In Vitro Effects of the Ionophore Lysocellin on Ruminal Fermentation and Microbial Populations1f2 L. Kung, Jr.*, R. S. Tung*, and L. L. Slytert
ABSTRACT: Batch and continuous culture techniques were used to evaluate the effect of the ionophore lysocellin on ruminal fermentation and microbial populations. In batch culture, .5 and 1 ppm (of the fluid) lysocellin markedly decreased (P e .011 the acetate:propionateratio without affecting fiber digestion, ammonia concentration, or culture pH. Greater concentrations of lysocellin had negative effects Ip c .05) on fiber digestion and increased CP e .051 culture pH. In continuous culture, a low level of lysocellin (33 ppm of the diet DM or about .7 ppm of the fluid) decreased pH Ip e .05) and methane (P c .05) production but had no effect on fiber digestion. Lysocellin tended to increase (P e .05) OM digestion in corn-based diets but decreased OM digestion in barley-based
diets htarch source x lysocellin interaction, P e .05). In addition, the molar proportion of propionate was increased more in barley- than in cornbased diets. Total anaerobes and amylolytic and lactate-utilizing microorganisms were not affected by the ionophore. In continuous culture, cellulolytic and lactate-producingorganisms were insensitive to lysocellin, but, in lysocellin-treated media, cellulolytic organisms were resistant, whereas lactic acid producers were sensitive to lysocellin at 4 ppm. In summary, the ionophore lysocellin alters ruminal fermentation by decreasing ruminal methane production and increasing the molar proportion of propionate; however, effects varied depending on whether corn or barley served as the primary starch source.
Key Words: Ionophores, Rumen Fermentation, Rumen Microorganisms J. Anim. Sci. 1992. 70:281-288
Introduction
also have been noted (Wolfi-om et al.,
Lysocellin is a divalent polyether antibiotic from Streptomyces cacaoci var. asoensis. When fed to growing cattle, it improves daily gain and feed efficiency Preston et al., 1985; Spears et al., 1989). Shifts in ruminal fermentation, namely decreased acetate and butyrate and increased propionate,
'Published
&B
1983;
Harvey et al., 1988). However, little information is available on the effect of lysocellin on ruminal microbial populations, fiber digestion and methane production. In addition, comparative effects of ionophores on ruminal metabolism in corn- vs barley-based diets are lacking; this is of interest because barley is fed in large quantities in Canada and some western areas of the United States. The objective of our study was to evaluate the effect of lysocellin on ruminal fermentation and fiber digestion in batch culture. We also evaluated the effect of lysocellin on ruminal fermentation and microbial populations from barley- or corn-based diets in continuous culture.
Misc. Paper No. 1388 of the Delaware Agric.
Em. Sta. 2Funding for this research was provided in part by (1.Unidel grant from the Univ. of Delaware. Lysocellin waa a giR from IMCPitman Moore, Terre Haute, IN. Received April 1, 1991. Accepted July 17, 1991.
281
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
*Delaware Agricultural Experiment Station, Department of Animal Science and Agricultural Biochemistry, College of Agricultural Science, University of Delaware, Newark 19717-1303 and WSDA-ARS, Ruminant Nutrition, Beltsville, MD 20705
KUNG ET AL.
282
Experimental Procedure
Diet? Item Feed composition, % Corn silage
Barley Corn Soybean meal, 48% CP Limestone n a c e mineral saltb Lysocellin, ppm of diet DM
1
2
3
4
27.6 65.0
27.6 65.0
26.6
26.6
6.1
6.1
1.1
1.1
65.0 7.2 1.0
.2
.2
65.0 7.2 1.0 .2 33 12.6 12.1 76.0 .45 .37 .l6 .80 .20
12.6 12.1 76.0 .45 .37 .I6 .80 .20
33
.2
Chemical composition, %
CP ADF TDN Ca
.37
13.7 14.4 75.0 .37
P Mg K
.40
.I5
.40
Na
13.7 14.4 75.0
1.01 .17
.15
1.01 .17
*Diet 4 was the basal diet used in batch culture fermentations. Diets 1 , 2 , 3, and 4 were used in the continuous culture fermentations. blO% P, 19% Ca,, 3%M g ,3% K,.75%S,.15%I, , 0 0 4 1 Co, .025% Cu,.035% Fe, .2% Mn,.75%Zn, .006% Se, 860,000 IU Vitamin A/ kg, 165,000 IU vitamin D/kg, and 2,475 IU Vitamin E&.
to inhibit microbial growth. Fermentors were stirred continuously at 75 rpm and temperature was held at 39°C. Twelve fermentors were filled to capacity with ruminal fluid from steers fed a 70% concentrate (corn and soybean meal) and 30% forage (corn silage) diet. Fermentors were purged with C02 before filling, after filling, and during feeding. Culture pH was maintained above 6.2 by infusing artificial saliva CMcDougall, 1948) modified to contain .117 M Na&03 and .0125 M urea instead of .117 M NaHC03. McDougall’s buffer and tap water (60:40) were infused continuously by peristaltic pumps to obtain a turnover rate of approximately 1.5 volumes per day. Gas was collected into butyl rubber bags. Fermentors were given 9.0 g twice daily of a I-mm grind of either a corn- (six fermentors) or a barley- (six fermentors) based diet (Table 1). Steady-state conditions were achieved after 5 d. At the start of d 6, 33 ppm of the ionophore lysocellin was included at each addition of feedstuffs to three fermentors given the corn diets and three fermentors given the barley diets. The start of d 6 marked the beginning of three 3-d sampling periods. The following procedures were used during collection and analyses of samples from each period: fermentor effluents were collected every 24 h and pH was recorded. Effluent was acidified with .1 mL of 5Ooh Has04 (vol/voll per 10
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Batch Fermentations. In vitro batch cultures with mixed ruminal microorganisms were established from fistdated steers fed a corn silage, corn, soybean meal, and alfalfa hay diet (forage:concentrate ratio of 50:50) to study the effect of lysocellin on ruminal fermentation. Ruminal fluid was collected from three steers at approximately 3 h after feeding. Fluid was strained through four layers of cheesecloth and processed for recovery of particulate-bound microorganisms under anaerobic conditionc (Craig et al., 1984). Twenty milliliters of strained ruminal fluid were combined with 20 mL of a prewarmed (30°C) phosphate-bicarbonate buffer (Goering and Van Soest, 1970) and added to 50-mL test tubes with .5 g of a diet consisting of 70% grain (corn, soybean meal, and minerals) and 30% forage (corn silage). Lysocellin was dissolved in ethanol and appropriate dilutions were made such that .5 mL supplied final concentrations of 0, .5,1, 2.5, 5, 10, 20, and 40 ppm (fluid basis). Four 50-mL test tubes were prepared for each steer at each dose. After 24 h of fermentation, pH ww determined in two incubation tubes for each steer and dose before addition of 1 mL of 25% nphosphoric acid to 5 mL of ruminal fluid. Ruminal fluid was analyzed for ammonia N (Okuda et al., 1965) and VFA by gas chromatography (Hewlett Packard Co.,Avondale, PA). The remaining two incubation tubes for each steer and dose were analyzed for residual ADF (AOAC, 1984). Acid detergent fiber digestion was calculated by subtracting the residual ADF from the amount of ADF present before digestion. Data were analyzed by analysis of variance techniques with main effects of steer and treatment (SAS, 1985) tested against the residual error term. Regressions on VFA data were not performed because the highest dose of lysocellin seemed toxic. Thus, treatment effects were evaluated by Scheffe’s (1953) test with specific comparisons of the 0 dose to each increasing dose level. Degrees of freedom for ADF digestion, pH, and ammonia N concentration were partitioned by multiple regression into linear, quadratic, and cubic effects. Continuous Culture Fermentation. The effect of the ionophore lysocellin on ruminal fermentation and microbial populations in continuous culture was studied. Treatments (Table 11 were diets consisting of 70% concentrate and 30% forage; the major variables were starch source (corn or barley) with and without 33 ppm dietary lysocellin (DM basis). Fermentors (Slyter et al., 1964) consisted of a closed system with 500-mL reaction vessels with a single overflow port. Effluent was collected in funnels with 16 mL of saturated HgC12
Table 1. Composition [%, DM basis) of experimental diets used in batch and continuous culture fermentations
283
LYSOCELLIN FERMENTATION
Data were analyzed by analysis of variance using the GLM procedure of SAS (1985). The
experimental design was a split plot with repeated measures (Gill and Hafs, 1971). Effect of treatment was tested in the main plot and period and period x treatment interaction were subplot treatments. The treatment effect was further separated into main effects for starch source (corn or barley), a lysocellin effect (0 or 33 ppml, and a starch x lysocellin interaction. Microbial data were log-transformed (base 10) before analyses. Means were tested using a F-protected (P < .05) pairwise comparison of the least squares means.
Results Batch Fermentations. The effect of various levels of lysocellin on ruminal fermentation was studied after 24 h of incubation. Low concentrations of lysocellin (.5 to 2.5 ppm, fluid basis) had no effect on culture pH, but greater concentrations caused a moderate increase in pH (Table 2). The 40 ppm level of lysocellin caused culture pH to increase above 7.5. Compared with untreated cultures, cultures with low levels (up to 2.5 ppml of lysocellin had greater ammonia N concentrations; however, when lysocellin addition was between 5 and 20 ppm, ammonia N content was less than in untreated cultures. The highest concentration of lysocellin (40 ppm) caused an increase in culture ammonia N concentration. Digestion of A D F was not affected by .5 ppm lysocellin but it decreased rapidly (P e .05) with increasing dose. Acid detergent fiber digestion was decreased by > 70% with doses of lysocellin > 10 ppm.
Table 2. Effect of lysocellin on pH, ammonia N and A D F digestion after 24 hours in ruminal batch culture ~~~~~
Lysocellin, PPm of the fluid 0
ADF
P* 6.29
.5
Ammonia N, mg/dLb
m=tim
25.2
47.0 48.5
6.24
29.8
1
6.30
28.6
34.7
2.5
6.37
28.0
34.8
5
6.56
24.6
28.8
132
10
6.76
22.2
20
6.73
22.2
7.6
40
7.53
32.4
5.0
.09
1.7
3.0
SEd
~~
*Linear and cubic effects [P < .OS) of lysocellin dose. bQuadratic effect (P .OS1 of lysocellin dose. ‘%inear and quadratic effects (P c .05) of lysocellin dose.
dn
= 3.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
mL of effluent, centrifuged at 12,000 x g for 15 min, and analyzed for VFA by gas chromatography as described previously. The 3-d period daily effluents composited by fermentor were sampled by using a wide-bore pipette while being constantly stirred in a squire blender. A portion of the composite effluent was lyophilized, ground to pass a I-mm mesh screen, and analyzed for N (Kjeldahl), ADP CAOAC, 19841, purines (Zinn and Owens, 19861, and ash CAOAC, 1984). On each day, approximately 300 mL of fluid effluent was treated with 60 mL of formaldehyde in .9% (wt/vol) saline and differentially centrifuged at 600 x g for 5 min, followed by 12,000 x g for 15 min to isolate ruminal bacteria. Bacteria were lyophilized,ground with a mortar and pestle, and analyzed for N (KjeldahU, purines (Zinn and Owens, 19861, and ash (5OO0C, 6 h). Bacterial protein synthesis was calculated from the ratio of purines in effluent to bacterial purine content and expressed as grams of bacterial N produced per kilogram of truly fermented OM. Fluid effluent also was analyzed for VFA by gas chromatography and for ammonia N (Okuda et al., 19651. Total gas production was measured at the end of each 3 d period and analyzed for CHI by gas chromatography (Fisher Gas Partitioner, Fisher Scientific, Pittsburgh, PA) (Slyter and Putnam, 1967). On the first day of Period 3, a representative sample from each fermentor was analyzed for total anaerobic bacteria (98-5 media, Bryant and Robinson, 19611, lactate-producing bacteria (Rogosa et al., 1951, as modified by Slyter et al., 19701, and lactate-utilizing bacteria (Slyter et al., 19761. Duplicate 98-5, lactate-producing and lactate-utilizing agar roll tube media were inoculated and incubated at 39°C for 4 , 2 , and 4 d, respectively. All colonies were counted in 98-5 tubes. All colonies medium-large to large were counted in lactate-producingagar tubes. All medium-smallor larger colonies were counted in lactateutilizing agar tubes. Tubes with broth that were used to determine total number of cellulolytic and amylolytic bacteria were incubated for 7 d at 39°C. The starch:cellulose medium of Bryant and Burkey (1953) was prepared as modified by Slyter et al. (1970). Disappearance of cellulose (observed visually) and starch (iodine test) in each of three tubes inoculated at three or more successive decimal dilutions and the most probable number (MPN) tables of Gdton et al. (19881 were used to detennine the number of cellulolytic and amylolytic bacteria, respectively, in the fermentor cultures. To determine proportions of ruminal bacteria that were sensitive or resistant to lysocellin, fermentor samples were inoculated into various agar and broth media containing 0 and 4 ~ 1 8of lysocellin/mL.
W N G ET AL.
284
Table 3. Effect of lysocellin on total concentration and moles/100 mole of volatile fatty acids after 24 hours in ruminal batch culture Lysocellin, ppm of the liquid 0
.5
SE'
mMe
C2b
104.5 104.9 101.5 97.0 91.5 84.2 79.8 57.3 3.8
82.7 58.5 59.8 60.1 60.3 60.4 59.7 87.2 .4
c3c
a d
c2:C3e
moI/lOO mol 17.9 29.8 30.7 30.8 31.4 30.8 30.9 20.4
15.8 8.1 6.5 6.5 5.5 5.9 6.1
3.50 1.98 1.94 1.95 1.92 1.96 1.94 3.32 .06
8.0
.3
.4
-
Total volatile fatty acids, 0 > 20, 40 (P c .05). bC2 acetate, 0 > . 5 < 40 (P < .05). cC3 = propionate, 0 c .5, 1, 2.5, 5, 10, 20, 40 (P < .05). dcq, = butyrate, 0 > all other doses (P c .05). 'C2:C3, 0 > .5, 1, 2.5, 5, 10, 20 (P < .Os). = 3.
tn
barley-based diets supplemented with lysocellin. The digestion of ADF was not affected by starch source or lysocellin treatment. Bacterial N synthesis (grams per kilogram of OM truly fermented) also was not affected by starch source but was less 8 c .01) with lysocellin. Fermentation end-products are shown in Table 6. Total VFA concentration was greater (P < .05) in corn- than in barley-based diets. Molar proportions of acetate (P < .05), butyrate (P .05), and valerate (P < .05) were decreased but propionate was increased (P < .05) with lysocellin treatment. Molar proportions of isobutyrate and butyrate were lower (P < .05) and the molar proportion of propionate was higher (P c .05) in corn- than in barley-based diets. An interaction (P -05) between starch source and lysocellin was noted for acetate, propionate, and isovalerate. Sensitivities of adapted and unadapted (to lysocellinl ruminal bacteria are shown in Table 7. Total anaerobes and amylolytic and lactateutilizing bacteria were not affected by lysocellin;
Total VFA concentration in batch culture was decreased (P < .05) with 20 and 40 ppm lysocellin (Table 3). The low level (.5 ppm) of lysocellin caused a reduction (P < .05) in molar proportion of acetate, whereas addition of 40 ppm of lysocel.05)proportion of acetate. All lin increased (P concentrations of lysocellin, except for the 40-ppm dose, decreased (P < .05) the acetate: propionate ratio. Molar proportions of butyrate were decreased (P < .05) by > 50% with the addition of lysocellin. Continuous CuIture Fermentations. Culture pH, ammonia N, and methane production are shown in Table 4. Culture pH and ammonia N were greater (P < .05) in barley- than in corn-based diets. Addition of lysocellin reduced (P < .05) methane production but had no effect on ammonia N. Organic matter and ADF digestion in continuous culture are shown in Table 5 . There was a starch x lysocellin interaction (P c .05) for apparent and true OM digestion; OM digestion increased in corn-based diets but decreased in
Table 4. Effect of starch source (corn or barley) and lysocellin on ruminal pH, ammonia N and methane in continuous culture corn Item
PH Ammonia N, mg/dL Methane. mmol/d
P-value
Barley
0s
33a
0s
6.5 1 10.2 5.8
6.47 13.8 4.7
6.75 14.9 8.8
aLysocellin at 0 or 33 ppm of diet DM. bn = 3. %ere were no starch x lysocellin interactions ~ N S= not statistically significant.
6.67 16.0
3.9
(P
> .lo).
SEk
Starch
LYsocellin
.02 1.42 .7
.05 .05 .09
.05 NSd .05
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
1
2.5 5 10 20 40
TVFA,
285
LYSOCELLIN FERMENTATION
Corn Item Digestion OM, %c OM, qgcd
ADF, % Bacterial N, a/kn OMde
Barley
oa
33a
08
33a
SEb
48.7 58.5 51.7
54.1 81.4 53.8
49.7 59.5 58.4
47.7 55.3 54.5
1.2 1.6 4.2
16.2
12.3
15.8
14.7
.8
aLysocellin at 0 or 33 ppm of diet DM. bn = 3.
%tarch x lysocellin interaction dCorrected for bacterial OM. 'Lysocellin effect Lp < ,011.
Ip
< .05).
however, numbers of cellulolytic bacteria unadapted to lysocellin in continuous culture were lower (P < .001 when grown in media containing 4 ppm of the ionophore. In contrast, cellulolytic bacteria adapted to lysocellin (in continuous culture) were unaffected by the ionophore in culture media. Lactate producers were sensitive to lysocellin in culture tubes regardless of whether they were adapted or unadapted to lysocellin in continuous culture (P < .01).
Discussion
1989).
Addition of lysocellin to batch cultures of mixed ruminal microorganisms caused changes in fermentation (Tables 2 and 3)similar to those noted with other ionophores (Thivend and Jouany, 1983;
Lysocellin decreased acetate:propionate ratios in batch culture (Table 3). Similar decreases in the acetate:propionate ratio observed in continuous culture were accompanied by decreased methane
Table 6. Effect of starch source (corn or barley] or lysocellin on ruminal VPA concentration Corn Item Total VFA, mM moll100 mol Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate: propionate
Barley
oa
338
145.2
139.8
127.2
52.7 30.0 .8 9.8 3.9 2.8
50.5 35.0
54.1 24.1 1.o 15.0 3.2 2.6
1.61
.9 7.0 4.2 2.5 1.59
aLysocellin at 0 or 33 ppm of diet DM.
bn
= 3.
'Starch effect of corn vs barley. dLysocellin effect of o vs 33 ppm. 'Starch x lysocellin interaction. ~ N S= not statistically significant.
P-value
SEb
Oa
2.27
130.5 48.5
34.0 1.0 9.5 4.5 2.5 1.55
' S
Ld
s
x Le
7.8
.05
.05
NSf
.7 1.0 .03 .7 .2 .1
NS
.05 .05
.05 .05
NS
NS NS
NS NS
.05 .05 .05
.07
.05
.05
.05 .05 .05
.05
NS .05
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Russell and Strobel, 1988). The lowest concentration of lysocellin (.5 ppm, fluid basis) decreased the acetate:propionate ratio without negatively affecting either total VFA concentrations or ADF digestion. This concentration was equivalent to about 40 ppm of feed, whereas monensin and other ionophores are fed to ruminants at concentrations of about 33 ppm. These findings agree with the in vivo lysocellin studies of Harvey et al. (1988). Surprisingly, concentrations of lysocellin that decreased fiber digestion (1 through 20 ppml had little effect on the molar proportion of acetate but markedly decreased the proportion of butyrate. This decrease was > 50% in several instances, and is greater than that usually observed with monensin. High pH and ammonia N and low ADF digestion and total VFA concentration suggest that the highest level of lysocellin was inhibitory to fermentation. The concentration of lysocellin used in continuous culture (33 ppm of the diet, DM basis) was equivalent to about .7 ppm of the fluid and was similar to the low dose (.5 ppm) used in the batch culture experiments. In continuous culture, the decrease in pH resulting from addition of lysocellin was small (Table 4). This finding agrees with our batch culture data (the lowest level of lysocelEn also tended to decrease pH1. In previous studies, ionophores have had various effects on ruminal pH, increasing it in one study (Schelling, 1984) but decreasing it in another (Bogaert et al.,
Table 5. Effect of starch source (corn or barley] and lysocellin on nutrient digestion and bacterial N synthesis in continuous culture
KUNG ET AL.
286
Table 7. Interaction between lysocellin in continuous culture and lysocellin in enumerating media on numbers of ruminal bacteria (loglo) per milliliter Lysocellin in fermentors, 33 DDma
-
-
+
+
Lysocellin in media, pg/m.L 0
4
0
4
SEb
Total anaerobes
8.6
Cellulolyticc Amylolytic Lactate utilizers Lactate producersd
6.1 9.3
8.5 4.7 9.3
6.9 6.0
6.5
8.5 7.7 9.4 7.5
4.0
6.8
8.8 7.4 9.4 7.5 4.4
.82 .99 .58 .TI .86
*Diet DM. bn = 6. 'Lyeocellin in fermentor x lysocellin in media interaction < .06). 'Lysocellin in media effect (P c .01).
(P
production (Table 4). Unexpectedly, lysocellin tended to increase the molar percentage of propionate more in the barley- than in the corn-based diet. Garrett (1976) conducted a study in which monensin was supplemented to either corn- or barley-based diets, but the main effects were pooled and it was not possible to differentiate monensin effects between grain sources. In other studies, steers fed high barley-based diets have responded to monensin supplementation (Horton et al.,1980, 1983). We hypothesize that ionophores could have variable effects on ruminal fermentation when added to diets with more r e d y fermentable carbohydrates (e.g., barley vs corn). In our barley diet supplemented with lysocellin, the greater change in acetate:propionate ratio was not consistent with lower OM digestion, but a greater proportion of starch (not measured) may have been fermented with barley vs corn. Low levels of lysocellin did not affect total VFA concentrations in either batch (Table 3) or continuous culture (Table 6); however, VFA concentrations were depressed when lysocellin was > 5 ppm in batch culture. Ionophores have had inconsistent effects on total VFA concentrations in other studies. Harvey et al. (1988) reported that doses of 11, 22, and 33 ppm (of the diet DM) lysocellin depressed total VFA. In contrast, Richardson et al. (1976) reported that low levels of monensin (similar to the levels of 1ysocelIj.n used in the current study)stimulated total VFA concentrations in batch culture. Effects of ionophores on fiber digestion are conflicting. Fiber digestion was decreased in in vitro studies using unadapted ruminal microorganisms (Simpson, 1978; Henderson et al., 1981; Russell and Strobel, 1988); however, in vivo studies have not substantiated these findings. For
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Bacterial populations
example, monensin decreased ADF digestion in lambs Boos et al., 19791, but the effect diminished after 40 d of treatment. Similarly, steers fed lysocellin had AVF digestibilities similar to those of control animals (Spears et al., 1989). In our batch study (with unadapted ruminal microorganisms in batch culture) lysocellin concentrations of > 2.5 ppm markedly decreased ADF digestion (Table 21. Although not directly compared in our study, the decrease in ADF digestion seems to be less than that obtained with similar levels of monensin (Simpson, 1978; Henderson et al., 19811, suggesting that adaptation may be less of a problem with lysocellin. Mackie et al. (1984) reported that both Gram-positive and Gramnegative organisms adapted to monensin and suggested an ionophore rotation program to maintain efficiency. Similar to monensin, we found that lysocellin reduced methane production; however, Carmean and Johnson (1990) r e ported that methane production was reduced in steers fed monensin after 2 d but not after 44 d of treatment, suggesting that the effects of monensin on ruminal fermentation may not persist through a typical finishing period. Recently, Morris et al. (1990) reported that a daily rotation regimen of monensin and lasalocid improved animal performance but that improvement was not a result of alterations in ruminal fermentation. Results from our continuous culture study support a microbial adaptation phenomenon (Table 7). Cellulolytic bacteria were decreased when unadapted cultures were enumerated in broth containing lysocellin; however, presence of lysocellin in the enumerating broth did not affect cellulolytic numbers if bacteria were from fermentors adapted to lysocellin. Ruminal lactate-producing organisms were sensitive to lysocellin, a result similar to the findings of Dennis et al. (19811 with monensin and lasalocid. However, unlike the cellulolytic bacteria, lactate-producing bacteria remained sensitive to lysocellin. It is unclear why lactate-producing bacteria remained sensitive to lysocellin, yet lysocellin addition to fermentors did not reduce the numbers of lactate-producing bacteria. We are unaware of in vivo studies that document direct microbial adaptation to ionophores over prolonged periods of time. Ionophores reduce protein degradation in the rumen (Schelling, 19841, and Hillaire et al. (1989) suggested that lysocellin markedly decreased bacterial ammonia uptake. The effect of lysocellin on ruminal ammonia in steers is inconclusive. Harvey et al. (19881 reported cubic effects of lysocellin on ruminal ammonia concentrations, whereas Spears et al. (1989) reported that lysocellin improved N digestion and absorption. In our batch culture study (Table 21, low concentrations
LYSOCELLIN FERMENTATION
Implications In vitro, low levels of lysocellin (.5 to 1 ppm of the fluid) altered ruminal fermentation by decreasing methane production and the acetate: propionate ratio. High concentrations of lysocellin ( > 5 ppm) decreased fiber digestion, but cellulolytic organisms were able to adapt to lysocellin in short-term continuous culture. Lactate-producing organisms were sensitive to lysocellin and not able to adapt. Lysocellin had a greater effect in altering the acetate:propionate ratio in barleythan in corn-based diets. Animal responses to ionophores may be affected by interactions with the diet.
Literature Cited AOAC. 1984. Official Methods of Analysis (14thEd.). Asso& tion of OaFicial Analytical Chemists, Arlington, VA. Bogaert, C., L. Gomez, J. P. Jouany, and G. Jeminet. 1989. Effects of the ionophore antibiotics h a l o c i d and c& tionomycin on ruminal fermentation in vitro (RUSlTEC). Anim. Feed Sci. Technol. 271. Bryant, M. P., and L. A. Burkey. 1953. Numbers and some predominant groups of bacteria in the rumen of cows fed different rations. J. Dairy Sci. 36:218. Bryant, M.P.,and I. M.Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446. Carmean, B. R., and D. E. Johnson. 1990. Persistence of monensin-induced changes in methane emissions and ruminal protozoa numbers in cattle. J. Anim. Sci. 68EuppL 11~517 (Abstr.). Craig. W. M.,B. J. Hong, G. A. Broderick, and R. J. Bula. 1984. In vitro inoculum enriched with particle associated microorganisms for determining rates of fiber digestion and
protein degradation J. Dairy Sci. 67:2902. Dennis, S.M., T. G. Nagaraja. and E. E. Bartley. 1981. Effects of lasalocid or monensin on lactate-producing or -using rumen bacteria. J. Anim. Sci. 52418. Galton, M. M., G. K. Morris,and W. T. Martin. 1968. Salmonellae in Foods and Feeds. NatL Communicable Disease Center, Atlanta, GA. Garrett, W. N. 1076. Innuence of rumensin on energy utilization of corn and barley diets. Proc.California Feeders Day Program, Holtville, CA. p 21. GiU, J. L., and H. D. Hafs. 1971. Analysis of repeated measuremente of RnimRdn. J. Adm. S c i 33:331. Goering, H.K., andP. J. Van Soest. 1970. Forage fiber analyses lapparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS,USDA, Washington, DC. Harvey, R.W., J. W. Spears, and D. E. Darden. 1988. Effects of lysocellin on perfomname, ruminal and plasma characteristics of growing cattle. J. Anim. Sci. 66:1036. Henderson, C., C. S.Stewart, and F. V. Nekrep. 1981. The effect of monensin on pure and mixed cultures of rumen bacteria. J. Appl. Bact. 51:159. Hillaire, M. C., J. P. Jouany, C. Gaboyard, and G. Jeminet. 1989. In vitro study of the effect of different ionophore antibiotics and certain derivatives on rumen fermentation and on protein nitrogen degradation. Reprod. Nutr. Dev. 29:247. Horton, G.M.J., K. A. Bassendowski, and E. H. Keeler. 1980. Digestion and metabolism in lambs and steers fed monensin with different levels of barley. J. Anim. Sci. 50:997. Horton, G.M.J., M.J. Farmer, K. A. Bassendowski, and G. M. Steacy. 1983. Rumen fermentation and digestibility in steers as influenced by level of intake and monensh Can. J. Anim. Sci. 63:855. Isichei. C. O., and W. G. Bergen. 1980. The effect of monensin on the composition of abomasalnitrogen flow in steers fed grain and silage rations. J. Anim. Sci. 51Guppl. 1):371 (Abstr.). Mackie, R. I., P. G. Bahrs, and J. J. Therion. 1984. Adaptation of rumen bacteria to sodium and monensin. Can. J. Anim. Sci. 84E~ppL):351(Ab&.). McDougall, E. I. 1948. Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 4399. Morris, F. E., M. E. Branine, M.L. Galyean, M.E. Hubbert, A. S. Freeman, and G. P. Lofgreen. 1990. Effect of rotating monensin plus tylosin and lasalocid on performance, ruminal fermentation, and site and extent of digestion in feedlot cattle. J. Anim. Sci. 68:3069. Okuda, H., S.Fuji, and Y.Kawashima. 1965. A direct colorimetric method for blood ammonia. Tokushima J. Exp. Med 12: 11.
PQOS,M.I., T. L. Hanson, and T. J. Klopfenstein. 1979. Monensin effects on diet digestibility, ruminalprotein bypass and microbial protein synthesis. J. Anim. Sci. 48:1516. Preston, Ft. L., Ft. H. Pritchard, and G. W. Wolfrom. 1985. Lysocellin effects on the grain, feed intake and efficiency of g r o w i n g - f i h h g cattle. J. Anim. Sci. Bl(Supp1. 1):493 (Abstr.). Richardson, L. F., A. P. Raun,E. L. Potter, C. 0. Cooley, and R. P. Rathmacher. 1976. Effect of monensin on rumen fermentation in vitro and in vivo. J. Anim. Sci. 43:657. Rogoaa. M.,J. A. Mitchell, and R. FL. Wiseman. 1951. A selective medium for the isolation and enumeration of oral and fecal lactobacilli. J. Bacteriol. 62:132. Russell, J. B. and H. J. Strobel. 1988. Effects of additives on in vitro ruminal fermentation A comparison of monensin and bacitracin, another gram-positive antibiotic. J. Anim. ScL 66:552. SAS. 1985. SAS User’s Guide Statistics. SAS Inst., Inc., G u y , NC. Scheffe, H. 1953. A method for judging all contrasts in the analysis of variaace. Biometrika 40:87. Schelling, G. T. 1984. Monensin mode of action in the rumen. J.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
of lysocellin increased ammonia N concentre tions, suggesting an increase in protein degrade tion and(or1a reduction in ammonia utilization by bacteria, but lysocellin had no effect on ammonia N concentrations in continuous culture (Table 41. Bacterial N synthesis (grams per kilogram of OM truly fermented) also was lower in continuous cultures treated with lysocellin, which agrees with data from Isichei and Bergen (1980) with monensin and Bogaert et al. (1989) with lasalocid. Lack of a decrease in the total anaerobic bacteria in continuous cultures treated with lysocellin compared to controls is taken as evidence that a greater proportion of the total bacteria grew in lysocellintreated cultures. The presence of lysocellin may have an influence similar to that of low pH, namely increasing the proportion-of bacteria that grow in nonselective medium (Slyter et al., 19661. Thus, bacterial protein would be better predicted by other methods (e.@;., the purine assay) than by bacterial count from nonselective medium.
287
288
KUNG ET AL. Slyter, L. L., and P. A. Putnam. 1987. In vivo vs. in vitro continuous culture of rumhid microbial populations. J. Anim. SCi. 261421. Spears, J. W., J. C. Burns, and G. W. Wolfrom. 1989. Lysocellin effects on growth performance, ruminal fermentation, nutrient digestibility and nitrogen metabolism in steers fed forage diets. J. Anim. Sci. 67:547. Thivend, P., and J. P. Jouany. 1983. Effect of lasalocid sodium on rumen fermentation and digestion in sheep. Reprod. Nutr. Dev. 23:817. Wolfrom, G. W., D. R Bright, P. J. Schalburg, and A. Clingerman. 1883. In vitro and in vivo effects of a new polyether antibiotic aysocellin) to enhance the feedlot performance of ruminants. In: Proc. 17th Conf. on Rumen Function.p l CAbstr.1. Chicago, IL. Zinn, R. A. and F.N.Owens.1886. Rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66:157.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Anim. Sci. 58:1518. Simpson, M. E., 1978. Effects of certain antibiotics on in vitro cellulose digestibility and volatile fatty acid 0 production by ruminal microorganisms. J. Anim. Sci. 47Suppl. 1): 439 (Abstr.1. Slyter, L. L., M. P. Bryant, and M. J. Wolin. 1866. Effect of pH on population and fermentation in a continuously cultured rumen ecosystem. Appl. Microbiol. 14:573. Slyter, L. L., D. L. Kern,and J. M. Weaver. 1976. Effect of pH on ruminal lactic acid utilization and accumulation in vitro. J. Anim. Sci. 43:333 (Abstr.). Slyter, L. L., W. 0. Nelson, andM. J. Wolin. 1864. Modification of a device for maintenance of the rumen microbial population in continuous culture. Appl. Microbiol. 12:374. Slyter, L. L., R. R. Oltjen, D. L. Kern, and F. C. Blank. 1970. Influence of type and level of grain and diethylstilbesterol on the rumen microbial populations of steers fed all-concentrate diets. J. Anim. Sci. 31:OOB.
ABSTRACT: Batch and continuous culture techniques were used to evaluate the effect of the ionophore lysocellin on ruminal fermentation and microbial populations. In batch culture, .5 and 1 ppm (of the fluid) lysocellin markedly decreased (P e .011 the acetate:propionateratio without affecting fiber digestion, ammonia concentration, or culture pH. Greater concentrations of lysocellin had negative effects Ip c .05) on fiber digestion and increased CP e .051 culture pH. In continuous culture, a low level of lysocellin (33 ppm of the diet DM or about .7 ppm of the fluid) decreased pH Ip e .05) and methane (P c .05) production but had no effect on fiber digestion. Lysocellin tended to increase (P e .05) OM digestion in corn-based diets but decreased OM digestion in barley-based
diets htarch source x lysocellin interaction, P e .05). In addition, the molar proportion of propionate was increased more in barley- than in cornbased diets. Total anaerobes and amylolytic and lactate-utilizing microorganisms were not affected by the ionophore. In continuous culture, cellulolytic and lactate-producingorganisms were insensitive to lysocellin, but, in lysocellin-treated media, cellulolytic organisms were resistant, whereas lactic acid producers were sensitive to lysocellin at 4 ppm. In summary, the ionophore lysocellin alters ruminal fermentation by decreasing ruminal methane production and increasing the molar proportion of propionate; however, effects varied depending on whether corn or barley served as the primary starch source.
Key Words: Ionophores, Rumen Fermentation, Rumen Microorganisms J. Anim. Sci. 1992. 70:281-288
Introduction
also have been noted (Wolfi-om et al.,
Lysocellin is a divalent polyether antibiotic from Streptomyces cacaoci var. asoensis. When fed to growing cattle, it improves daily gain and feed efficiency Preston et al., 1985; Spears et al., 1989). Shifts in ruminal fermentation, namely decreased acetate and butyrate and increased propionate,
'Published
&B
1983;
Harvey et al., 1988). However, little information is available on the effect of lysocellin on ruminal microbial populations, fiber digestion and methane production. In addition, comparative effects of ionophores on ruminal metabolism in corn- vs barley-based diets are lacking; this is of interest because barley is fed in large quantities in Canada and some western areas of the United States. The objective of our study was to evaluate the effect of lysocellin on ruminal fermentation and fiber digestion in batch culture. We also evaluated the effect of lysocellin on ruminal fermentation and microbial populations from barley- or corn-based diets in continuous culture.
Misc. Paper No. 1388 of the Delaware Agric.
Em. Sta. 2Funding for this research was provided in part by (1.Unidel grant from the Univ. of Delaware. Lysocellin waa a giR from IMCPitman Moore, Terre Haute, IN. Received April 1, 1991. Accepted July 17, 1991.
281
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
*Delaware Agricultural Experiment Station, Department of Animal Science and Agricultural Biochemistry, College of Agricultural Science, University of Delaware, Newark 19717-1303 and WSDA-ARS, Ruminant Nutrition, Beltsville, MD 20705
KUNG ET AL.
282
Experimental Procedure
Diet? Item Feed composition, % Corn silage
Barley Corn Soybean meal, 48% CP Limestone n a c e mineral saltb Lysocellin, ppm of diet DM
1
2
3
4
27.6 65.0
27.6 65.0
26.6
26.6
6.1
6.1
1.1
1.1
65.0 7.2 1.0
.2
.2
65.0 7.2 1.0 .2 33 12.6 12.1 76.0 .45 .37 .l6 .80 .20
12.6 12.1 76.0 .45 .37 .I6 .80 .20
33
.2
Chemical composition, %
CP ADF TDN Ca
.37
13.7 14.4 75.0 .37
P Mg K
.40
.I5
.40
Na
13.7 14.4 75.0
1.01 .17
.15
1.01 .17
*Diet 4 was the basal diet used in batch culture fermentations. Diets 1 , 2 , 3, and 4 were used in the continuous culture fermentations. blO% P, 19% Ca,, 3%M g ,3% K,.75%S,.15%I, , 0 0 4 1 Co, .025% Cu,.035% Fe, .2% Mn,.75%Zn, .006% Se, 860,000 IU Vitamin A/ kg, 165,000 IU vitamin D/kg, and 2,475 IU Vitamin E&.
to inhibit microbial growth. Fermentors were stirred continuously at 75 rpm and temperature was held at 39°C. Twelve fermentors were filled to capacity with ruminal fluid from steers fed a 70% concentrate (corn and soybean meal) and 30% forage (corn silage) diet. Fermentors were purged with C02 before filling, after filling, and during feeding. Culture pH was maintained above 6.2 by infusing artificial saliva CMcDougall, 1948) modified to contain .117 M Na&03 and .0125 M urea instead of .117 M NaHC03. McDougall’s buffer and tap water (60:40) were infused continuously by peristaltic pumps to obtain a turnover rate of approximately 1.5 volumes per day. Gas was collected into butyl rubber bags. Fermentors were given 9.0 g twice daily of a I-mm grind of either a corn- (six fermentors) or a barley- (six fermentors) based diet (Table 1). Steady-state conditions were achieved after 5 d. At the start of d 6, 33 ppm of the ionophore lysocellin was included at each addition of feedstuffs to three fermentors given the corn diets and three fermentors given the barley diets. The start of d 6 marked the beginning of three 3-d sampling periods. The following procedures were used during collection and analyses of samples from each period: fermentor effluents were collected every 24 h and pH was recorded. Effluent was acidified with .1 mL of 5Ooh Has04 (vol/voll per 10
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Batch Fermentations. In vitro batch cultures with mixed ruminal microorganisms were established from fistdated steers fed a corn silage, corn, soybean meal, and alfalfa hay diet (forage:concentrate ratio of 50:50) to study the effect of lysocellin on ruminal fermentation. Ruminal fluid was collected from three steers at approximately 3 h after feeding. Fluid was strained through four layers of cheesecloth and processed for recovery of particulate-bound microorganisms under anaerobic conditionc (Craig et al., 1984). Twenty milliliters of strained ruminal fluid were combined with 20 mL of a prewarmed (30°C) phosphate-bicarbonate buffer (Goering and Van Soest, 1970) and added to 50-mL test tubes with .5 g of a diet consisting of 70% grain (corn, soybean meal, and minerals) and 30% forage (corn silage). Lysocellin was dissolved in ethanol and appropriate dilutions were made such that .5 mL supplied final concentrations of 0, .5,1, 2.5, 5, 10, 20, and 40 ppm (fluid basis). Four 50-mL test tubes were prepared for each steer at each dose. After 24 h of fermentation, pH ww determined in two incubation tubes for each steer and dose before addition of 1 mL of 25% nphosphoric acid to 5 mL of ruminal fluid. Ruminal fluid was analyzed for ammonia N (Okuda et al., 1965) and VFA by gas chromatography (Hewlett Packard Co.,Avondale, PA). The remaining two incubation tubes for each steer and dose were analyzed for residual ADF (AOAC, 1984). Acid detergent fiber digestion was calculated by subtracting the residual ADF from the amount of ADF present before digestion. Data were analyzed by analysis of variance techniques with main effects of steer and treatment (SAS, 1985) tested against the residual error term. Regressions on VFA data were not performed because the highest dose of lysocellin seemed toxic. Thus, treatment effects were evaluated by Scheffe’s (1953) test with specific comparisons of the 0 dose to each increasing dose level. Degrees of freedom for ADF digestion, pH, and ammonia N concentration were partitioned by multiple regression into linear, quadratic, and cubic effects. Continuous Culture Fermentation. The effect of the ionophore lysocellin on ruminal fermentation and microbial populations in continuous culture was studied. Treatments (Table 11 were diets consisting of 70% concentrate and 30% forage; the major variables were starch source (corn or barley) with and without 33 ppm dietary lysocellin (DM basis). Fermentors (Slyter et al., 1964) consisted of a closed system with 500-mL reaction vessels with a single overflow port. Effluent was collected in funnels with 16 mL of saturated HgC12
Table 1. Composition [%, DM basis) of experimental diets used in batch and continuous culture fermentations
283
LYSOCELLIN FERMENTATION
Data were analyzed by analysis of variance using the GLM procedure of SAS (1985). The
experimental design was a split plot with repeated measures (Gill and Hafs, 1971). Effect of treatment was tested in the main plot and period and period x treatment interaction were subplot treatments. The treatment effect was further separated into main effects for starch source (corn or barley), a lysocellin effect (0 or 33 ppml, and a starch x lysocellin interaction. Microbial data were log-transformed (base 10) before analyses. Means were tested using a F-protected (P < .05) pairwise comparison of the least squares means.
Results Batch Fermentations. The effect of various levels of lysocellin on ruminal fermentation was studied after 24 h of incubation. Low concentrations of lysocellin (.5 to 2.5 ppm, fluid basis) had no effect on culture pH, but greater concentrations caused a moderate increase in pH (Table 2). The 40 ppm level of lysocellin caused culture pH to increase above 7.5. Compared with untreated cultures, cultures with low levels (up to 2.5 ppml of lysocellin had greater ammonia N concentrations; however, when lysocellin addition was between 5 and 20 ppm, ammonia N content was less than in untreated cultures. The highest concentration of lysocellin (40 ppm) caused an increase in culture ammonia N concentration. Digestion of A D F was not affected by .5 ppm lysocellin but it decreased rapidly (P e .05) with increasing dose. Acid detergent fiber digestion was decreased by > 70% with doses of lysocellin > 10 ppm.
Table 2. Effect of lysocellin on pH, ammonia N and A D F digestion after 24 hours in ruminal batch culture ~~~~~
Lysocellin, PPm of the fluid 0
ADF
P* 6.29
.5
Ammonia N, mg/dLb
m=tim
25.2
47.0 48.5
6.24
29.8
1
6.30
28.6
34.7
2.5
6.37
28.0
34.8
5
6.56
24.6
28.8
132
10
6.76
22.2
20
6.73
22.2
7.6
40
7.53
32.4
5.0
.09
1.7
3.0
SEd
~~
*Linear and cubic effects [P < .OS) of lysocellin dose. bQuadratic effect (P .OS1 of lysocellin dose. ‘%inear and quadratic effects (P c .05) of lysocellin dose.
dn
= 3.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
mL of effluent, centrifuged at 12,000 x g for 15 min, and analyzed for VFA by gas chromatography as described previously. The 3-d period daily effluents composited by fermentor were sampled by using a wide-bore pipette while being constantly stirred in a squire blender. A portion of the composite effluent was lyophilized, ground to pass a I-mm mesh screen, and analyzed for N (Kjeldahl), ADP CAOAC, 19841, purines (Zinn and Owens, 19861, and ash CAOAC, 1984). On each day, approximately 300 mL of fluid effluent was treated with 60 mL of formaldehyde in .9% (wt/vol) saline and differentially centrifuged at 600 x g for 5 min, followed by 12,000 x g for 15 min to isolate ruminal bacteria. Bacteria were lyophilized,ground with a mortar and pestle, and analyzed for N (KjeldahU, purines (Zinn and Owens, 19861, and ash (5OO0C, 6 h). Bacterial protein synthesis was calculated from the ratio of purines in effluent to bacterial purine content and expressed as grams of bacterial N produced per kilogram of truly fermented OM. Fluid effluent also was analyzed for VFA by gas chromatography and for ammonia N (Okuda et al., 19651. Total gas production was measured at the end of each 3 d period and analyzed for CHI by gas chromatography (Fisher Gas Partitioner, Fisher Scientific, Pittsburgh, PA) (Slyter and Putnam, 1967). On the first day of Period 3, a representative sample from each fermentor was analyzed for total anaerobic bacteria (98-5 media, Bryant and Robinson, 19611, lactate-producing bacteria (Rogosa et al., 1951, as modified by Slyter et al., 19701, and lactate-utilizing bacteria (Slyter et al., 19761. Duplicate 98-5, lactate-producing and lactate-utilizing agar roll tube media were inoculated and incubated at 39°C for 4 , 2 , and 4 d, respectively. All colonies were counted in 98-5 tubes. All colonies medium-large to large were counted in lactate-producingagar tubes. All medium-smallor larger colonies were counted in lactateutilizing agar tubes. Tubes with broth that were used to determine total number of cellulolytic and amylolytic bacteria were incubated for 7 d at 39°C. The starch:cellulose medium of Bryant and Burkey (1953) was prepared as modified by Slyter et al. (1970). Disappearance of cellulose (observed visually) and starch (iodine test) in each of three tubes inoculated at three or more successive decimal dilutions and the most probable number (MPN) tables of Gdton et al. (19881 were used to detennine the number of cellulolytic and amylolytic bacteria, respectively, in the fermentor cultures. To determine proportions of ruminal bacteria that were sensitive or resistant to lysocellin, fermentor samples were inoculated into various agar and broth media containing 0 and 4 ~ 1 8of lysocellin/mL.
W N G ET AL.
284
Table 3. Effect of lysocellin on total concentration and moles/100 mole of volatile fatty acids after 24 hours in ruminal batch culture Lysocellin, ppm of the liquid 0
.5
SE'
mMe
C2b
104.5 104.9 101.5 97.0 91.5 84.2 79.8 57.3 3.8
82.7 58.5 59.8 60.1 60.3 60.4 59.7 87.2 .4
c3c
a d
c2:C3e
moI/lOO mol 17.9 29.8 30.7 30.8 31.4 30.8 30.9 20.4
15.8 8.1 6.5 6.5 5.5 5.9 6.1
3.50 1.98 1.94 1.95 1.92 1.96 1.94 3.32 .06
8.0
.3
.4
-
Total volatile fatty acids, 0 > 20, 40 (P c .05). bC2 acetate, 0 > . 5 < 40 (P < .05). cC3 = propionate, 0 c .5, 1, 2.5, 5, 10, 20, 40 (P < .05). dcq, = butyrate, 0 > all other doses (P c .05). 'C2:C3, 0 > .5, 1, 2.5, 5, 10, 20 (P < .Os). = 3.
tn
barley-based diets supplemented with lysocellin. The digestion of ADF was not affected by starch source or lysocellin treatment. Bacterial N synthesis (grams per kilogram of OM truly fermented) also was not affected by starch source but was less 8 c .01) with lysocellin. Fermentation end-products are shown in Table 6. Total VFA concentration was greater (P < .05) in corn- than in barley-based diets. Molar proportions of acetate (P < .05), butyrate (P .05), and valerate (P < .05) were decreased but propionate was increased (P < .05) with lysocellin treatment. Molar proportions of isobutyrate and butyrate were lower (P < .05) and the molar proportion of propionate was higher (P c .05) in corn- than in barley-based diets. An interaction (P -05) between starch source and lysocellin was noted for acetate, propionate, and isovalerate. Sensitivities of adapted and unadapted (to lysocellinl ruminal bacteria are shown in Table 7. Total anaerobes and amylolytic and lactateutilizing bacteria were not affected by lysocellin;
Total VFA concentration in batch culture was decreased (P < .05) with 20 and 40 ppm lysocellin (Table 3). The low level (.5 ppm) of lysocellin caused a reduction (P < .05) in molar proportion of acetate, whereas addition of 40 ppm of lysocel.05)proportion of acetate. All lin increased (P concentrations of lysocellin, except for the 40-ppm dose, decreased (P < .05) the acetate: propionate ratio. Molar proportions of butyrate were decreased (P < .05) by > 50% with the addition of lysocellin. Continuous CuIture Fermentations. Culture pH, ammonia N, and methane production are shown in Table 4. Culture pH and ammonia N were greater (P < .05) in barley- than in corn-based diets. Addition of lysocellin reduced (P < .05) methane production but had no effect on ammonia N. Organic matter and ADF digestion in continuous culture are shown in Table 5 . There was a starch x lysocellin interaction (P c .05) for apparent and true OM digestion; OM digestion increased in corn-based diets but decreased in
Table 4. Effect of starch source (corn or barley) and lysocellin on ruminal pH, ammonia N and methane in continuous culture corn Item
PH Ammonia N, mg/dL Methane. mmol/d
P-value
Barley
0s
33a
0s
6.5 1 10.2 5.8
6.47 13.8 4.7
6.75 14.9 8.8
aLysocellin at 0 or 33 ppm of diet DM. bn = 3. %ere were no starch x lysocellin interactions ~ N S= not statistically significant.
6.67 16.0
3.9
(P
> .lo).
SEk
Starch
LYsocellin
.02 1.42 .7
.05 .05 .09
.05 NSd .05
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
1
2.5 5 10 20 40
TVFA,
285
LYSOCELLIN FERMENTATION
Corn Item Digestion OM, %c OM, qgcd
ADF, % Bacterial N, a/kn OMde
Barley
oa
33a
08
33a
SEb
48.7 58.5 51.7
54.1 81.4 53.8
49.7 59.5 58.4
47.7 55.3 54.5
1.2 1.6 4.2
16.2
12.3
15.8
14.7
.8
aLysocellin at 0 or 33 ppm of diet DM. bn = 3.
%tarch x lysocellin interaction dCorrected for bacterial OM. 'Lysocellin effect Lp < ,011.
Ip
< .05).
however, numbers of cellulolytic bacteria unadapted to lysocellin in continuous culture were lower (P < .001 when grown in media containing 4 ppm of the ionophore. In contrast, cellulolytic bacteria adapted to lysocellin (in continuous culture) were unaffected by the ionophore in culture media. Lactate producers were sensitive to lysocellin in culture tubes regardless of whether they were adapted or unadapted to lysocellin in continuous culture (P < .01).
Discussion
1989).
Addition of lysocellin to batch cultures of mixed ruminal microorganisms caused changes in fermentation (Tables 2 and 3)similar to those noted with other ionophores (Thivend and Jouany, 1983;
Lysocellin decreased acetate:propionate ratios in batch culture (Table 3). Similar decreases in the acetate:propionate ratio observed in continuous culture were accompanied by decreased methane
Table 6. Effect of starch source (corn or barley] or lysocellin on ruminal VPA concentration Corn Item Total VFA, mM moll100 mol Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate: propionate
Barley
oa
338
145.2
139.8
127.2
52.7 30.0 .8 9.8 3.9 2.8
50.5 35.0
54.1 24.1 1.o 15.0 3.2 2.6
1.61
.9 7.0 4.2 2.5 1.59
aLysocellin at 0 or 33 ppm of diet DM.
bn
= 3.
'Starch effect of corn vs barley. dLysocellin effect of o vs 33 ppm. 'Starch x lysocellin interaction. ~ N S= not statistically significant.
P-value
SEb
Oa
2.27
130.5 48.5
34.0 1.0 9.5 4.5 2.5 1.55
' S
Ld
s
x Le
7.8
.05
.05
NSf
.7 1.0 .03 .7 .2 .1
NS
.05 .05
.05 .05
NS
NS NS
NS NS
.05 .05 .05
.07
.05
.05
.05 .05 .05
.05
NS .05
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Russell and Strobel, 1988). The lowest concentration of lysocellin (.5 ppm, fluid basis) decreased the acetate:propionate ratio without negatively affecting either total VFA concentrations or ADF digestion. This concentration was equivalent to about 40 ppm of feed, whereas monensin and other ionophores are fed to ruminants at concentrations of about 33 ppm. These findings agree with the in vivo lysocellin studies of Harvey et al. (1988). Surprisingly, concentrations of lysocellin that decreased fiber digestion (1 through 20 ppml had little effect on the molar proportion of acetate but markedly decreased the proportion of butyrate. This decrease was > 50% in several instances, and is greater than that usually observed with monensin. High pH and ammonia N and low ADF digestion and total VFA concentration suggest that the highest level of lysocellin was inhibitory to fermentation. The concentration of lysocellin used in continuous culture (33 ppm of the diet, DM basis) was equivalent to about .7 ppm of the fluid and was similar to the low dose (.5 ppm) used in the batch culture experiments. In continuous culture, the decrease in pH resulting from addition of lysocellin was small (Table 4). This finding agrees with our batch culture data (the lowest level of lysocelEn also tended to decrease pH1. In previous studies, ionophores have had various effects on ruminal pH, increasing it in one study (Schelling, 1984) but decreasing it in another (Bogaert et al.,
Table 5. Effect of starch source (corn or barley] and lysocellin on nutrient digestion and bacterial N synthesis in continuous culture
KUNG ET AL.
286
Table 7. Interaction between lysocellin in continuous culture and lysocellin in enumerating media on numbers of ruminal bacteria (loglo) per milliliter Lysocellin in fermentors, 33 DDma
-
-
+
+
Lysocellin in media, pg/m.L 0
4
0
4
SEb
Total anaerobes
8.6
Cellulolyticc Amylolytic Lactate utilizers Lactate producersd
6.1 9.3
8.5 4.7 9.3
6.9 6.0
6.5
8.5 7.7 9.4 7.5
4.0
6.8
8.8 7.4 9.4 7.5 4.4
.82 .99 .58 .TI .86
*Diet DM. bn = 6. 'Lyeocellin in fermentor x lysocellin in media interaction < .06). 'Lysocellin in media effect (P c .01).
(P
production (Table 4). Unexpectedly, lysocellin tended to increase the molar percentage of propionate more in the barley- than in the corn-based diet. Garrett (1976) conducted a study in which monensin was supplemented to either corn- or barley-based diets, but the main effects were pooled and it was not possible to differentiate monensin effects between grain sources. In other studies, steers fed high barley-based diets have responded to monensin supplementation (Horton et al.,1980, 1983). We hypothesize that ionophores could have variable effects on ruminal fermentation when added to diets with more r e d y fermentable carbohydrates (e.g., barley vs corn). In our barley diet supplemented with lysocellin, the greater change in acetate:propionate ratio was not consistent with lower OM digestion, but a greater proportion of starch (not measured) may have been fermented with barley vs corn. Low levels of lysocellin did not affect total VFA concentrations in either batch (Table 3) or continuous culture (Table 6); however, VFA concentrations were depressed when lysocellin was > 5 ppm in batch culture. Ionophores have had inconsistent effects on total VFA concentrations in other studies. Harvey et al. (1988) reported that doses of 11, 22, and 33 ppm (of the diet DM) lysocellin depressed total VFA. In contrast, Richardson et al. (1976) reported that low levels of monensin (similar to the levels of 1ysocelIj.n used in the current study)stimulated total VFA concentrations in batch culture. Effects of ionophores on fiber digestion are conflicting. Fiber digestion was decreased in in vitro studies using unadapted ruminal microorganisms (Simpson, 1978; Henderson et al., 1981; Russell and Strobel, 1988); however, in vivo studies have not substantiated these findings. For
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Bacterial populations
example, monensin decreased ADF digestion in lambs Boos et al., 19791, but the effect diminished after 40 d of treatment. Similarly, steers fed lysocellin had AVF digestibilities similar to those of control animals (Spears et al., 1989). In our batch study (with unadapted ruminal microorganisms in batch culture) lysocellin concentrations of > 2.5 ppm markedly decreased ADF digestion (Table 21. Although not directly compared in our study, the decrease in ADF digestion seems to be less than that obtained with similar levels of monensin (Simpson, 1978; Henderson et al., 19811, suggesting that adaptation may be less of a problem with lysocellin. Mackie et al. (1984) reported that both Gram-positive and Gramnegative organisms adapted to monensin and suggested an ionophore rotation program to maintain efficiency. Similar to monensin, we found that lysocellin reduced methane production; however, Carmean and Johnson (1990) r e ported that methane production was reduced in steers fed monensin after 2 d but not after 44 d of treatment, suggesting that the effects of monensin on ruminal fermentation may not persist through a typical finishing period. Recently, Morris et al. (1990) reported that a daily rotation regimen of monensin and lasalocid improved animal performance but that improvement was not a result of alterations in ruminal fermentation. Results from our continuous culture study support a microbial adaptation phenomenon (Table 7). Cellulolytic bacteria were decreased when unadapted cultures were enumerated in broth containing lysocellin; however, presence of lysocellin in the enumerating broth did not affect cellulolytic numbers if bacteria were from fermentors adapted to lysocellin. Ruminal lactate-producing organisms were sensitive to lysocellin, a result similar to the findings of Dennis et al. (19811 with monensin and lasalocid. However, unlike the cellulolytic bacteria, lactate-producing bacteria remained sensitive to lysocellin. It is unclear why lactate-producing bacteria remained sensitive to lysocellin, yet lysocellin addition to fermentors did not reduce the numbers of lactate-producing bacteria. We are unaware of in vivo studies that document direct microbial adaptation to ionophores over prolonged periods of time. Ionophores reduce protein degradation in the rumen (Schelling, 19841, and Hillaire et al. (1989) suggested that lysocellin markedly decreased bacterial ammonia uptake. The effect of lysocellin on ruminal ammonia in steers is inconclusive. Harvey et al. (19881 reported cubic effects of lysocellin on ruminal ammonia concentrations, whereas Spears et al. (1989) reported that lysocellin improved N digestion and absorption. In our batch culture study (Table 21, low concentrations
LYSOCELLIN FERMENTATION
Implications In vitro, low levels of lysocellin (.5 to 1 ppm of the fluid) altered ruminal fermentation by decreasing methane production and the acetate: propionate ratio. High concentrations of lysocellin ( > 5 ppm) decreased fiber digestion, but cellulolytic organisms were able to adapt to lysocellin in short-term continuous culture. Lactate-producing organisms were sensitive to lysocellin and not able to adapt. Lysocellin had a greater effect in altering the acetate:propionate ratio in barleythan in corn-based diets. Animal responses to ionophores may be affected by interactions with the diet.
Literature Cited AOAC. 1984. Official Methods of Analysis (14thEd.). Asso& tion of OaFicial Analytical Chemists, Arlington, VA. Bogaert, C., L. Gomez, J. P. Jouany, and G. Jeminet. 1989. Effects of the ionophore antibiotics h a l o c i d and c& tionomycin on ruminal fermentation in vitro (RUSlTEC). Anim. Feed Sci. Technol. 271. Bryant, M. P., and L. A. Burkey. 1953. Numbers and some predominant groups of bacteria in the rumen of cows fed different rations. J. Dairy Sci. 36:218. Bryant, M.P.,and I. M.Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446. Carmean, B. R., and D. E. Johnson. 1990. Persistence of monensin-induced changes in methane emissions and ruminal protozoa numbers in cattle. J. Anim. Sci. 68EuppL 11~517 (Abstr.). Craig. W. M.,B. J. Hong, G. A. Broderick, and R. J. Bula. 1984. In vitro inoculum enriched with particle associated microorganisms for determining rates of fiber digestion and
protein degradation J. Dairy Sci. 67:2902. Dennis, S.M., T. G. Nagaraja. and E. E. Bartley. 1981. Effects of lasalocid or monensin on lactate-producing or -using rumen bacteria. J. Anim. Sci. 52418. Galton, M. M., G. K. Morris,and W. T. Martin. 1968. Salmonellae in Foods and Feeds. NatL Communicable Disease Center, Atlanta, GA. Garrett, W. N. 1076. Innuence of rumensin on energy utilization of corn and barley diets. Proc.California Feeders Day Program, Holtville, CA. p 21. GiU, J. L., and H. D. Hafs. 1971. Analysis of repeated measuremente of RnimRdn. J. Adm. S c i 33:331. Goering, H.K., andP. J. Van Soest. 1970. Forage fiber analyses lapparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS,USDA, Washington, DC. Harvey, R.W., J. W. Spears, and D. E. Darden. 1988. Effects of lysocellin on perfomname, ruminal and plasma characteristics of growing cattle. J. Anim. Sci. 66:1036. Henderson, C., C. S.Stewart, and F. V. Nekrep. 1981. The effect of monensin on pure and mixed cultures of rumen bacteria. J. Appl. Bact. 51:159. Hillaire, M. C., J. P. Jouany, C. Gaboyard, and G. Jeminet. 1989. In vitro study of the effect of different ionophore antibiotics and certain derivatives on rumen fermentation and on protein nitrogen degradation. Reprod. Nutr. Dev. 29:247. Horton, G.M.J., K. A. Bassendowski, and E. H. Keeler. 1980. Digestion and metabolism in lambs and steers fed monensin with different levels of barley. J. Anim. Sci. 50:997. Horton, G.M.J., M.J. Farmer, K. A. Bassendowski, and G. M. Steacy. 1983. Rumen fermentation and digestibility in steers as influenced by level of intake and monensh Can. J. Anim. Sci. 63:855. Isichei. C. O., and W. G. Bergen. 1980. The effect of monensin on the composition of abomasalnitrogen flow in steers fed grain and silage rations. J. Anim. Sci. 51Guppl. 1):371 (Abstr.). Mackie, R. I., P. G. Bahrs, and J. J. Therion. 1984. Adaptation of rumen bacteria to sodium and monensin. Can. J. Anim. Sci. 84E~ppL):351(Ab&.). McDougall, E. I. 1948. Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 4399. Morris, F. E., M. E. Branine, M.L. Galyean, M.E. Hubbert, A. S. Freeman, and G. P. Lofgreen. 1990. Effect of rotating monensin plus tylosin and lasalocid on performance, ruminal fermentation, and site and extent of digestion in feedlot cattle. J. Anim. Sci. 68:3069. Okuda, H., S.Fuji, and Y.Kawashima. 1965. A direct colorimetric method for blood ammonia. Tokushima J. Exp. Med 12: 11.
PQOS,M.I., T. L. Hanson, and T. J. Klopfenstein. 1979. Monensin effects on diet digestibility, ruminalprotein bypass and microbial protein synthesis. J. Anim. Sci. 48:1516. Preston, Ft. L., Ft. H. Pritchard, and G. W. Wolfrom. 1985. Lysocellin effects on the grain, feed intake and efficiency of g r o w i n g - f i h h g cattle. J. Anim. Sci. Bl(Supp1. 1):493 (Abstr.). Richardson, L. F., A. P. Raun,E. L. Potter, C. 0. Cooley, and R. P. Rathmacher. 1976. Effect of monensin on rumen fermentation in vitro and in vivo. J. Anim. Sci. 43:657. Rogoaa. M.,J. A. Mitchell, and R. FL. Wiseman. 1951. A selective medium for the isolation and enumeration of oral and fecal lactobacilli. J. Bacteriol. 62:132. Russell, J. B. and H. J. Strobel. 1988. Effects of additives on in vitro ruminal fermentation A comparison of monensin and bacitracin, another gram-positive antibiotic. J. Anim. ScL 66:552. SAS. 1985. SAS User’s Guide Statistics. SAS Inst., Inc., G u y , NC. Scheffe, H. 1953. A method for judging all contrasts in the analysis of variaace. Biometrika 40:87. Schelling, G. T. 1984. Monensin mode of action in the rumen. J.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
of lysocellin increased ammonia N concentre tions, suggesting an increase in protein degrade tion and(or1a reduction in ammonia utilization by bacteria, but lysocellin had no effect on ammonia N concentrations in continuous culture (Table 41. Bacterial N synthesis (grams per kilogram of OM truly fermented) also was lower in continuous cultures treated with lysocellin, which agrees with data from Isichei and Bergen (1980) with monensin and Bogaert et al. (1989) with lasalocid. Lack of a decrease in the total anaerobic bacteria in continuous cultures treated with lysocellin compared to controls is taken as evidence that a greater proportion of the total bacteria grew in lysocellintreated cultures. The presence of lysocellin may have an influence similar to that of low pH, namely increasing the proportion-of bacteria that grow in nonselective medium (Slyter et al., 19661. Thus, bacterial protein would be better predicted by other methods (e.@;., the purine assay) than by bacterial count from nonselective medium.
287
288
KUNG ET AL. Slyter, L. L., and P. A. Putnam. 1987. In vivo vs. in vitro continuous culture of rumhid microbial populations. J. Anim. SCi. 261421. Spears, J. W., J. C. Burns, and G. W. Wolfrom. 1989. Lysocellin effects on growth performance, ruminal fermentation, nutrient digestibility and nitrogen metabolism in steers fed forage diets. J. Anim. Sci. 67:547. Thivend, P., and J. P. Jouany. 1983. Effect of lasalocid sodium on rumen fermentation and digestion in sheep. Reprod. Nutr. Dev. 23:817. Wolfrom, G. W., D. R Bright, P. J. Schalburg, and A. Clingerman. 1883. In vitro and in vivo effects of a new polyether antibiotic aysocellin) to enhance the feedlot performance of ruminants. In: Proc. 17th Conf. on Rumen Function.p l CAbstr.1. Chicago, IL. Zinn, R. A. and F.N.Owens.1886. Rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66:157.
Downloaded from https://academic.oup.com/jas/article-abstract/70/1/281/4631891 by Iowa State University user on 21 January 2019
Anim. Sci. 58:1518. Simpson, M. E., 1978. Effects of certain antibiotics on in vitro cellulose digestibility and volatile fatty acid 0 production by ruminal microorganisms. J. Anim. Sci. 47Suppl. 1): 439 (Abstr.1. Slyter, L. L., M. P. Bryant, and M. J. Wolin. 1866. Effect of pH on population and fermentation in a continuously cultured rumen ecosystem. Appl. Microbiol. 14:573. Slyter, L. L., D. L. Kern,and J. M. Weaver. 1976. Effect of pH on ruminal lactic acid utilization and accumulation in vitro. J. Anim. Sci. 43:333 (Abstr.). Slyter, L. L., W. 0. Nelson, andM. J. Wolin. 1864. Modification of a device for maintenance of the rumen microbial population in continuous culture. Appl. Microbiol. 12:374. Slyter, L. L., R. R. Oltjen, D. L. Kern, and F. C. Blank. 1970. Influence of type and level of grain and diethylstilbesterol on the rumen microbial populations of steers fed all-concentrate diets. J. Anim. Sci. 31:OOB.
