Trop Anim Health Prod (2015) 47:921–926 DOI 10.1007/s11250-015-0809-4
REGULAR ARTICLES
Improving rumen ecology and microbial population by dried rumen digesta in beef cattle Anusorn Cherdthong 1 & Metha Wanapat 1 & Anuthida Saenkamsorn 1 & Chanadol Supapong 1 & Nirawan Anantasook 2 & Pongsatorn Gunun 3
Received: 25 February 2015 / Accepted: 25 March 2015 / Published online: 8 April 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Four Thai native beef cattle with initial body weight (BW) of 91.8±4.75 kg were randomly assigned according to a 4×4 Latin square design to receive four concentrates replacement levels of soybean meal (SBM) by dried rumen digesta (DRD) at 0, 33, 67, and 100 % on dry matter (DM) basis. All cattle were fed rice straw ad libitum while additional concentrate was fed at 0.5 % BW daily. The experiment was conducted for four periods of 21 days. Rumen fluid was analyzed for predominant cellulolytic bacterial population by using real-time PCR technique. Increasing levels of DRD did not alter total feed intake, ruminal pH and temperature, and plasma urea nitrogen (P>0.05). Protozoa and fungal population were not differed by DRD supplementation while population of bacteria at 4 h post feeding was increased when SBM was replaced with DRD at 66 and 100 % DM. Population of total bacteria and R. flavefaciens at 4 h post feeding were significantly highest with inclusion of 100 % of DRD in the ration. The experimental diets has no effect on excretion and absorption of purine derivatives (P>0.05), while microbial crude protein and efficiency of microbial N synthesis were significantly increased with DRD inclusion in the diet and highest with 100 % DRD replacement (P>0.05). Replacement of
* Anusorn Cherdthong [email protected] 1
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand
2
Program in Animal Production Technology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani 41000, Thailand
3
Department of Animal Science, Faculty of Natural Resources, Rajamangala University of Technology-Isan, Sakon Nakhon Campus, Phangkhon 47160, Sakon Nakhon, Thailand
SBM by DRD at 100 % DM improved the rumen ecology and microbial population in beef cattle fed on rice straw. Keywords Manipulation . Microbial protein synthesis . Real-time PCR . Rumen ecology . Slaughterhouse by-product
Introduction Ruminants are herbivores that utilize a symbiotic relationship with the rumen microorganisms to exploit fiber feeds as a source of nutrients (Hungate 1966; Infascelli et al. 2005). Rumen content contains a high number of microorganisms, including bacteria, protozoa, and fungi. Bacteria are the most numerous of these microorganisms and play a major role in the biological degradation of dietary fiber. Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the predominant cellulolytic bacterial species found in the rumen (Koike and Kobayashi 2001; Calabrò et al. 2012). In recent years, there has been increasing research directed towards rumen ecology and rumen manipulation. Microbial populations change with feed type as well as with other environmental influences such as the inclusion of feed additives in the diet (Infascelli et al. 2005; Calabrò et al. 2014). Dried rumen digesta (DRD) are abundantly available as slaughterhouse by-product and mainly considered as a waste material creating environmental pollution (Cherdthong and Wanapat 2013). With appropriate preparation, DRD could provide a great source of nutrients when included in diets of animals (Okpanachi et al. 2010). DRD is plant material at various stages of digestion rich in microbial cells and crude protein (19.6 % crude protein (CP); Cherdthong and Wanapat 2013). In addition, it contains amino acids, vitamins, and volatile fatty acids (VFA), which beneficially affects in the rumen and also improves the potential for ruminal microbial activity
922
factors (Okpanachi et al. 2010; Cherdthong and Wanapat 2013). Mohammed et al. (2013) reported that DRD could be incorporated into the diets of growing rabbits without compromising the health status and digestibility of nutrients. Moreover, Cherdthong and Wanapat (2013) revealed that supplementation of DRD in concentrate diets resulted in improved in vitro rumen fermentation and in vitro dry matter (DM) digestibility in buffalo rumen fluid. Therefore, the objective of this experiment was to study the effects of replacement soybean meal (SBM) by DRD on rumen ecology, rumen microbial population, and microbial protein synthesis in Thai native beef cattle fed on rice straw as roughage source.
Materials and methods Dietary, animals, experimental design, and feeding Four Thai native beef cattle with initial body weight (BW) of 91.8±4.75 kg were randomly assigned according to a 4×4 Latin square design to receive four concentrates replacement levels of soybean meal (SBM) by dried rumen digesta (DRD) at 0, 33, 67, and 100 % on dry matter (DM) basis. Fresh rumen digesta were obtained from a local slaughterhouse and sundried for 2 days, then ground to pass a 1-cm sieve (Cyclotech Mill, Tecator, Sweden). All animals were fed rice straw ad libitum as roughage source while additional concentrate was fed at 0.5 % BW daily and offered in two equal meals per day at 0700 and 1600 h. The proportions of concentrate ingredients and the chemical composition of the concentrates, DRD, and rice straw are shown in Table 1. All cattle were kept in individual pens while clean fresh water and feed blocks were available at all times. Individual intakes of rice straw and concentrate were recorded daily by weighing the offered and refused feeds during the morning feeding. The experiment was conducted for four periods of 21 days. The first 14 days were for adaptation period and the last 7 days were for samples collection as animals were moved to metabolism crates and fed the straw at 90 % DM of the previous voluntary feed intake. Concentrate was still offered at 0.5 % DM BW daily, and feed blocks were available at all times for the whole experiment. Sampling procedures and chemical analysis Feces and an aliquot of 10 % of the urine were collected daily and stored in a freezer at −20 °C until the end of the experiment. Urine was collected in a container with 5 % H2SO4 solution to maintain the pH below 3. Feed offered, refusals, and fecal samples were collected during the last 7 days of each period. The samples were dried at 60 °C and ground (1 mm screen using a Cyclotech Mill, Tecator, Sweden) and analyzed using the AOAC (1995) method for DM, crude protein (CP),
Trop Anim Health Prod (2015) 47:921–926
ash, and acidic detergent fiber (ADF). Neutral detergent fiber (NDF) in samples was estimated according to Van Soest et al. (1991). Urine samples were analyzed for total N (AOAC 1995), and allantoin in urine was determined by HPLC as described by Chen and Gomes (1995). The amount of microbial purines absorbed was calculated from purine derivative excretion based on the relationship derived by Chen and Gomes (1995). Jugular vein blood samples were collected at 0 and 4 h after feeding on the last day of each period for determination of plasma urea nitrogen (PUN). All samples were taken using a 21-ga needle, and the tubes containing 12 mg of EDTA as anticoagulant and plasma was separated by centrifugation at 500×g for 10 min at 4 °C and stored at −20 °C until used. At the same time, 45 ml of rumen fluid was taken from the rumen by a stomach tube connected to a vacuum pump. Ruminal pH and temperature were determined using a portable pH and temperature meter (HANNA Instruments HI 8424 microcomputer, Singapore). Rumen fluid samples were then filtered through four layers of cheesecloth and used for microbial analysis. A first portion was fixed with 10 % formalin solution in sterilized 0.9 % saline solution. The total direct count of bacteria, protozoa, and fungal zoospores were made by the methods of Galyean (1989) based on the use of a hemocytometer (Boeco, Hamburg, Germany). The second portion was stored at −20 °C for DNA extraction by the RBB+C method (Yu and Morrison 2004). The primers used for the real-time PCR are as follows: primers for F. succinogenes, Fs219f (5′-GGT ATG GGA TGA GCT TGC-3′) and Fs654r (5′-GCC TGC CCC TGA ACT ATC-3′). R. albus primers are Ra1281f (5′-CCC TAA AAG CAG TCT TAG TTC G-3′) and Ra1439r (5′ CCT CCT TGC GGT TAG AAC A-3′) (175-bp product). R. flavefaciens primers, Rf154f (5′-TCT GGA AAC GGA TGG TA-3′) and Rf425r (5′-CCT TTA AGA CAG GAG TTT ACA A-3′), were also selected to allow species amplification (295 bp). Regular PCR conditions for F. succinogenes were as follows: 30 s at 94 °C for denaturing, 30 s at 60 °C for annealing and 30 s at 72 °C for extension (48 cycles), except for 9 min denaturation in the first cycle and 10 min extension in the last cycle. Amplification of 16S rRNA for the other two species was carried out similarly except an annealing temperature of 55 °C was used. Quantification of total bacterial population, primer, and condition was previously published by Wanapat and Cherdthong (2009). Four samplederived standards were prepared from a treatment pool set of community DNA. The regular PCR was used to generate sample-derived DNA standards for each realtime PCR assay. Then, the PCR product was purified using a QIA quick PCR purification kit (QIAGEN, Inc., Valencia, CA) and quantified using a spectrophotometer.
Trop Anim Health Prod (2015) 47:921–926 Table 1 Ingredient and chemical composition of diets used in the experiment
923
Ingredients
Replacement levels
DRD
Rice straw
98.4 91.3 8.7 40.5 20.1 19.6
95.4 86.3 13.4 77.0 57.1 2.3
(DRD replacing SBM, %DM) 0
33
67
100
Cassava chips Soybean meal (SBM) Dried rumen digesta (DRD) Rice bran Coconut meal
55.6 11.0 0.0 10.5 8.9
55.6 7.4 3.7 10.3 8.9
55.6 3.6 7.4 10.2 8.9
55.6 0.0 11.1 10.1 8.6
Palm kernel meal Urea Salt Sulfur powder Mineral and vitaminsa Molasses Chemical composition Dry matter, % Organic matter, %DM Ash, %DM aNeutral detergent fiber, %DM Acid detergent fiber, %DM Crude protein, %DM
7.3 1.0 1.0 1.0 1.0 2.7
7.1 1.3 1.0 1.0 1.0 2.7
7.0 1.6 1.0 1.0 1.0 2.7
7.0 1.9 1.0 1.0 1.0 2.7
96.5 93.3 6.7 18.7 6.9 13.1
95.4 92.1 8.9 20.2 8.7 13.3
95.4 91.0 8.0 22.4 11.4 13.2
94.2 90.6 9.4 24.2 13.1 13.4
a
Minerals and vitamins (each kg contains): vitamin A 10,000,000 IU, vitamin E 70,000 IU, vitamin D 1,600,000 IU, Fe 50 g, Zn 40 g, Mn 40 g, Co 0.1 g, Cu 10 g, Se 0.1 g, and I 0.5 g
For each sample-derived standard, copy number concentration was calculated based on the length of the PCR product and the mass concentration. A tenfold serial dilution was made in Tri-EDTA prior to real-time PCR (Yu
et al. 2005). In total, four real-time PCR standards were prepared. The conditions of the real-time PCR assays of target genes were the same as those of the regular PCR described above. Biotools QuantiMix EASY SYG KIT
Table 2 Replacing soybean meal (SBM) with dried rumen digesta (DRD) in concentrate diets on intake, rumen ecology, and blood metabolite
Table 3 Dried rumen digesta (DRD) as a substitute for soybean meal (SBM) in concentrate diets on microbial population in the rumen of cattle
Item
Item
Replacement levels SEM P value (DRD replacing SBM, %DM) 0
Total DM intake, %BW 3.1 Ruminal pH 0 h post feeding 7.0 4 h post feeding 6.7 Mean 6.9 Ruminal temperature, °C 0 h post feeding 38.7 4 h post feeding 39.0 Mean 38.9 Plasma urea nitrogen, mg/dl 0 h post feeding 9.3 4 h post feeding 15.6 Mean 12.5
33
67
Replacement levels (DRD replacing SBM, %DM) 0
SEM
P value
33
67
100
9.4 11.6a 10.5
8.7 13.5ab 11.1
8.4 14.7b 11.6
0.55 0.90 0.76
0.39 0.03 0.67
5.4 8.7 7.1
4.7 9.3 7.0
5.9 9.0 7.5
0.44 0.86 0.75
0.35 0.89 0.67
3.0 5.4 4.2
2.9 5.4 4.2
2.8 5.9 4.4
0.20 0.41 0.33
0.11 0.37 0.23
100
3.2
3.3
3.2
1.21
0.14
6.9 6.6 6.8
6.8 6.6 6.7
6.9 6.7 6.8
1.12 0.87 0.99
0.32 0.27 0.29
38.9 39.3 39.1
39.1 39.8 39.5
38.2 38.9 38.6
1.87 2.22 2.01
0.38 0.40 0.39
9.7 14.4 12.1
10.2 15.2 12.7
9.9 14.4 12.2
0.38 1.43 0.98
0.21 0.31 0.30
Ruminal microbes, cell/ml Bacteria, ×1011 0 h post feeding 9.8 4 h post feeding 11.4a Mean 10.6 6 Protozoa, ×10 0 h post feeding 6.8 4 h post feeding 8.7 Mean 7.8 Fungal zoospore, ×104 0 h post feeding 2.4 4 h post feeding 4.5 Mean 3.5 a,b
Means in the same row with different superscripts differ (P
REGULAR ARTICLES
Improving rumen ecology and microbial population by dried rumen digesta in beef cattle Anusorn Cherdthong 1 & Metha Wanapat 1 & Anuthida Saenkamsorn 1 & Chanadol Supapong 1 & Nirawan Anantasook 2 & Pongsatorn Gunun 3
Received: 25 February 2015 / Accepted: 25 March 2015 / Published online: 8 April 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Four Thai native beef cattle with initial body weight (BW) of 91.8±4.75 kg were randomly assigned according to a 4×4 Latin square design to receive four concentrates replacement levels of soybean meal (SBM) by dried rumen digesta (DRD) at 0, 33, 67, and 100 % on dry matter (DM) basis. All cattle were fed rice straw ad libitum while additional concentrate was fed at 0.5 % BW daily. The experiment was conducted for four periods of 21 days. Rumen fluid was analyzed for predominant cellulolytic bacterial population by using real-time PCR technique. Increasing levels of DRD did not alter total feed intake, ruminal pH and temperature, and plasma urea nitrogen (P>0.05). Protozoa and fungal population were not differed by DRD supplementation while population of bacteria at 4 h post feeding was increased when SBM was replaced with DRD at 66 and 100 % DM. Population of total bacteria and R. flavefaciens at 4 h post feeding were significantly highest with inclusion of 100 % of DRD in the ration. The experimental diets has no effect on excretion and absorption of purine derivatives (P>0.05), while microbial crude protein and efficiency of microbial N synthesis were significantly increased with DRD inclusion in the diet and highest with 100 % DRD replacement (P>0.05). Replacement of
* Anusorn Cherdthong [email protected] 1
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand
2
Program in Animal Production Technology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani 41000, Thailand
3
Department of Animal Science, Faculty of Natural Resources, Rajamangala University of Technology-Isan, Sakon Nakhon Campus, Phangkhon 47160, Sakon Nakhon, Thailand
SBM by DRD at 100 % DM improved the rumen ecology and microbial population in beef cattle fed on rice straw. Keywords Manipulation . Microbial protein synthesis . Real-time PCR . Rumen ecology . Slaughterhouse by-product
Introduction Ruminants are herbivores that utilize a symbiotic relationship with the rumen microorganisms to exploit fiber feeds as a source of nutrients (Hungate 1966; Infascelli et al. 2005). Rumen content contains a high number of microorganisms, including bacteria, protozoa, and fungi. Bacteria are the most numerous of these microorganisms and play a major role in the biological degradation of dietary fiber. Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the predominant cellulolytic bacterial species found in the rumen (Koike and Kobayashi 2001; Calabrò et al. 2012). In recent years, there has been increasing research directed towards rumen ecology and rumen manipulation. Microbial populations change with feed type as well as with other environmental influences such as the inclusion of feed additives in the diet (Infascelli et al. 2005; Calabrò et al. 2014). Dried rumen digesta (DRD) are abundantly available as slaughterhouse by-product and mainly considered as a waste material creating environmental pollution (Cherdthong and Wanapat 2013). With appropriate preparation, DRD could provide a great source of nutrients when included in diets of animals (Okpanachi et al. 2010). DRD is plant material at various stages of digestion rich in microbial cells and crude protein (19.6 % crude protein (CP); Cherdthong and Wanapat 2013). In addition, it contains amino acids, vitamins, and volatile fatty acids (VFA), which beneficially affects in the rumen and also improves the potential for ruminal microbial activity
922
factors (Okpanachi et al. 2010; Cherdthong and Wanapat 2013). Mohammed et al. (2013) reported that DRD could be incorporated into the diets of growing rabbits without compromising the health status and digestibility of nutrients. Moreover, Cherdthong and Wanapat (2013) revealed that supplementation of DRD in concentrate diets resulted in improved in vitro rumen fermentation and in vitro dry matter (DM) digestibility in buffalo rumen fluid. Therefore, the objective of this experiment was to study the effects of replacement soybean meal (SBM) by DRD on rumen ecology, rumen microbial population, and microbial protein synthesis in Thai native beef cattle fed on rice straw as roughage source.
Materials and methods Dietary, animals, experimental design, and feeding Four Thai native beef cattle with initial body weight (BW) of 91.8±4.75 kg were randomly assigned according to a 4×4 Latin square design to receive four concentrates replacement levels of soybean meal (SBM) by dried rumen digesta (DRD) at 0, 33, 67, and 100 % on dry matter (DM) basis. Fresh rumen digesta were obtained from a local slaughterhouse and sundried for 2 days, then ground to pass a 1-cm sieve (Cyclotech Mill, Tecator, Sweden). All animals were fed rice straw ad libitum as roughage source while additional concentrate was fed at 0.5 % BW daily and offered in two equal meals per day at 0700 and 1600 h. The proportions of concentrate ingredients and the chemical composition of the concentrates, DRD, and rice straw are shown in Table 1. All cattle were kept in individual pens while clean fresh water and feed blocks were available at all times. Individual intakes of rice straw and concentrate were recorded daily by weighing the offered and refused feeds during the morning feeding. The experiment was conducted for four periods of 21 days. The first 14 days were for adaptation period and the last 7 days were for samples collection as animals were moved to metabolism crates and fed the straw at 90 % DM of the previous voluntary feed intake. Concentrate was still offered at 0.5 % DM BW daily, and feed blocks were available at all times for the whole experiment. Sampling procedures and chemical analysis Feces and an aliquot of 10 % of the urine were collected daily and stored in a freezer at −20 °C until the end of the experiment. Urine was collected in a container with 5 % H2SO4 solution to maintain the pH below 3. Feed offered, refusals, and fecal samples were collected during the last 7 days of each period. The samples were dried at 60 °C and ground (1 mm screen using a Cyclotech Mill, Tecator, Sweden) and analyzed using the AOAC (1995) method for DM, crude protein (CP),
Trop Anim Health Prod (2015) 47:921–926
ash, and acidic detergent fiber (ADF). Neutral detergent fiber (NDF) in samples was estimated according to Van Soest et al. (1991). Urine samples were analyzed for total N (AOAC 1995), and allantoin in urine was determined by HPLC as described by Chen and Gomes (1995). The amount of microbial purines absorbed was calculated from purine derivative excretion based on the relationship derived by Chen and Gomes (1995). Jugular vein blood samples were collected at 0 and 4 h after feeding on the last day of each period for determination of plasma urea nitrogen (PUN). All samples were taken using a 21-ga needle, and the tubes containing 12 mg of EDTA as anticoagulant and plasma was separated by centrifugation at 500×g for 10 min at 4 °C and stored at −20 °C until used. At the same time, 45 ml of rumen fluid was taken from the rumen by a stomach tube connected to a vacuum pump. Ruminal pH and temperature were determined using a portable pH and temperature meter (HANNA Instruments HI 8424 microcomputer, Singapore). Rumen fluid samples were then filtered through four layers of cheesecloth and used for microbial analysis. A first portion was fixed with 10 % formalin solution in sterilized 0.9 % saline solution. The total direct count of bacteria, protozoa, and fungal zoospores were made by the methods of Galyean (1989) based on the use of a hemocytometer (Boeco, Hamburg, Germany). The second portion was stored at −20 °C for DNA extraction by the RBB+C method (Yu and Morrison 2004). The primers used for the real-time PCR are as follows: primers for F. succinogenes, Fs219f (5′-GGT ATG GGA TGA GCT TGC-3′) and Fs654r (5′-GCC TGC CCC TGA ACT ATC-3′). R. albus primers are Ra1281f (5′-CCC TAA AAG CAG TCT TAG TTC G-3′) and Ra1439r (5′ CCT CCT TGC GGT TAG AAC A-3′) (175-bp product). R. flavefaciens primers, Rf154f (5′-TCT GGA AAC GGA TGG TA-3′) and Rf425r (5′-CCT TTA AGA CAG GAG TTT ACA A-3′), were also selected to allow species amplification (295 bp). Regular PCR conditions for F. succinogenes were as follows: 30 s at 94 °C for denaturing, 30 s at 60 °C for annealing and 30 s at 72 °C for extension (48 cycles), except for 9 min denaturation in the first cycle and 10 min extension in the last cycle. Amplification of 16S rRNA for the other two species was carried out similarly except an annealing temperature of 55 °C was used. Quantification of total bacterial population, primer, and condition was previously published by Wanapat and Cherdthong (2009). Four samplederived standards were prepared from a treatment pool set of community DNA. The regular PCR was used to generate sample-derived DNA standards for each realtime PCR assay. Then, the PCR product was purified using a QIA quick PCR purification kit (QIAGEN, Inc., Valencia, CA) and quantified using a spectrophotometer.
Trop Anim Health Prod (2015) 47:921–926 Table 1 Ingredient and chemical composition of diets used in the experiment
923
Ingredients
Replacement levels
DRD
Rice straw
98.4 91.3 8.7 40.5 20.1 19.6
95.4 86.3 13.4 77.0 57.1 2.3
(DRD replacing SBM, %DM) 0
33
67
100
Cassava chips Soybean meal (SBM) Dried rumen digesta (DRD) Rice bran Coconut meal
55.6 11.0 0.0 10.5 8.9
55.6 7.4 3.7 10.3 8.9
55.6 3.6 7.4 10.2 8.9
55.6 0.0 11.1 10.1 8.6
Palm kernel meal Urea Salt Sulfur powder Mineral and vitaminsa Molasses Chemical composition Dry matter, % Organic matter, %DM Ash, %DM aNeutral detergent fiber, %DM Acid detergent fiber, %DM Crude protein, %DM
7.3 1.0 1.0 1.0 1.0 2.7
7.1 1.3 1.0 1.0 1.0 2.7
7.0 1.6 1.0 1.0 1.0 2.7
7.0 1.9 1.0 1.0 1.0 2.7
96.5 93.3 6.7 18.7 6.9 13.1
95.4 92.1 8.9 20.2 8.7 13.3
95.4 91.0 8.0 22.4 11.4 13.2
94.2 90.6 9.4 24.2 13.1 13.4
a
Minerals and vitamins (each kg contains): vitamin A 10,000,000 IU, vitamin E 70,000 IU, vitamin D 1,600,000 IU, Fe 50 g, Zn 40 g, Mn 40 g, Co 0.1 g, Cu 10 g, Se 0.1 g, and I 0.5 g
For each sample-derived standard, copy number concentration was calculated based on the length of the PCR product and the mass concentration. A tenfold serial dilution was made in Tri-EDTA prior to real-time PCR (Yu
et al. 2005). In total, four real-time PCR standards were prepared. The conditions of the real-time PCR assays of target genes were the same as those of the regular PCR described above. Biotools QuantiMix EASY SYG KIT
Table 2 Replacing soybean meal (SBM) with dried rumen digesta (DRD) in concentrate diets on intake, rumen ecology, and blood metabolite
Table 3 Dried rumen digesta (DRD) as a substitute for soybean meal (SBM) in concentrate diets on microbial population in the rumen of cattle
Item
Item
Replacement levels SEM P value (DRD replacing SBM, %DM) 0
Total DM intake, %BW 3.1 Ruminal pH 0 h post feeding 7.0 4 h post feeding 6.7 Mean 6.9 Ruminal temperature, °C 0 h post feeding 38.7 4 h post feeding 39.0 Mean 38.9 Plasma urea nitrogen, mg/dl 0 h post feeding 9.3 4 h post feeding 15.6 Mean 12.5
33
67
Replacement levels (DRD replacing SBM, %DM) 0
SEM
P value
33
67
100
9.4 11.6a 10.5
8.7 13.5ab 11.1
8.4 14.7b 11.6
0.55 0.90 0.76
0.39 0.03 0.67
5.4 8.7 7.1
4.7 9.3 7.0
5.9 9.0 7.5
0.44 0.86 0.75
0.35 0.89 0.67
3.0 5.4 4.2
2.9 5.4 4.2
2.8 5.9 4.4
0.20 0.41 0.33
0.11 0.37 0.23
100
3.2
3.3
3.2
1.21
0.14
6.9 6.6 6.8
6.8 6.6 6.7
6.9 6.7 6.8
1.12 0.87 0.99
0.32 0.27 0.29
38.9 39.3 39.1
39.1 39.8 39.5
38.2 38.9 38.6
1.87 2.22 2.01
0.38 0.40 0.39
9.7 14.4 12.1
10.2 15.2 12.7
9.9 14.4 12.2
0.38 1.43 0.98
0.21 0.31 0.30
Ruminal microbes, cell/ml Bacteria, ×1011 0 h post feeding 9.8 4 h post feeding 11.4a Mean 10.6 6 Protozoa, ×10 0 h post feeding 6.8 4 h post feeding 8.7 Mean 7.8 Fungal zoospore, ×104 0 h post feeding 2.4 4 h post feeding 4.5 Mean 3.5 a,b
Means in the same row with different superscripts differ (P
