Short communication
The effects of feeding monensin and yeast culture,
alone or in combination, on the concentration and
generic composition of rumen protozoa in steers
fed on low-quality pasture supplemented with
increasing levels of concentrate
L.C. Arakaki
a, R.C. Stahringer
b, J.E. Garrett
c, B.A. Dehority
d,*aInstituto de Patobiologia, INTA-CICVyA, CC77, (1708), Moron, Argentina bEEA-INTA, Colonia Benitez, Chaco, Argentina
cDiamond V Mills, Cedar Rapids, IA 52407, USA
dDepartment of Animal Sciences, Ohio Agricultural Research and Development Center,
The Ohio State University, Wooster, OH 44691-4096, USA
Received 7 May 1999; received in revised form 6 January 2000; accepted 26 January 2000
Abstract
Sixteen zebu-cross steers, kept under grazing conditions, were supplemented with increasing levels of concentrate (0.4, 0.7 and 1.0% of body weight) over three periods of 41, 41 and 42 days, respectively. The animals were divided into four groups, a control plus groups supplemented with either monensin, yeast culture or a combination of monensin and yeast culture. Samples of rumen contents were taken on Day 0 and at the end of each period to determine the concentration and generic composition of rumen protozoa. After 124 days on treatment, protozoal concentrations increased in the controls (p<0.01), yeast culture treatment (p<0.03) and monensin treatment (p<0.06). However, protozoal concentrations did not change in the steers fed the combination of monensinyeast culture. No marked differences were observed in the generic composition of protozoa among the four treatments.Entodiniumwas the predominant genus in all groups: control (87.7%); monensin (78.1%); yeast culture (69.6%) and the combination of both, (89.3%). The lower percentage ofEntodiniumin the monensin and yeast culture treatments was primarily replaced by an increase in the percentage ofDasytricha. The rest of the ciliate population, which belonged to9 genera, was observed in similar
proportions for all treatment groups.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Ionophores; Monensin; Protozoa; Rumen; Yeast culture 84 (2000) 121±127
*Corresponding author. Tel.:1-330263-3909; fax:1-330-263-3949.
E-mail address: dehority.1@osu.edu (B.A. Dehority)
1. Introduction
Protozoa represent the second-most abundant population of micro-organisms within the rumen. They are present in almost all domestic ruminants; however, they can differ in both, the species present and generic distribution, depending on the type of feed and geographical location (Dehority, 1979; Dehority and Orpin, 1988). Protozoa are predatory upon the rumen bacteria and have strong proteolytic activity. Although the protozoa are not essential in the rumen fermentation, many of them participate in ®ber digestion, help avoid abrupt drops in ruminal pH with high concentrate diets and although the biological value of protozoal and bacterial protein are similar, digestibility of protozoal protein is signi®cantly higher (Hungate, 1966; Dehority, 1986). However, recent studies indicate that the amount of protozoal protein passing on down the intestinal tract may be limited (Ankrah et al., 1990). When the substrate is not limiting, the protozoa might also increase bacterial growth rate by increasing the `dilution rate' of the bacterial population through ingestion and degradation of the bacteria (Dehority, 1986). Strategies suggested to manipulate the rumen fermentation and improve diet digestibility generally involve some type of modi®cation or alteration of the rumen protozoal population. At the present time, both yeast culture and monensin appear to be capable of modifying the rumen fermentation and microbial composition. Monensin, an ionophore antibiotic, improves energetic ef®ciency in beef cattle (Spears and Harvey, 1984) as well as reduces the severity of bloat in grazing cattle (Katz et al., 1986). It has also been reported to have a selective inhibitory effect on Gram-positive bacteria (Van Nevel and Demeyer, 1988) and toxic effects on rumen ciliates (Hino, 1981; Thivend and Jouany, 1983). In contrast, yeast culture has been shown to improve and stabilize fermentation patterns as well as to stimulate bacterial growth (Wiedmeier et al., 1987; Dawson et al., 1990; Mathieu et al., 1996). Spedding (1990) reported an increase in protozoal concentrations when yeast culture was fed, while Mathieu et al. (1996) did not observe any signi®cant effect. However, Mathieu et al. (1996) suggested that there was a tendency for both,Epidinium and total protozoal concentrations to increase. Since it is known that diet can affect the protozoal population (Hungate, 1966; Dehority, 1986), the response of protozoa to feed additives may vary with the animal's diet.
The present study had two main objectives. First, to determine the effect of yeast culture and monensin, both alone and in combination, on rumen protozoal concentration and generic composition and, second, the effect of varying concentrate levels upon this response.
2. Materials and methods
2.1. Animals and feed
divided into three periods of 41, 41 and 42 days each. The steers were allowed to feed on a pasture of Dicantium caricosum (common name in Argentina is Dicantio; in other regions, it is called Nadi bluegrass) and supplemented with increasing amounts of concentrate in each successive period, i.e. 0.4, 0.7 and 1% of the body weight, respectively. The concentrate consisted of 80% rice polishings and 20% cotton seed meal. Composition of the dietary ingredients is listed in Table 1. Pastures were sampled four times spread over the entire experimental period by randomly cutting small 0.5 m0.5 m areas at soil level with a scissors. Approximately 20±30 samples were taken from each plot.
All steers were allowed to graze standing pastures, but were divided into four treatment groups. Each group was fed an allotment of concentrate daily, with the level of concentrate increasing for each period as listed above. The control group received no feed additives, the monensin group received 200 mg monensin per head per day, the yeast culture group received 30 g yeast culture per head per day and the monensin plus yeast culture group received 200 mg monensin and 30 g yeast culture per head per day. The additives were added to the concentrate and fed at the same level across all three periods. During the ®rst three-to-four days in Period 1, the steers were fed concentrate at 0.2% of body weight in order to facilitate their adaptation to the new diet. The amount of concentrate was then increased daily until the assigned level, 0.4%, was being consumed at the end of the ®rst week. The diets were well accepted by the steers and no adverse effects on consumption were observed.
The yeast culture, Diamond V `XP' yeast culture (Diamond V Mills, P.O. Box 74570, Cedar Rapids, IA), contained dehydratedSaccharomyces cerevisiaegrown on a media of ground yellow corn, hominy feed, corn gluten feed, wheat middlings, rye middlings, diastatic malt, corn syrup and cane molasses. The product is guaranteed to contain not <12% crude protein and 3% crude fat, and not >6.5% crude ®ber.
2.2. Rumen sampling
Samples of rumen contents were collected through an esophageal cannula 3-to-4 h after concentrate feeding, and ®ltered through two layers of gauze. Equal parts of rumen ¯uid and a saline±formalin solution (0.85% NaCl in 20% formalin solution) were mixed and stored for protozoa counting and characterization. Prior to the analyses, a 2 ml aliquot of the ®xed rumen sample was stained with 2 ml of methyl green±formalin
Table 1
Percentage composition of the different dietary ingredients on a dry matter basis
CPa NDFb ADFc LIGd
Cotton seed meal 21.8 34.6 22.9 7.4
Rice bran 8.3 13.5 12.3 3.8
Dicanthium caricosum 1.9 65.7 45.5 6.5
solution (Ogimoto and Imai, 1981). Protozoal concentrations and generic composition were determined using a 1-ml counting chamber (Hausser Scienti®c Partnership, Cat. No. 3800), following the procedures described by Dehority (1993).
2.3. Statistical analysis
Responses were analyzed by two-way, (treatment and time), ANOVA using GLM of SAS (1987). Time effect (which is confounded with the concentrate effect) was considered as a repeated measure, using the Greenhouse and Geisser corrections (Greenhouse and Geisser, 1959) for the signi®cance level. Post-ANOVA contrasts were made to test the signi®cance of the total protozoa increase. Comparison of the generic protozoa composition between groups were made by the non-parametric analysis of variance or the Kruskal±Wallis test, (Conever, 1980).
3. Results
The mean rumen protozoal concentrations determined in the steers on Day 0 and the last day of each feeding period, are presented in Table 2. Over the entire 124-day period, an increase in protozoal concentrations was observed in the control steers (p<0.01), steers supplemented with yeast culture (p<0.03) and steers supplemented with monensin (p<0.06). Although a marked increase in protozoal concentration was observed during the ®rst feeding period, between days 0 and 41, when yeast culture was fed, it was not signi®cant (p>0.15). In contrast, the increase in protozoal concentration in the monensin group occurred almost entirely in Period 2, between days 41 and 84 (p<0.02).The control group tended to increase during both the periods, i.e. between days 0 and 41 and between days 41 and 82. However, the increase was only signi®cant over the entire 124-day period (p<0.01). Protozoal concentrations remained fairly stable over all 124 days in the yeast culture plus monensin treatment group.
Table 2
Mean rumen protozoal concentrations in steers on pasture plus concentrate, without feed additives or supplemented with either monensin, yeast culture or monensin plus yeast culture
Day sampleda Protozoal concentrations (number105/ml)b
Control Monensin Yeast culture Monensinyeast culture
0 1.2 1.0 1.6 1.7
41 1.7 1.0 2.8 1.7
82 2.9 2.5 2.8 1.7
124 3.3 2.4 3.3 1.8
(p<0.01)c (p<0.06)c (p<0.03)c NS
aLevel of concentrate fed daily as percent of body weight was 0.4% between days 0±41, 0.7% between days
41±82 and 1.0% between days 82±124.
bStandard error of the means (SEM)0.5.
Generic distribution of protozoa in the four treatment groups after 124 days on experiment is shown in Table 3. Twelve genera were observed, with a fairly similar occurrence in all treatment groups.Entodinium percentages were higher in the control and monensin plus yeast culture groups (p<0.05). The lower percentage ofEntodiniumin the monensin and yeast culture treatment groups was primarily balanced by an increase (p<0.05) inDasytricha.
4. Discussion
The increase in protozoal concentration in the control group can probably be explained on the basis of an increase in the level of concentrates in the diet. Feeding 0.7 or 1.0% of body weight corresponds to 30 up to 50% concentrate in the diet, which normally results in a marked increase in protozoal concentrations (Dehority and Orpin, 1988). Protozoal concentrations in the monensin group appeared to follow a somewhat similar pattern, i.e. responding to higher concentrate levels. Although concentrations were somewhat lower, they did not differ from the control group on days 41, 82 or 124. Monensin did not appear to have a toxic effect as previously reported by Hino (1981) and Thivend and Jouany (1983). The present data would appear to support the study by Dennis et al. (1986) which suggested that inhibition by ionophore antibiotics is only transient and that continued feeding of ionophores could result in the selection of a resistant protozoal population, perhaps similar to that seen in Table 3.
The marked increase in protozoal concentration as a result of feeding yeast culture agrees with the results reported by Spedding (1990). A possible explanation might be that
Table 3
Percent generic distribution of rumen protozoa in steers after 124 days on pasture plus concentrate, without feed additives or supplemented with either monensin, yeast culture or monensin plus yeast culture
Genus Percenta
Entodinium 87.7 (79.6±96.0) bb 78.1 (76.2±83.0) c 69.6(56.0±89.0) c 89.3 (87.0±95.0) b
Diplodinium ± ± ± 0.1 (0±0.5)
Eremoplastron 0.2 (0±0.5) 1.0 (0±3.7) 0.4 (0±1.0) 0.4 (0±0.5) Eudiplodinium 1.0 (0±1.5) 1.0 (0±1.8) 1.2 (0±5.0) 0.2 (0±0.7) Ostracodinium 2.2 (1.3±3.2) 2.7 (0.9±5.5) 4.3 (1.0±6.2) 1.2 (0±2.5) Diploplastron 0.8 (0±1.4) 1.6 (0±3.5) 2.1 (1.0±3.4) 1.4 (0±5.0) Metadinium 1.2 (0.5±1.8) 0.7 (0±2.0) 0.8 (0±3.0) 0.7 (0±1.8)
Enoploplastron ± ± 0.2 (0±1.0) ±
Elytroplastron 1.1 (0±2.5) ± 0.6 (0±2.0) 0.5 (0±1.3) Epidinium 2.1 (0±3.3) 2.9 (0±7.0) 1.8 (1.0±2.5) 1.3 (0±2.1) Isotricha 0.6 (0±1.7) 2.2 (0±5.5) 3.2 (0±6.8) 2.0 (0±3.9) Dasytricha 2.7 (0.7±4.4) b 9.4 (6.0±11.5) c 15.5 (6.0±20.0) c 2.9 (0.7±6.0) b Unidentified 0.4 (0±1.0) 0.4 (0±0.7) 0.3 (0±0.6) 0.2 (0±0.5)
aMean (range).
the addition of yeast culture resulted in an increase in total bacterial numbers, which were then used as both, a protein and energy source by the protozoa (Dehority, 1986; Dehority and Orpin, 1988). In the present study, an increase in the total viable number and amylolytic bacteria with yeast culture supplementation were observed, (unpublished results). Other possible explanations are that yeast culture stabilized the rumen pH, increased dry-matter intake, or both. However, these responses have been variable between studies (Kamalamma Krishnamoorthy and Krishnappa, 1996; Mathieu et al., 1996; Putnam et al., 1997; Roa et al., 1997).
The complete lack of response when monensin and yeast culture were combined was unexpected, since protozoal concentrations increased with either supplement alone. There is no obvious explanation for protozoal concentrations to have remained the same over the entire 124 days. A similar discrepancy between the two supplements alone and in combination was observed with percentages ofEntodiniumandDasytricha.
Mathieu et al. (1996) observed a tendency forEpidiniumto increase with yeast culture supplementation. However, the percentage of this genus in the present study tended to be lower in the yeast culture supplement group, as compared with the control and monensin groups.
In conclusion, results of this work suggest that further studies in which a lower level of concentrate is fed on pasture (0.4% or below) might separate out the effects of yeast culture and monensin. Yeast culture appeared to have a distinct effect on protozoal concentrations in Period 1, when the low level of concentrate was fed.
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