Factors affecting the concentration and cellulolytic activity of sheep rumen fungi

N E Obispo* and B A Dehority

Department of Animal Sciences

Ohio Agricultural Research and Development Center,

The Ohio State University, Wooster 44691-4096

Dehority.1@osu.edu

* Centro Nacional de Investigaciones Agropecuarias (CENIAP) del INIA, Venezuela.
nobispo@reacciun.ve

Abstract

Fungal concentrations were determined in a sheep fed its daily ration at three different feeding frequencies, once (1x), six (6x) and twenty-four (24x) times a day.  Concomitantly, at each sampling time in vitro cellulolytic activity was measured. 

Fungal numbers were affected by feeding frequency with the highest concentrations occurring with 6x feeding per day.  Cellulose digestion was increased at all feeding frequencies when the insoluble fraction of rumen fluid was added to the basal fermentation medium.  Neither fungal numbers nor feeding frequency were correlated with cellulose digestion in vitro; however, a feeding frequency x medium interaction was observed. 

The results suggest an equilibrium exists in the rumen between an inhibitory factor(s) and growth factor(s) which serves to control fungal concentrations.

Key words:  Feeding frequency, rumen fungi, cellulose digestion, fungal numbers

Introduction

Although it has been determined that the anaerobic fungi are normal inhabitants of the foregut and hindgut of herbivores, their significance to the in vivo fermentation is still uncertain.  Despite the ability of rumen fungi to degrade the structural components of the plant cell wall in vitro, the major portion of such degradation in the rumen is still attributed to several bacterial species (Windhan and Akin 1984).  The extensive digestion of plant material by fungi in vitro (Bauchop 1981, 1989; Orpin and Hart 1980; Theodorou et al 1989) is due mainly to their large array of enzymes which can degrade the major structural components of the plant cell walls (Barichievich and Calza 1990; Lowe et al 1987; Pearce and Bauchop 1985.

Cellulose digestion is increased when the fungi are co-cultured with methanogens or lactate-utilizing bacteria (Joblin et al 1989).  In contrast, inhibition of cellulolysis occurs in co-culture with the cellulolytic bacteria Ruminococcus albus and R. flavefaciens (Irvine and Stewart 1991; Richardson et al 1986).  No inhibition has been observed when the fungi are grown in co-culture with Bacteroides succinogenes (Richardson et al 1986).  Stewart et al (1992) and later Bernalier et al (1993) found an extracellular, thermo-labile protein produced by R. flavefaciens and R. albus, which inhibits cellulase activity of Neocallimastix frontalis.  Inhibition was only observed with an insoluble substrate like cellulose, suggesting the inhibitory mechanism dealt with attachment of the fungi.  More recently, Dehority and Tirabasso (1993) found that mixed rumen bacteria produce a heat stable compound(s) in vitro, which markedly inhibits growth of the rumen fungi.  Similar inhibitory activity was shown to occur in the fluid fraction of rumen contents.  These same authors were later able to demonstrate the presence of a fungal growth factor(s) in the insoluble portion of rumen fluid (Dehority and Tirabasso 1994, unpublished).

It seems probable that these antagonistic effects between the bacteria and fungi must also occur in the rumen, and such interactions could exert regulatory effects on the size and activity of the fungal population.  The present study was undertaken in an attempt to evaluate the potential significance of these inhibitory factors on the growth and cellulolytic activity of the rumen fungi in vivo.  Assuming the inhibitory factor(s) is produced by the bacteria, it was reasoned that feeding frequency, or availability of substrate, should affect its production and concentration.

Material and methods

Animals and diets

A fistulated mature Targhee wether (BW 80 kg) fed pelleted alfalfa (1.5 kg/day) plus vitamins and minerals with free access to water, was used as source of inoculum for this experiment.  During three consecutive 15 d periods (10 d adaptation and 5 d sampling), the daily ration was fed in one of three different feeding frequencies: one (1x), six (6x) and twenty-four (24x) times a day.  For the 6x and 24x feeding schedules, an individual automatic feeder was used to deliver the correct amount of feed, at the desired intervals.  Samples were collected from several locations in the rumen just prior to 8:00 a.m.,  the normal time of once a day feeding, and composited for the subsequent microbial studies.  

Microbial and in vitro Digestion Assays

Anaerobic cultural techniques were similar to those described by Hungate (1950), as modified by Dehority (1969).  Total and cellulolytic fungal numbers were estimated by the procedure described by Obispo and Dehority (1992). At each sample time, two ml of the 10^-1 dilution were used as inoculum for measuring fungal cellulose digestion in vitro.  The fermentation medium, without rumen fluid, was used either "as is" or with an added growth factor (Dehority and Tirabasso 2000).  To inhibit bacterial growth in the medium, the antibiotics penicillin and streptomycin were added to tubes to give a final concentration of 2,000 and 300 U per ml, respectively.  Five tubes of each fermentation medium were inoculated and one of them was immediately frozen to stop the fermentation process and serve as a control.  The remaining four tubes were incubated at 39°C for 7 days.  After fermentation, fungal numbers were determined in each tube (Obispo and Dehority 1992).  Fungal growth factor was prepared by extracting lyophilized rumen fluid (LRF) powder with 5.0 ml distilled water for one hour on an automatic shaker.  The quantity of LRF to be extracted was chosen so that the growth factor activity added to each fermentation tube was equivalent to the concentration which would be present in 100% rumen fluid.  After centrifugation at 12,000 rpm for 20 min and decanting the supernatant, the remaining pellet was suspended in 4.0 ml distilled water and added to the medium in each tube.  

Chemical Analysis

After incubation in the fermentation tubes, cellulose digestion was determined according to the procedure described by Hiltner and Dehority (1983). 

Statistical Analysis

The MPN number was transformed to Log₁₀ to reduce variation according to Cochran (1950).  The data were tested by analysis of variance in a 3 x 2 factorial design (three feeding frequencies; two fermentation media).  The data were analyzed using the SAS general linear models procedure (SAS 1988).  Least-square means were separated with a protected least-significant-difference test when significant treatment main effects were observed.  Fungal numbers were analyzed by ANOVA with mean separation by Duncan’s Multiple Range test.

Results and Discussion

Concentrations of anaerobic fungi (fungi g^-1 of rumen contents) were found to vary with feeding frequency (P < 0.001).  Higher concentrations occurred when the feed was offered six times a day (15.1 x 10⁴) as compared with once (6.4 x 10⁴) or twenty-four (6.2 x 10⁴) times a day.

The digestion of cellulose in vitro was not significantly correlated with either fungal numbers or feeding frequency.  The addition of growth factor(s) to the control medium (Table 1) increased (P < 0.001) the average amount of cellulose digested by two-fold (32.8% as compared with 16.2%).  Cellulose digestion was higher in the growth factor medium for all three feeding frequencies; however, there was a feeding frequency x medium interaction (P < 0.05) and the separate means for these data are shown.  The percent cellulose digestion in growth factor medium inoculated with rumen contents when the sheep was fed 6x was significantly higher than the 1x feeding schedule.

In previous studies (Obispo and Dehority 1992) it was found that rumen fungal concentrations remained more or less constant over a period of 24 h, and numbers did not appear to be influenced by feeding once, twice or three times daily.  Based on reports in the literature, frequent feeding appears to increase the rate of turnover in the rumen (Evans 1981; Robinson and Sniffen 1985; Yang and Varga 1989). Suprisingly though, turnover rates appear fastest around 4x to 6x feeding daily and decrease with a greater number of feedings (Nocek and Braund 1985).  The present data show a marked increase in fungal concentrations at 6x feeding per day which would not necessarily be expected if turnover rate is faster with this feeding schedule. 

Orpin (1977) has shown that an increase in zoospore concentrations generally occurs shortly after feeding, which he associated with the presence of some component(s) of the diet that triggers the release of zoospores from the sporangia.  Feeding every 4 h (6x) may maximize the reproductive cycle of the rumen fungi, i.e., encystment, growth of sporangia and release of zoospores.  Thus, the presence of stimulatory components and release of zoospores become important regulatory factors of the population size of these microorganisms.

On the other hand, it seems possible that an equilibrium between inhibitory and growth factors produced by the bacterial population in the rumen may also exert important regulatory effects on the fungal population and/or their enzymatic activity.  The inhibitory activity in rumen fluid has been shown to be concentration dependent (Dehority and Tirabasso 1993); therefore, rate of growth of the bacterial population in vivo, and concurrent production of inhibitor(s), probably depends on both quantity of feed and feeding frequency.  Superimposed on this would be turnover rate which would serve to dilute the inhibitor.  Thus, if 6x feeding has the most rapid turnover, it might be expected to have the lowest concentration of inhibitor.  No information is available on the optimum concentration of growth factor activity in the rumen fluid.

Thus, at the present time, there is not a ready explanation for the mechanisms involved in the equilibrium, if any, between inhibitory and growth factors in the rumen to support fungal growth and development.  However, a feeding frequency x medium interaction could be an indication that both inhibitory and growth factors influence regulation of fungal development in the rumen.  Further studies, in which inhibitor(s) and growth factor(s) concentrations are assayed in rumen fluid at various feeding frequencies, would appear warranted.

Conclusions

An equilibrium between inhibitory and growth factors produced by rumen bacteria may be a major regulator of the fungal population in the rumen.  Apparent changes in the concentration of these factors in the rumen, as a result of dilution caused by an increased turnover rate, would appear to explain the differences observed at the various feeding frequencies.

Acknowledgements

Salaries and research support were provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center (OARDC), The Ohio State University. Manuscript no. 127-95.

References

Barichievich E and Calza R E 1990  Media carbon induction of extracellular cellulase activities in Neocallimastix frontalis isolate EB188.  Current Microbiol. 20:265-271.

Bauchop T 1981 The anaerobic fungi in fibre digestion. Agric. Environ. 6:339-348.

Bauchop T 1989 Rumen anaerobic fungi of cattle and sheep. Appl.. Environ. Microbiol. 38:148-158.

Bernalier, A, Fonty G, Bonnemoy F and Gouet Ph 1993  Inhibition of cellulolytic activity of Neocallimastix frontalis by Ruminococcus flavefaciens. J. Gen. Microbiol. 139:873-880.

Cochran W G 1950 Estimation of bacterial densities by means of the most probable number.  Biometrics. 6:105-116.

Dehority B A and Tirabasso P A 2000 Antibiosis between ruminal bacteria and rumen fungi. Appl. Environ. Microbiol. 66: 2921-2927

Dehority B A and Tirabasso P A 1993 Antibiosis between rumen bacteria and fungi. Twenty Second Conference on Rumen Function. Abstracts.

Dehority B A 1969  Pectin-fermenting bacteria isolated from the bovine rumen. J. Bacteriol. 99:189-196.

Evans E 1981 An evaluation on the relationships between dietary parameters and the rumen liquid turnover rate.  Can. J. Anim. Sci. 61:91-96.

Hiltner P and Dehority B A 1983  Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.  Appl. Environ. Microbiol. 46:642-648.

Hungate R E 1950  The anaerobic mesophilic cellulolytic bacteria.  Bacteriol. Rev. 14:1.

Irvine H L and Stewart C S 1991 Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii.  Ltrs. in Appl. Microbiol. 12:62-64.

Joblin K N, Campbell G P, Richardson A J and Stewart C S 1989 Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens.  Ltrs. in Appl. Microbiol. 19:195-197.

Lowe S. E, Theodorou M K and Trinci A P J 1987  Cellulases and xylanases of an anaerobic rumen fungus grown on wheat straw, wheat straw hollocellulose, cellulose, and xylan.  Appl. Environ. Microbiol. 53:1216-1223.

Nocek J E and Braund D G 1985  Effect of feeding frequency on diurnal dry matter and water consumption, liquid dilution rate, and milk yield in the first lactation.  J. Dairy Sci. 68:2238-2247.

Obispo N E and Dehority B A 1992  A most probable number method for enumeration of rumen fungi with studies on factors affecting their concentrations in the rumen. J. Microbiol. Methods. 16:259-270.

Orpin C G and Hart Y 1980  Digestion of plant particles by rumen phycomycete fungi. J. Appl. Bacteriol. 49:X.

Orpin C G 1977  On the induction of zoosporogenesis in the phycomycetes Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis.  J. Gen. Microbiol. 101:181-189.

Pearce P D and T Bauchop 1985  Glycosidases of the rumen anaerobic fungi Neocallimastix frontalis grown on cellulosic substrates.  Appl. Environ. Microbiol. 49:1265-1269.

Richardson A J, Stewart C S, Campbell G P, Wilson A B and Joblin K J 1986  Influence of co-culture with rumen bacteria on the lignocelluloytic activity of phycomycetous fungi from the rumen. XIV International Congress of Microbiology, Microbial Ecology Section, 7-13 Sept., Manchester, England. PG2-24, 233. 

Robinson P H and Sniffen C J 1985.  Forestomach and whole tract digestibility for lactating dairy cows as influenced by feeding frequency. J. Dairy Sci. 68:857-867.

SAS/STAT^® 1988  User’s Guide release 6.03. SAS Institute, Inc. Cary, NC.

Stewart C S, Duncan S H, Richardson A J, Backwell C and Begbie R 1992  The inhibition of fungal cellulolysis by cell-free preparations from ruminococci.  FEMS Microbiol. Ltrs. 97:83-88.

Theodorou M K, Longland A C, Dhanoa M G, Lowe S E and Trinci A P 1989 Growth of Neocallimastix sp R1 on Italian rygrass hay: removal of neutral sugars from plant cell walls.  Appl. Environ. Microbiol. 55:1363-1367.

Windhan W R and Akin D E 1984  Rumen fungi and forage fiber degradation.  Appl. Environ. Microbiol. 48:473-476.

Yang C M and Varga C G 1989  Effect of three concentrate feeding frequencies on the rumen protozoa, rumen digesta kinetics, and milk yield in dairy cows.  J. Dairy Sci. 72-950-957.

Received 22 August 2002

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