APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1990, p. 3174-3178
Vol. 56, No. 10
0099-2240/90/103174-05$02.00/0 Copyright © 1990, American Society for Microbiology
Dynamics of Ruminal Ciliated Protozoa in Feedlot Cattlet GENE TOWNE,'* T. G. NAGARAJA,1 ROBERT T. BRANDT, JR.,' AND KEN E. KEMP2 Department of Animal Sciences and Industry, Call Hall,' and Department of Statistics,2 Kansas State University, Manhattan, Kansas 66506-1600 Received 1 June 1990/Accepted 31 July 1990
Fluctuations in ciliated protozoan concentrations were monitored in 40 individually fed crossbred heifers that were stepped up to an 85% concentrate diet either slowly (12 days) or rapidly (3 days), with or without monensin (30 ppm). Ruminal fluid was withdrawn from all animals by stomach tube at the start of the study, after each group reached full feed, and at 14-day intervals thereafter throughout the finishing period until termination (day 119). Neither monensin nor speed of step-up affected (P > 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P < 0.05), suggesting an adaptation to ruminal conditions. Concentrations of Isotricha spp. were higher (P < 0.05) on the final two sampling dates than at any other time. After day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Protozoa were not detected in 11 heifers on day 42 and day 56, but only two animals were defaunated on day 119, indicating either exogenous or endogenous refaunation. Average protozoan concentrations were not different (P > 0.25) between ruminal samples collected by stomach tube the day before slaughter (2.8 x 105/g) and digesta samples collected the next day (1.6 x 105/g). In feedlot cattle, defaunation apparently is transitory and individual animals harbor a dynamic protozoan population that fluctuates in response to changing ruminal conditions.
rapid-step-up schedule were fed a 70% concentrate diet for 3 days before receiving the final diet. The final diet consisted of 85% cracked corn, 10% roughage (dehydrated alfalfa and sorghum silage), and 5% supplement. The cattle were individually fed once daily in amounts sufficient to allow ad libitum intake. Sampling. To reference ruminal sampling with time of feeding, all animals were denied access to feed 3 h before sample collection. Approximately 500 ml of ruminal contents was collected by stomach tube and vacuum pump from all 40 animals on the day preceding the trial (day 0), after the fast-step-up group reached full feed (day 5), after the slowstep-up group reached full feed (day 14), and at 14-day intervals thereafter until termination (day 119). The sampling tube and hose were rinsed in 5% (vol/vol) formalin following each use to prevent artificial inoculation of viable protozoa among animals. Immediately after the ruminal fluid was collected, pH was measured. Sample portions were acidified with 8% (vol/vol) perchloric acid and frozen for subsequent lactate analysis. For protozoan samples, approximately 20 ml of mixed ruminal fluid was pipetted with a wide-orifice pipette into a preweighed flask containing 10 ml of 10% (vol/vol) formalin (pH 6.8). The flasks were later reweighed, and additional formalin was added to obtain a 1:1 (wt/wt) dilution of ruminal
When ruminants are switched from a high-forage to a high-grain diet, the ruminal microbial population undergoes a dramatic shift. Under ad libitum feeding, ciliated protozoa reputedly either are rapidly eliminated or are reduced to extremely low levels (3, 7, 12, 13, 23, 26). However, we have detected high protozoan numbers in the ruminal contents of feedlot cattle at slaughter (25), indicating that successional patterns in protozoan populations differ from what the literature suggests. How quickly animals are stepped up to a high-grain diet could influence ciliated protozoan concentrations. Microbial changes have been followed for short periods in sheep adapted from low- to high-concentrate diets (8, 14, 15); however, intake was restricted in those studies, and the ruminal conditions were not analogous to those of feedlot cattle fed ad libitum. Lyle et al. (13) examined protozoan changes during a 28-day adaptation in cattle fed all-concentrate diets, but information on protozoan dynamics throughout the entire finishing period is lacking. Therefore, our objectives were to systematically monitor ruminal protozoan populations in cattle from the beginning of a finishing diet until slaughter and to determine whether protozoan concentrations are ultimately influenced by speed of step-up. MATERIALS AND METHODS Animals and diet. Forty Hereford-Angus heifers (average weight, 308 kg), previously fed a sorghum silage diet, were randomly allotted to one of four treatments in a 2 x 2 factorial arrangement. The main variables were speed of step-up (slow or rapid) and monensin (without or with 30 ppm). Cattle in the slow-step-up program were fed a 25, 40, 55, and 70% corn diet in successive increments (3 days for each step) before being placed on the final diet. Cattle in the
contents. To determine whether protozoan numbers from the siphoned ruminal samples were representative of whole ruminal contents, digesta from the cattle were collected at slaughter, 1 day after the final stomach tube sample. The time interval between removal from feed and water and slaughter was approximately 8 h. After evisceration, the rumen was slit and about 950 ml of digesta was collected. Ruminal pH was measured, and approximately 20 g of the mixed sample was preserved and diluted as described above. Analytical procedures. For protozoan enumeration, a portion of each preserved sample was pipetted into individual
* Corresponding author. t Contribution no. 90-539-J of the Kansas Agricultural Experiment Station.
3174
PROTOZOAN DYNAMICS IN FEEDLOT CATTLE
VOL. 56, 1990
e
3175
Fast Step-up
1o6. '
I
0~)
o \"\~~~~~*_ NA~~~~~~,_S~~~~~
I--
0-~~~~0-
40-
0 N 0 0-I
0
x-Slow Step-up
|0 -
o3102I
1I
U
1
0
5
14
28
42
56
70
98
84
112
119
Sampling Day FIG. 1. Semilog plot of transformed represents 20 observations.
protozoan concentrations in feedlot cattle
tubes by using a wide-orifice pipette. A phosphate buffer containing methyl green and 30% (vol/vol) glycerol was added to each tube, and the contents were mixed. After staining, the diluted samples were pipetted into a SedgwickRafter counting chamber. The minimal dilution for counting protozoa was 1:15, but higher dilutions were frequently required to provide 20 to 40 cells per microscopic field. Ciliated protozoa were counted in 30 microscopic fields at x100 magnification. Protozoan genera were classified according to Hungate's scheme (11). L-(+)-Lactate was determined enzymatically after the acidified samples were thawed and centrifuged (9). Statistical analysis. Because many animals either were defaunated or possessed low concentrations of protozoa, whereas other animals had extremely high protozoan concentrations, we used a logarithmic transformation [log1o (X + 100)] to equalize the variances and reduce skewness in the data (27). The transformed protozoan concentrations, ruminal pH, and lactic acid concentrations were analyzed as a split plot, with the whole-plot layout being a completely randomized design and sampling time being the subplot factor. The whole-plot factors of step-up speed, monensin, and the speed x monensin interaction were tested by using the animal (speed x monensin) mean square as the wholeplot error term, whereas the subplot effects of time and time x treatment interactions were tested with the residual mean square as the error term. Because any effects from speed of step-up likely would be equilibrated following an adaptation period, the data also were analyzed by using only the first four sample dates (days 0, 5, 14, and 28). However, conclusions based on the F test were similar between the two analyses, and statistics for the entire data set were used unless otherwise noted. The frequencies of defaunation among treatments were compared by using chi-square contingency analysis. Ruminal pH and protozoan concentrations from the final stomach tube sample were compared with ruminal contents collected at slaughter by using the paired t test.
over
time
as
influenced by speed of step-up. Each value
RESULTS
Neither speed of step-up nor monensin affected (P > 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Likewise, no speed of step-up by monensin interactions (P > 0.10) was detected. Protozoan concentrations decreased from day 0 to day 5 in the fast-step-up program but increased in the slow-step-up program, producing a speed x time interaction (P < 0.09; Fig. 1). After day 5, protozoan concentrations in both groups followed similar fluctuations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P < 0.05). Concentrations of Isotricha spp. were higher (P < 0.05) on the final two sampling dates than at any other time (Tables 1 and 2). In contrast, Polyplastron multivesiculatum concentrations peaked on day 5 and then stabilized at low levels throughout the remainder of the trial. At all sampling times except day 0, one to six animals possessed >106 protozoa per g of ruminal contents (Table 3). The number of defaunated animals ranged from 1 on day 14 to 11 on day 42 and day 56, and then dropped to 2 on day 119; however, speed of step-up did not affect (P > 0.10) the number of defaunated animals at any sampling time. At the beginning of the study, the cattle possessed various concentrations of 11 protozoan genera, but after day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Entodinium spp. normally were the prevalent protozoa; however, 51 faunated ruminal samples (13.1%) were devoid of entodinia and contained only Isotricha spp. or P. multivesiculatum or both. Average protozoan concentrations did not differ (P > 0.25) between samples collected by stomach tube on day 119 (2.8 x 105/g) and digesta samples collected the following day after slaughter (1.6 x 105/g). However, ruminal pH was higher (P < 0.01) in samples collected by stomach tube (6.72) than in samples collected at slaughter (5.67). Although analysis of the entire data set indicated that
3176
APPL. ENVIRON. MICROBIOL.
TOWNE ET AL.
Genus
Isotricha Entodinium Polyplastron
Total
TABLE 1. Log-transformed count of total protozoa and surviving genera from feedlot cattle over time Log-transformed count on sampling day: 0 5 14 28 42 56 70 84 98 112 119 3.052 3.278 2.817 2.770 2.770 2.712b 2.984 3.126 3.331 3.504b 3.724" 5.108 5.350 4.259b 3.387b 2.897b 2.772b 3.250b 3.423b 4.482b 4.024b 4.039b 2.664 3.551b 2.296b 2.014b 2.029b 2.084b 2.034b 2.069b 2.071b 2.031b 2.232b 5.177 5.402 3.566b 3.294b 3.249b 3.660b 3.848b 4.671b 4.439b 4.492b 4.422"
SEa
0.1128 0.1562 0.0651
0.1368
n = 40. Antilogarithmic transformation to the geometric means will yield asymmetrical confidence limits. b Within each row, the mean is different from day 0 (P < 0.05).
a
TABLE 2. Raw counts of total protozoa and surviving genera from feedlot cattle over time Raw count (1031g)a on sampling day:
Genus
Isotricha Entodinium Polyplastron Total a
0
5
14
28
42
56
70
84
3.4 144.5 0.7 164.4
7.2 548.3 12.4 590.0
1.9 320.2 1.1 325.3
3.1 131.7
Vol. 56, No. 10
0099-2240/90/103174-05$02.00/0 Copyright © 1990, American Society for Microbiology
Dynamics of Ruminal Ciliated Protozoa in Feedlot Cattlet GENE TOWNE,'* T. G. NAGARAJA,1 ROBERT T. BRANDT, JR.,' AND KEN E. KEMP2 Department of Animal Sciences and Industry, Call Hall,' and Department of Statistics,2 Kansas State University, Manhattan, Kansas 66506-1600 Received 1 June 1990/Accepted 31 July 1990
Fluctuations in ciliated protozoan concentrations were monitored in 40 individually fed crossbred heifers that were stepped up to an 85% concentrate diet either slowly (12 days) or rapidly (3 days), with or without monensin (30 ppm). Ruminal fluid was withdrawn from all animals by stomach tube at the start of the study, after each group reached full feed, and at 14-day intervals thereafter throughout the finishing period until termination (day 119). Neither monensin nor speed of step-up affected (P > 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P < 0.05), suggesting an adaptation to ruminal conditions. Concentrations of Isotricha spp. were higher (P < 0.05) on the final two sampling dates than at any other time. After day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Protozoa were not detected in 11 heifers on day 42 and day 56, but only two animals were defaunated on day 119, indicating either exogenous or endogenous refaunation. Average protozoan concentrations were not different (P > 0.25) between ruminal samples collected by stomach tube the day before slaughter (2.8 x 105/g) and digesta samples collected the next day (1.6 x 105/g). In feedlot cattle, defaunation apparently is transitory and individual animals harbor a dynamic protozoan population that fluctuates in response to changing ruminal conditions.
rapid-step-up schedule were fed a 70% concentrate diet for 3 days before receiving the final diet. The final diet consisted of 85% cracked corn, 10% roughage (dehydrated alfalfa and sorghum silage), and 5% supplement. The cattle were individually fed once daily in amounts sufficient to allow ad libitum intake. Sampling. To reference ruminal sampling with time of feeding, all animals were denied access to feed 3 h before sample collection. Approximately 500 ml of ruminal contents was collected by stomach tube and vacuum pump from all 40 animals on the day preceding the trial (day 0), after the fast-step-up group reached full feed (day 5), after the slowstep-up group reached full feed (day 14), and at 14-day intervals thereafter until termination (day 119). The sampling tube and hose were rinsed in 5% (vol/vol) formalin following each use to prevent artificial inoculation of viable protozoa among animals. Immediately after the ruminal fluid was collected, pH was measured. Sample portions were acidified with 8% (vol/vol) perchloric acid and frozen for subsequent lactate analysis. For protozoan samples, approximately 20 ml of mixed ruminal fluid was pipetted with a wide-orifice pipette into a preweighed flask containing 10 ml of 10% (vol/vol) formalin (pH 6.8). The flasks were later reweighed, and additional formalin was added to obtain a 1:1 (wt/wt) dilution of ruminal
When ruminants are switched from a high-forage to a high-grain diet, the ruminal microbial population undergoes a dramatic shift. Under ad libitum feeding, ciliated protozoa reputedly either are rapidly eliminated or are reduced to extremely low levels (3, 7, 12, 13, 23, 26). However, we have detected high protozoan numbers in the ruminal contents of feedlot cattle at slaughter (25), indicating that successional patterns in protozoan populations differ from what the literature suggests. How quickly animals are stepped up to a high-grain diet could influence ciliated protozoan concentrations. Microbial changes have been followed for short periods in sheep adapted from low- to high-concentrate diets (8, 14, 15); however, intake was restricted in those studies, and the ruminal conditions were not analogous to those of feedlot cattle fed ad libitum. Lyle et al. (13) examined protozoan changes during a 28-day adaptation in cattle fed all-concentrate diets, but information on protozoan dynamics throughout the entire finishing period is lacking. Therefore, our objectives were to systematically monitor ruminal protozoan populations in cattle from the beginning of a finishing diet until slaughter and to determine whether protozoan concentrations are ultimately influenced by speed of step-up. MATERIALS AND METHODS Animals and diet. Forty Hereford-Angus heifers (average weight, 308 kg), previously fed a sorghum silage diet, were randomly allotted to one of four treatments in a 2 x 2 factorial arrangement. The main variables were speed of step-up (slow or rapid) and monensin (without or with 30 ppm). Cattle in the slow-step-up program were fed a 25, 40, 55, and 70% corn diet in successive increments (3 days for each step) before being placed on the final diet. Cattle in the
contents. To determine whether protozoan numbers from the siphoned ruminal samples were representative of whole ruminal contents, digesta from the cattle were collected at slaughter, 1 day after the final stomach tube sample. The time interval between removal from feed and water and slaughter was approximately 8 h. After evisceration, the rumen was slit and about 950 ml of digesta was collected. Ruminal pH was measured, and approximately 20 g of the mixed sample was preserved and diluted as described above. Analytical procedures. For protozoan enumeration, a portion of each preserved sample was pipetted into individual
* Corresponding author. t Contribution no. 90-539-J of the Kansas Agricultural Experiment Station.
3174
PROTOZOAN DYNAMICS IN FEEDLOT CATTLE
VOL. 56, 1990
e
3175
Fast Step-up
1o6. '
I
0~)
o \"\~~~~~*_ NA~~~~~~,_S~~~~~
I--
0-~~~~0-
40-
0 N 0 0-I
0
x-Slow Step-up
|0 -
o3102I
1I
U
1
0
5
14
28
42
56
70
98
84
112
119
Sampling Day FIG. 1. Semilog plot of transformed represents 20 observations.
protozoan concentrations in feedlot cattle
tubes by using a wide-orifice pipette. A phosphate buffer containing methyl green and 30% (vol/vol) glycerol was added to each tube, and the contents were mixed. After staining, the diluted samples were pipetted into a SedgwickRafter counting chamber. The minimal dilution for counting protozoa was 1:15, but higher dilutions were frequently required to provide 20 to 40 cells per microscopic field. Ciliated protozoa were counted in 30 microscopic fields at x100 magnification. Protozoan genera were classified according to Hungate's scheme (11). L-(+)-Lactate was determined enzymatically after the acidified samples were thawed and centrifuged (9). Statistical analysis. Because many animals either were defaunated or possessed low concentrations of protozoa, whereas other animals had extremely high protozoan concentrations, we used a logarithmic transformation [log1o (X + 100)] to equalize the variances and reduce skewness in the data (27). The transformed protozoan concentrations, ruminal pH, and lactic acid concentrations were analyzed as a split plot, with the whole-plot layout being a completely randomized design and sampling time being the subplot factor. The whole-plot factors of step-up speed, monensin, and the speed x monensin interaction were tested by using the animal (speed x monensin) mean square as the wholeplot error term, whereas the subplot effects of time and time x treatment interactions were tested with the residual mean square as the error term. Because any effects from speed of step-up likely would be equilibrated following an adaptation period, the data also were analyzed by using only the first four sample dates (days 0, 5, 14, and 28). However, conclusions based on the F test were similar between the two analyses, and statistics for the entire data set were used unless otherwise noted. The frequencies of defaunation among treatments were compared by using chi-square contingency analysis. Ruminal pH and protozoan concentrations from the final stomach tube sample were compared with ruminal contents collected at slaughter by using the paired t test.
over
time
as
influenced by speed of step-up. Each value
RESULTS
Neither speed of step-up nor monensin affected (P > 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Likewise, no speed of step-up by monensin interactions (P > 0.10) was detected. Protozoan concentrations decreased from day 0 to day 5 in the fast-step-up program but increased in the slow-step-up program, producing a speed x time interaction (P < 0.09; Fig. 1). After day 5, protozoan concentrations in both groups followed similar fluctuations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P < 0.05). Concentrations of Isotricha spp. were higher (P < 0.05) on the final two sampling dates than at any other time (Tables 1 and 2). In contrast, Polyplastron multivesiculatum concentrations peaked on day 5 and then stabilized at low levels throughout the remainder of the trial. At all sampling times except day 0, one to six animals possessed >106 protozoa per g of ruminal contents (Table 3). The number of defaunated animals ranged from 1 on day 14 to 11 on day 42 and day 56, and then dropped to 2 on day 119; however, speed of step-up did not affect (P > 0.10) the number of defaunated animals at any sampling time. At the beginning of the study, the cattle possessed various concentrations of 11 protozoan genera, but after day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Entodinium spp. normally were the prevalent protozoa; however, 51 faunated ruminal samples (13.1%) were devoid of entodinia and contained only Isotricha spp. or P. multivesiculatum or both. Average protozoan concentrations did not differ (P > 0.25) between samples collected by stomach tube on day 119 (2.8 x 105/g) and digesta samples collected the following day after slaughter (1.6 x 105/g). However, ruminal pH was higher (P < 0.01) in samples collected by stomach tube (6.72) than in samples collected at slaughter (5.67). Although analysis of the entire data set indicated that
3176
APPL. ENVIRON. MICROBIOL.
TOWNE ET AL.
Genus
Isotricha Entodinium Polyplastron
Total
TABLE 1. Log-transformed count of total protozoa and surviving genera from feedlot cattle over time Log-transformed count on sampling day: 0 5 14 28 42 56 70 84 98 112 119 3.052 3.278 2.817 2.770 2.770 2.712b 2.984 3.126 3.331 3.504b 3.724" 5.108 5.350 4.259b 3.387b 2.897b 2.772b 3.250b 3.423b 4.482b 4.024b 4.039b 2.664 3.551b 2.296b 2.014b 2.029b 2.084b 2.034b 2.069b 2.071b 2.031b 2.232b 5.177 5.402 3.566b 3.294b 3.249b 3.660b 3.848b 4.671b 4.439b 4.492b 4.422"
SEa
0.1128 0.1562 0.0651
0.1368
n = 40. Antilogarithmic transformation to the geometric means will yield asymmetrical confidence limits. b Within each row, the mean is different from day 0 (P < 0.05).
a
TABLE 2. Raw counts of total protozoa and surviving genera from feedlot cattle over time Raw count (1031g)a on sampling day:
Genus
Isotricha Entodinium Polyplastron Total a
0
5
14
28
42
56
70
84
3.4 144.5 0.7 164.4
7.2 548.3 12.4 590.0
1.9 320.2 1.1 325.3
3.1 131.7
