± 0.85 1.28b ± 1.15 0.23b ± 0.18 0.08b ± 0.03 0.04a ± 0.02 Lig. B – 6.99c ± 0.04 0.58c ± 0.38 0.49a ± 0.39 0.09c ± 0.09 – – 0.01b ± 0.01 – 6.77c ± 0.10 1.57c ± 0.79 0.80c ± 0.50 0.64c ± 0.63 0.02c ± 0.01 0.11b ± 0.01 0.01b ± 0.01
I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 3 Gas amount (ml/g), pH of the fermented substrate, L-lactate amounts (mmol/l or kg) and volatile fatty acids (mmol/l or kg) produced after incubation of the substrates with buffer and feces/excreta of swine/turkeys for 24 h, regardless the consumed diets.
4
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
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I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 4 Chemical composition of swine diets used in digestibility trial (on fresh matter basis).
Chemical analyses (g/kg) ME (MJ/kg)* Dry matter Crude ash Crude protein Crude fat Crude fiber Nitrogen free extract Starch Sugar Macrominerals (g/kg) Calcium Magnesium Phosphorus Sodium Potassium Chlorine Sulphur Trace elements (mg/kg) Copper Zinc Iron Manganese Selenium Amino acids (g/kg) Threonine Glutamic Glycine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine
Control diet
Experimental diet
13.7 899 51.1 175.8 46.1 39.3 587 382.6 41.6
13.3 897 51.5 174.2 42.9 46.4 582 359.4 43.6
10.5 2.4 5.7 2.7 8.5 4.9 2.2
9.0 2.3 5.5 2.4 8.3 4.7 2.1
19.6 145.8 174.8 120.9 0.5
14.4 136.4 222.0 111.5 0.5
6.1 40.4 7.9 2.7 2.8 6.9 12.5 5.9 8.3 4.8 10.6
6.5 39.6 7.8 2.6 2.8 6.6 12.2 5.7 8.2 4.8 10.5
4. Discussion
* Metabolizable energy (MJ/kg) = 0.021503 CP + 0.032497 crude fat + 0.016309 starch + 0.014701 [organic matter-(CP + crude fat + starch + CF)] 0.021071 CF (Kamphues et al., 2009).
Table 5 DM, OM and CF digestibility rates (%) of swine diets as well as of lignocellulose A ingredient per se (n = 3).
Digestibility (%): Dry matter Organic matter Crude fiber
lignocellulose A resulted in greater amounts of total VFAs as well as of acetic, propionic, butyric and valeric acids when compared to the product B. Concerning the L-lactate amounts produced from the substrates, lignocellulose A was found to produce higher concentration than lignocellulose B but did not differ with the commercial diet. Using the caecum and colon contents of turkeys as inoculums, the same findings of swine were reported in turkeys. It was noticed that lignocellulose A substrate was well fermented than lignocellulose B via producing greater amounts of VFAs and gas. This was reflected by a lower pH value in lignocellulose A. However, the amounts of L-lactate produced from lignocellulose A were nearly equal to the product B. Moreover, the commercial turkey diet produced higher amounts of gas and VFAs compared to lignocellulose products, but no effect on L-lactate was observed.
Diet ( )1
Diet (+)2
Pure lign. A3
80.3 ± 0.71 83.2 ± 0.73 28.4 ± 6.63
79.6 ± 0.36 82.3 ± 0.31 30.8 ± 6.65
50.1 ± 14.7 47.0 ± 12.2 48.0 ± 23.9
1
Control: animals fed on a commercial swine diet. Experimental group: animals fed on a commercial swine diet containing 2.0% lignocellulose A. 3 Nutrient digestibility of lignocellulose A was estimated by difference between control and experimental diet. 2
The tested substrates were chemically analysed (The data are presented in another contribution, under preparation). It was found that the crude fiber content was about 38.2, 21.4, 560, and 612 g/kg for commercial swine diet, turkey P6 diet, lignocellulose A and B, respectively. Comparing lignocellulose products, lignocellulose A was noticed to contain higher CP (32.0 vs. 7.95 g/kg), ether extract (13.1 vs. 2.65 g/kg), and nitrogen free extract (267 vs. 260 g/kg) than lignocellulose B. In the present study, faces of swine or excreta of turkeys can be used as a microbial inoculum to measure the fermentation rate of feedstuffs. Successful use of a liquid suspension of feces to determine digestibility of different temperate feedstuffs has been reported (El-Shaer et al., 1987). Also, Jones and Barnes (1996) found that the use of feces may be easier rather than to collect rumen fluid as an inoculum source for in vitro dry matter digestibility. Lowman et al. (1999) noticed that equine feces were suitable inoculum sources for gas production assessment in the horse. In addition, Lattimer et al. (2007) reported that equine feces can be used successfully for in vitro experiments using the Daisy II incubator. Feces have been recognized as an inexpensive and readily available source of microorganisms that could easily be collected from several animals at a time (Earing et al., 2014). The average total gastrointestinal tract retention time in swine has been reported to be about 45 h (Wilfart et al., 2007). Therefore, the incubation period used in the Daisy system was 48 h to simulate the normal condition in vivo. Moreover, the retention time in the hindgut of swine is about 24 h, thus the incubation period included in the gas production technique was 24 h. The mean retention time in total tract or hindgut of turkeys is shorter than swine, but the incubation lengths used in this study were comparable to that of swine to facilitate the comparison between both species.
Table 6 Amount (g fresh matter/kg BW), pH values and DM (%) of the digesta in the different parts of swine digestive tract as well as fecal DM content (%) (n = 3). Amount of digesta (g fresh matter/kg BW)
Stomach Small intestine Caecum Colon Feces 1 2 a,b
Digesta pH
DM content (%)
Diet ( )1
Diet (+)2
Diet ( )
Diet (+)
Diet ( )
Diet (+)
31.2a ± 2.27 9.26a ± 0.85 2.81b ± 0.55 15.6b ± 0.65 –
26.7b ± 1.47 8.17b ± 2.91 4.70a ± 0.69 16.9a ± 1.00 –
3.89a ± 0.28 6.76a ± 0.17 6.14a ± 0.20 6.34a ± 0.31 –
3.91a ± 0.07 6.45a ± 0.16 6.18a ± 0.15 6.06a ± 0.34 –
16.5a ± 4.49 13.4a ± 2.34 11.5b ± 1.01 18.2b ± 1.75 28.99a ± 1.22
17.4a ± 1.08 12.0a ± 1.63 12.7a ± 0.21 20.1a ± 1.02 30.02a ± 3.55
Control: animals fed on a commercial swine diet. Experimental group: animals fed on a commercial swine diet containing 2.0% ligocellulose A. Means within the same row (in each parameter) with different superscripts are significantly different (p < 0.05).
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
L-lactate C5 C4 C3 C2 P VFA pH
a
pH
a
Gas
Note: C2: acetic, C3: propionic, C4: butyric, C5: valeric. 1 n = 3 animals 4 samples per substrate. 2 n = 3 birds 4 samples per substrate. a,b,c Means within the same column with different superscripts are significantly different (p < 0.05).
a
Gas C2 VFA
Substrate
Swine (n = 12)1
c
P
a
a
C3
a
C4
a
C5
a
L-lactate
Turkeys (n = 12)2
c
Commercial 22.6 ± 7.91 5.62 ± 0.25 41.9 ± 8.99 22.2 ± 2.01 8.48 ± 2.98 7.32 ± 3.30 3.94 ± 2.10 0.54 ± 0.71 29.8 ± 1.26 5.44 ± 0.04 30.5a ± 1.96 15.75a ± 0.98 9.49a ± 1.09 2.62a ± 0.49 2.64a ± 1.13 0.03a ± 0.01 diet Lig. A 2.50b ± 1.05 6.52b ± 0.03 4.01b ± 2.04 3.16b ± 1.63 0.55b ± 0.42 0.18b ± 0.18 0.12b ± 0.06 0.78a ± 0.61 4.0b ± 1.15 6.43b ± 0.02 3.66b ± 2.90 1.12b ± 0.74 2.26b ± 2.04 0.10b ± 0.08 0.18b ± 0.06 0.07a ± 0.03 Lig. B 1.0c ± 0.01 6.65a ± 0.03 1.46c ± 0.71 1.13c ± 0.64 0.18c ± 0.09 0.09c ± 0.08 0.06c ± 0.04 0.07b ± 0.08 1.0c ± 0.00 6.57a ± 0.01 1.49c ± 1.17 0.97c ± 0.30 0.44c ± 0.38 – 0.08c ± 0.07 0.05a ± 0.05
I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx Table 7 Gas amount (ml/g), pH of the fermented substrate, L-lactate amounts (mmol/l or kg) and volatile fatty acids (mmol/l or kg) produced after incubation of the substrates with buffer and caecum/hind gut contents of swine/turkeys for 24 h, regardless the consumed diets.
6
The type of diet fed to animals did not influence the nature of inoculum or bacteria as indicated by similar in vitro DM digestibility of ingredients or fermentation products among the animals fed different diets. The low fiber content-feedstuffs were highly digested than fiber rich ingredients, as observed in commercial diets compared to lignocellulose products. However, the in vitro DM digestibility of lignocellulose A was higher than B, indicating that the lignocellulose A contains higher amounts of fermentable fiber. The in vitro DM digestibility of swine diet and lignocellulose A was about 75.4 and 34.3%, respectively. These in vitro results are consistent with that found in vivo as the DM digestibility of swine diet and lignocellulose A per se was determined in swine to be about 80 and 50%, respectively. Moreover, the digestibility rates of organic matter and crude fiber of lignocellulose A were nearly similar to that of DM (about 48%). Thus, the findings of Daisy II technique in this study are supported with that obtained in vivo. Therefore, the Daizy II incubator is appropriate for the determination of in vitro digestibility of nutrients using fecal liquor. This finding is confirmed with the results of the previous studies (Holden, 1999; Mabjeesh et al., 2000; Laudadio et al., 2009). In vivo, no effect of diet type on fermentation was also detected as indicated by similar results in digestibility of DM, organic matter, and crude fiber between control and experimental animals. Moreover, lignocellulose A could improve the quality of feces as indicated by its role in increasing the fecal DM content by about 1% than the control (30.02 vs 28.99%). Thus, lignocelluloses can be used to provide a better consistency of feces or used to prevent loose or watery fecal matter. No effect of diet type consumed by the animals on digestibility, gas production, and fermentation products was determined. The same finding was observed by Earing et al. (2014) who found no effect of donor diet on in vitro digestibility. This assumed that the fiber source did not influence the number, nature or activity of fibrolytic bacteria in GIT or the amount added to the diet (2.0%) was not enough to encourage its multiplication. In spite of no gas production in lignocellulose B, very little fermentation occurred. This could be due to the pressure of gas formed in this substrate was lower than that of atmospheric pressure, thus it cannot be measured by the gas meter. The fermentation of substrates with fecal or excreta inocula showed higher production of gas and VFAs in lignocellulose A than B. This could be attributed to the higher contents of organic nutrients (CP, ether extract and nitrogen free extract) in lignocellulose A. Furthermore, the fiber of lignocellulose A was well fermented by caecum inocula and resulted in higher amounts of acetic, propionic, butyric and valeric acids in comparison to lignocellulose B. Thus, lignocellulose A can be used to stimulate the integrity and health of the intestinal lining. It was reported that lignocellulose had a positive effect on performance in swine (Kroismayr et al., 2008a; Bernscherer et al., 2011) and in broilers (Kroismayr et al., 2008b). Increasing the amount of digesta in caecum and colon of swine fed diets containing lignocellulose A indicating the higher fermentation of this product. Nevertheless, commercial swine or turkey diets were easily fermented by feces or caecum inoculum due to the higher starch contents (McDonald et al., 2010). In using fecal/excreta or caecal inocula in swine or turkeys, the more the gas production the lower pH values of substrates. This could be due to production of VFAs in these fermented substrates (Johnston et al., 2003). The obtained results revealed that the findings of using swine feces as an inoculum were coordinated with that resulted from using turkey excreta – inoculum. The same observations were found in caecal inocula of swine and turkeys. In addition, all over swine and turkeys, the fermentation results of the substrates with the caecum inocula were nearly parallel to that obtained by fecal or excreta inoculums.
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006
I.M.I. Youssef, J. Kamphues / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
5. Conclusions The present results indicate that the fecal or excreta inoculum can be used to determine the in vitro DM digestibility or fermentation of feeds. In addition, Daisy II method can be used to predict in vitro digestibility with relatively small variation. The new lignocellulose products differ markedly regarding their microbial degradability. Therefore, their effects on the hind gut flora (and its fermentation), the gut fill and the feces quality should be different. Regarding the product B mechanical effects might be dominant whereas for the product A further advantages (fermentation) could be expected. Moreover, the digestibility of DM, organic matter and crude fiber of lignocellulose A per se was estimated to be about 50%, 47.0%, and 48.0%, respectively. Therefore, lignocellulose A can be used as a good source of fiber in diets of monogastric animals in order to maintain the gut health and to improve the feces or excreta quality.
References AOAC, 2005. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists, Gaithersburg, MD. Bach Knudsen, K.E., Jorgensen, H., 2001. Intestinal degradation of dietary carbohydrates – from birth to maturity. In: Lindberg, J.E., Ogle, B., (Eds.). Digestive Physiology of Pigs. CABI Publishing, New York, p. 109. Bernscherer, M., Mair, C., Schedle, K., 2011. Einfluss eines Lignozellulosezusatzes auf die Mast- und Schlachtleistung bei Schweinen. 10. BOKU-Symposium Tierernaehrung, Wein. Earing, J.E., Cassill, B.D., Hayes, S.H., Vanzant, E.S., Lawrence, L.M., 2014. Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses. J. Anim. Sci. 88, 3954–3963. El-Shaer, H.M., Omed, H.M., Chamberlain, A.G., 1987. Use of faecal organisms from sheep for the in vitro determination of digestibility. J. Agric. Sci. Cambridge 109, 257–259. Govers, M.J., Gannon, N.J., Dunshea, F.R., Gibson, P.R., Muir, J.G., 1999. Wheat bran affects the site of fermentation of resistant starch and luminal indexes related to colon cancer risk: a study in pigs. Gut 45 (6), 840–847. Holden, L.A., 1999. Comparison of methods of in vitro dry matter digestibility for ten feeds. J. Dairy Sci. 82, 1791–1794. Johnston, L.J., Noll, S., Renteria, A., Shurson, J., 2003. Feeding by-products high in concentration of fiber to nonruminants. The Third National Symposium on Alternative Feeds for Livestock and Poultry held in Kansas City, MO on November 4.
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Jones, R.J., Barnes, P., 1996. In vitro digestibility assessment of tropical shrubs legumes using rumen fluid or faecal fluid as the inoculum source. Tropical Grasslands 30, 374–377. Kamphues, J., Kienzle, E., Coenen, M., Pallauf, J., Wanner, M., Simon, O., Zentek, J., 2009. Supplemente Zu Vorlesungen Und Übungen in Der Tierernährung. M. & H. Schaper, Hannover, Germany. Krieg, R., Schüle, S., Dohms, J., 2008. Lignocellulose als sichere Rohfaserquelle zur Leistungsstabilisierung bei Häsinnen und Jungtieren in der Kaninchenzucht. In: 7.BOKU-Symposium Tierernährung: Wien, Austria, pp. 136–145. Krieg, R., Martienssen, M., Zentek, J., 2012. Effect of the ratio lignin to cellulose (adfadl) on caecal fermentation, gut morphology and performance of rabbits around weaning. In: Proceedings 10th World Rabbit Congress – September 3–6, Sharm El- Sheikh –Egypt, 685–689. Kroismayr, A., Neufeld, K., Affentranger, P., 2008a. Einfluss einer neuartigen Lignocellulose auf Leistungsparameter in der Ferkelaufzucht. 7. BOKUSymposium Tierernaehrung, Wein. Kroismayr, A., Neufeld, N., Neufeld, K., 2008b. Einfluss einer neuen Lignocellulose Formulierung auf die Leistungsdaten beim Broiler. 7. BOKU-Symposium Tierernaehrung, Wein. Lattimer, J.M., Cooper, S.R., Freeman, D.W., Lalman, D.L., 2007. Effect of yeast culture on in vitro fermentation of a high concentrate or high-fiber diet using equine fecal inoculums in a DaisyII incubator. J. Anim. Sci. 85, 2484–2491. Laudadio, V., Lacalandra, G.M., Monaco, D., Khorchani, T., Hammadi, M., Tufarelli, V., 2009. Faecal liquor as alternative microbial inoculum source for in vitro (DaisyII) technique to estimate the digestibility of feeds for camels. J. Camelid Sci. 2, 01–07. Lowman, R.S., Theodorou, M., Hyslop, J.J., Dhanoa, M.S., Cuddeford, D., 1999. Evaluation of an in vitro batch culture technique for estimating the in vivo digestibility and digestible energy content of equine feeds using equine faeces as the source of microbial inoculum. Anim. Feed Sci. Technol. 80, 11–27. Mabjeesh, S.J., Cohen, M., Arieli, A., 2000. In vitro methods for measuring the dry matter digestibility of ruminant feedstuffs: comparison of methods and inoculum source. J. Dairy Sci. 83, 2289–2294. Marten, G.C., Barnes, R.F., 1980. Prediction of energy digestibility of forages with in vitro rumen fermentation and fungal enzyme systems. In: Pigden, W.J., Balch, C.C., Graham, M., (Eds.). Standardization of Analytical Methodology for Feeds. Int. Dev. Res. Center, Ottawa, Canada, 61–128. McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., Morgan, C.A., Sinclair, L.A., Wilkinson, R.G., 2010. Animal Nutrition, Seventh Edition, Longman Scientific and Technical. John Wiley & Sons Inc, New York, USA. Sarandan H, Nina Neufeld, Neufeld K. Effects of a new lignocellulose product for fibre supplementation on MMA symptoms, reproductive performance and faecal quality in a pig farm with evident mma problems in Romania. Lucra˘ri Stiinłifice Medicina˘ Veterinara˘ Vol. Xli, 2008, Timisoara., 851–858. SAS Institute, 2002. SASÒ User’s Guide: Statistics. Version 9.1. SAS Institute, Cary, NC. Wilfart, A., Montagne, L., Simmins, H., Noblet, J., van Milgen, J., 2007. Digesta transit in different segments of the gastrointestinal tract of pigs as affected by insoluble fibre supplied by wheat bran. Br. J. Nutr. 98, 54–62.
Please cite this article in press as: Youssef, I.M.I., Kamphues, J. Fermentation of lignocellulose ingredients in vivo and in vitro via using fecal and caecal inoculums of monogastric animals (swine/turkeys). Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.05.006