The bacterial population adherent to plant particles in the rumen of reindeer fed lichen , timothy hay or silage

Male reindeer (Rangifer tarandus tarandus) calves taken from a narural winter pasture were given ad lib. access to lichen (» = 3), timothy silage (» = 3) and hay (« = 3) for 7 weeks. Median numbers of viable anaerobic bacteria adherent to the plant particles (cells/g wet weight of rumen solids), growing on a habitat simulating medium (M8V), were significantly higher (P = 0.05) in the rumen of reindeer fed lichen (26.5 x 10'53.0 x 10') and hay (4.0 x 10'40.5 x 10'), compared to reindeer fed silage (1.15 x 10' 3.25 x 10'). Anaerobic bacterial strains (w = 551) from the plant particles obtained from the rumen of the nine reindeer examined, were isolated using an acid swollen cellulose medium (M8SC) and tested for their ability to hydrolyse carboxymethyl cellulose (CMC). The proportion of CMC hydrolysing adherent bacteria isolated from M8SC was significantly higher in reindeer fed hay (21.5%) compared ro animals fed lichen (5.3%) and silage (2.7%) (P = 0.05). The CMC hydrolysing bacterial srrains (»=42) isolated from reindeer fed hay where characterised as non-cellulolytic Butyrivibrio fibrisohens (9.5%), cellulolytic B. fibrisohens (50.0%), Clostridium sp. (2.4%) and unknowns (38.1%), while CMC hydrolysing strains (»=11) isolated from animals fed lichen and strains (»=4) isolated from animals fed silage where all characterised as B. fibrisohens. None of the bacterial strains isolated from the rumen solids of reindeer fed lichen or silage were found to be cellulolytic. This study suggests that both lichen and timothy silage have a negative influence, compared to hay, on the numbers of cellulolytic bacteria adherent to the plant particles in the rumen of reindeer.


Introduction
Semi-domesticated reindeer {Rangifer tarandus tarandus) in northern Norway are intermediate, mixed feeders (Hofmann, 1985), exposed to large seasonal changes in vegetation types, climate and day length.High quality vascular plants are selected in summer, while lichens, primarily Cladonia sp., may constitute a large proportion of the winter diet.Lichen is mainly composed of hemicellulose, but also contain lichenin (lichen starch), which is in [3-1,4 and 0-1,3 linkages (Hale, 1961;Culberson, 1969).Compared to grass, lichens like Cladonia stellaris contains very little cellulose, constituting only 1.7% of its dry matter (DM) (Person et al., 1980).Dehority (1975) isolated bacterial strains (#=21) from the rumen fluid of an Alaskan reindeer fed dried lichen, but these isolates were unable to digest cellulose.Likewise, Aagnes et al. (1995) reported that bacterial strains (# = 90) isolated from the rumen fluid from two lichen-fed reindeer were unable to hydrolyse carboxymethyl cellulose (CMC).Hence, prolonged feeding on a pure lichen diet seems to have a negative influence on the numbers of cellulolytic bacteria in the reindeer rumen.Rumen fluid of a lichen-fed reindeer may therefore also express less ability to digest cellulose compared to rumen fluid from a reindeer fed a grass diet.In winter, crusts of snow and ice often cover the pasture, exposing the reindeer for periods of starvation, during which supplementary feeding can prevent loss of animals.Different qualities of grass silage have been evaluated as food for reindeer (Aagnes & Mathiesen, 1995;Olsen et al, 1995;Aagnes et al, 1996;Moen et al. 1998), revealing pronounced morphophysiological limitations towards digestion of fibrous timothy (Phleurn pra tense) silage (Aagnes et al., 1996).Reindeet fed leaf rich silage made of Phleurn pratense, Agrostis tenuis and Poa spp., demonstrated low in vivo apparent digestibility coefficients (62-66%) and in vitro dry matter digestibility (IVDMD) of the silage (only 67-71% after 72 h incubation in rumen fluid), with correspondingly low ruminal VFA concentrations (38-53 mM) and high pH (6.95-7.17),indicating a limitation in the ability of the reindeer and its microbiota to utilise this grass silage (Aagnes & Mathiesen, 1995).Depressed ability of the rumen microbiota to digest cellulose when fed grass silage (Olsen et al.. 1995) may have been caused by natural periods of starvation prior to the feeding experiment.Total numbers of rumen bacteria decrease dramatically during starvation and the composition of the bacterial population is changed (Mathiesen et al., 1984;Aagnes et al., 1995).Such changes in the ruminal ecosystem may reduce numbers of one or several of the synergistic bacteria acting together on fibre digestion, causing problems in adjusting to a new diet when re-fed.The unusual structure and chemistry of the lichen may also influence the composition of the rumen microbiota and its ability to digest timothy silage.Both the physical and chemical properties of the grass are changed during silage-making, when soluble carbohydrates are fermented to organic acids and nitrogenous compounds.Grass silage may therefore also in itself effect the bacterial population in the reindeer rumen in a different way than hay, even when harvested from the same crop.Studies of the bacterial population in the rumen fluid of reindeer fed two different qualities of timothy silage, however, revealed no correlation between the composition of the bacterial strains isolated and the ability of the rumen fluid to ferment cellulose in vitro (Olsen 56 et al., 1997).Cellulolytic bacteria were isolated from the tumen fluid of only one of the twelve silage-fed reindeer investigated (Olsen et al, 1997).Cellulolytic bacterial species are, however, found in close association with the plant material in the rumen (Cheng et al, 1977;Akin & Barton, 1983).Consequently, the present study was undertaken to investigate the bacterial population closely associated or adherent to the plant particles in the reindeer rumen, and how this population is influenced by a pure lichen diet, and by timothy hay and silage hatvested from the same crop.

Animals
Nine male reindeer calves (age 6 months, body mass (BM) 43.5 -54.5 kg) were rounded up in November on a natural early winter pasture in northern Norway (68°N, 17°E) and brought to the Department of Arctic Biology at Tromsø.Three of the calves were given ad lib.access to lichen (47 days), and compared with three calves fed regrowth timothy with 79% leaves preserved as hay (50 days) and three calves fed regrowth timothy from the same crop, but preserved as silage (50 days) (Øksendal, 1994;Moen et al., 1998).The chemical composition of the diets are presented in Table 1.Routine treatment against parasites were administered to all animals (Aagnes et al., 1996).To allow expression of their normal seasonal appetite (Ryg & Jacobsen, 1982;Larsen et al.. 1985), all the reindeet calves were exposed to natural photo period in temperature (4 °C) regulated rooms.The animals were slaughtered at their regular feeding-time.The gastrointestinal tract was removed allowing incisions through the rumen wall for sampling of contents within 15 minutes of death.

Viable-cell counts
Numbers of anaerobic bacteria adhering to and living close to the rumen plant particles of reindeer fed lichen, timothy hay and silage were estimated according to Aagnes et al. (1995).Rumen solids were obtained by removing all rumen fluid through two layets of muslin.The dry rumen solids (2.5 g) were suspended for 30 min at room temperature in a M8 buffer solution (47.5 ml) (Olsen et al, 1994) containing 0.1% (w/v) methyl cellulose (catalogue no.M 0262; Sigma) to detach the adherent bacteria from the food particles (Kudo et al, 1987).The solution with the rumen solids were homogenised in cortex tubes using a Polytron PT 10 OD homogenizer (Kinematica, GMB, Luzern, Schweiz) for 30-60 sec at a speed setting of 2. The homogenised rumen solids were diluted serially in ten fold steps in M8 buffer (Olsen et al, 1994) in Hungate anaerobic culture tubes (catalogue no.2047/16-125; Bellco, Vineland; N.J.), by techniques of Hungate (1950).Cultures of bacteria present at dilution's of 10 '' to 10"' were made viable by inoculating 1 ml from each dilution in five parallels in petri-dishes in an anaerobic glovebox (COY Laboratory products, Inc., Ann Arbor, Michigan, U.S.A.) maintained with an atmosphere in the box of N2, CO,, and H2, and a palladium catalyst present to remove O,.Melted anaerobic rumen culture medium (M8V) or M8SC medium were added to the petri-dishes and mixed thoroughly with the inoculum.Viable count determinations were made in an anaerobic rumen culture medium (M8V) (Orpin et al, 1985) designated to support the growth of a wide range of bacterial species based on a M8 anaerobic buffer (Olsen et al, 1994).The M8V medium in addition to M8 buffer contained the carbohydrates: glucose, sucrose, maltose, cellobiose, starch, pectin and xylan, each at a concentration of 0.2% (w/v).It was solidified using 2.0% (w/v) agar.Vitamins (10.0 ml per 1 medium) (Roche et al., 1973) were sterile-filtered through a Millex R-GS single use filter unit (0.22 um) (Millipore S.A., Molsheim, France) and added to the medium before use.M8SC medium were used to select for and isolate the cellulolytic bacteria.The preparation of the M8SC medium was similar to that of the M8V medium in all respects, except that the carbohydrates were replaced by acid Rangifer, 18 (2), 1998 swollen cellulose (0.2% v/v) (Schellhorn & Forsberg, 1984).Petri-dishes containing inoculated M8V or M8SC were incubated at 39 °C in anaerobic jars, gassed with C02 inside the anaerobic chamber for 48 h and 5-6 days, respectively.

Isolation of bacteria
A sector comprising 1/8 or 1/16 (depending on the density of viable bacteria) of the area of the petridish with the inoculated M8SC was marked, and all the bacterial colonies present within this sector were selected using sterile glass Pasteur pipettes.All the bacterial colonies within the sector was isolated on 4-5 of the parallels of M8SC containing the 10 7 or 10 s dilution's.The bacteria were isolated and purified by sneaking and plating repeatedly onto M8SC medium in new petri-dishes at 48 h intervals in the anaerobic chamber.Most of the bacterial strains were pure when first isolated, but some isolates required several transfers before pure.The bacterial strains were subsequently plated on M8 buffer containing 0.2% (w/v) cellobiose and 0.1% (w/v) low-viscosity carboxy methyl cellulose (CMC), incubated for 24 h at 39 °C, before staining the M8-CMC plates with Congo red (Teather & Wood, 1982).CMC-hydrolysing isolates showed a zone of clearing, unstained by Congo red, around the colony, and were transferred to individual Hungate tubes containing a slope of M8SC, incubated for 24¬ 48 h and stored at -80 °C until analysis.

Identification of bacteria
Strains positive for the Congo red stain test, were identified by standard microbiological techniques (Holt etal, 1994;Krieg & Holt, 1984;Ogimoto & Imai, 1981;Sneath et al., 1986), by examination of morphology and motility in liquid M8C (M8 buffer added 0.2% (w/v) cellobiose) or M8G (M8 buffer added 0.2% (w/v) glucose), by Gram staining and identification of acidic fermentation products after growth in the same liquid media, and by spore formation.The concentrations of volatile fatty acids, lactate and succinate were determined by gas liquid chromatography (S0rmo et al, 1994).Bacterial isolates were grown (24 h) in Hungate-tubes containing 9 ml liquid M8-medium added 0.2% (w/v) cellobiose and for strains not willing to grow on cellobiose, 0.2% glucose (w/v) was used instead.One ml of this culture was inoculated into another 9 ml liquid M8-media with the same carbohydrate added as in the previous growth tubes for an additional 24 h incubation period under standardised conditions at 39 °C.Fermentation products were determined after acidification of the liquid phase (S0rmo et al. 1994).Aerobic gtowth was tested on Nutrient agat (Difco Laboratories, Detroit, Michigan, U.S.A.) containing 0.5% (w/v) glucose at 39 °C.

Statistical methods
Colonies of viable bacteria growing on M8V and M8SC media were counted and results presented as median (range) number of bacteria per gram wet weight of rumen solids.Median numbers of viable bacterial cells in the different groups of reindeer fed different diets were compared by the Wilcoxen rank-sum test for comparison of two treatments (Johnson & Bhattacharyya, 1992).The null hypothesis was rejected at P >0.05.

Discussion
Fibre-digesting bacteria interact and coexist with non-fibrolytic bacteria in the rumen ecosystem, and this synergistic interaction among the bacterial strains contributes to increased fibre digestion compared to the individual strains when grown in monocultures (Scheifinger & Wolin, 1973;Latham & Wolin, 1977;Fondevila & Dehority, 1994).The composition of the symbiotic bacterial population of the bovine rumen is influenced by the diet eaten by the host (Hungate, 1966).Starvation is also known to both change the composition and to reduce the numbers of bacteria in the reindeer rumen (Mathiesen et al, 1984;Aagnes et al., 1995).A natural lichen diet combined with periods of starvation during winter may effect the ruminal microbial ecosystem of the reindeer in such a way that when fed silage or hay, the rumen microbiota is less capable of cellulose digestion.Lichen is very low in protein (Table 1), and even though reindeer have a very efficient recycling of urea (Hove & Jacobsen, 1975) reindeer fed lichen alone will be in a negative nitrogen (N) balance (Jacobsen & Skjenneberg, 1975).Microbial synthesis may be reduced at very low levels of N. Aagnes et al. (1995) characterised the rumen fluid bacterial population from two adult female reindeer with ad lib.access to lichen, finding strains of Streptococcus and Clostridium to be the most dominant.The low cellulose content of the lichen (Person et al, 1980;Øksendal, 1994: Table 4) may influence the composition of the rumen microbiota.In fact, Dehority (1975) and Aagnes et al. (1995) report that no cellulolytic bacterial strains were isolated from the rumen fluid of reindeer fed lichen.In the current study the bacterial population adherent to plant particles in reindeer calves (#=3) fed lichen were investigated.Total numbers of viable adherent bacteria (g wet weight of rumen solids) growing on M8V medium was higher in reindeer calves examined in this paper (Table 2) , than in adult reindeer (n = 2) (21.0 x 10 s -38.0 x 10 8 ) fed lichen (Aagnes et al, 1995).Only 11 of the bacterial strains (# = 209) isolated from the rumen solids from lichen-fed reindeer using an acid swollen cellulose medium were CMC active, but none of these were found to be cellulolytic (Table 2-3).
Similarly, cellulolytic bacteria appeared to be absent or depressed in Orkney sheep eating a diet of seaweed low in cellulose (Orpin et al, 1995b).The CMCactive strains isolated from lichen-fed reindeer were gram negative, motile, curved rods producing butyric acid as their main fermentation product (Table 3) , and they were characterised as B. fibrisolvens (Bryant & Small, 1956).Dehority (1975) also isolated Butyrivibrio-like strains from the rumen fluid of reindeer fed lichen, but Aagnes et al. (1995) report no such findings.Strains of B. fibrisolvens have been found to solubilize hemicellulose more extensively than cellulose (Dehority & Scott, 1967; Table 4. Dry matter (DM) food intake, reticulo-rumen wet weight relative to body mass (BM), ruminal pH, rotal concentration of volatile fatty acids (VFA), NH4-N content and the in vitro DM digestibility (% IVDMD) of pure cellulose (Whatman filterpaper no. 1) after 48 h incubation in rumen fluid from reindeer calves fed lichen (Cladonia stellaris), timothy (Phleumpratense) hay or silage.Values are given as ranges, » = 3 in each group.

Intake
Wet weight pH VFA NH4-N % IVDMD (g/kg BM/day) (% of BM) (mM) (mg/1) of cellulose fraction of Cladonia stellaris has not been characterised.Hemicellulose are complex plant polysaccharides with (3-1,4-linked xylose residues in the main core polymer (xylan) as a common factor, but with branches containing a variety of other glucosidic linkages (Van Soest, 1994).Degradation of hemicellulose therefore requires different enzymes capable of cleaving a variety of linkages.The similarities of P-l,4-xylosidic linkages of the hemicellulose and the P-l,4-glucosidic linkages of the cellulose may perhaps explain why many of the cellulolytic strains isolated from the rumen all seem able to degrade hemicellulose as well as cellulose (Dehorithy, 1965;Coen & Dehority, 1970;Kock & Kistner, 1969;Morris & van Gylswyk, 1980).A low intake of cellulose in lichen-fed reindeer may have suppressed the ability of the fibre-digesting bacteria to ferment cellulose, while the high intake of hemicellulose might have stimulated the production of enzymes cleaving hemicellulose.Hence, the lichen seems to have a negative influence on the numbers of cellulolytic bacteria present both in dilutions of the rumen fluid (Dehority, 1975;Aagnes et al, 1995) and on the rumen solids (Table 2-3) in reindeer.Rumen fluid from the lichen-fed calves (animal 1-3) were allowed to ferment cellulose in vitro, and did not demonstrate a depressed cellulolytic activity (Øksendal, 1994: Table 4).This may be explained by the fact that the rumen fluid used in the digestibility trial was not diluted, hence cellulolytic microorganisms present in low concentrations may have been stimulated by the presence of the substrate resulting in a digestion of cellulose during the 48 h incubation period.
Grazers like cattle are known to have a higher DM intake of hay compared to silage prepared from the same crop (Campling, 1966;Uden, 1984).Recently this has also been demonstrated for reindeer (Moen et al, 1998;Table 4).Total concentrations of VFA in the rumen fluid was significantly lower and ruminal pH significantly higher in reindeer fed silage compared to hay, indicating a higher fermentation activity in the rumen of reindeer fed hay (Moen et al, 1998;Table 4).Although the DM intake of hay is considerably higher than that of silage, this does not seem to have effected the rumen size of the animals fed the two different diets (Moen et al, 1998;Table 4).Ruminal clearance rate is limited by plant particle density and size, which is influenced by mastication and microbial digestion (Lechner-Doll et al, 1991).Rough timothy silage Rangifer, 18 (2), 1998 with a high fibre contents and stem/leaf ratio seems to accumulate in the reindeer rumen, and the capacity of the reticulo-rumen increases to compensate for this delay in digestion (Aagnes et al, 1996).Even when fed silage prepared from leafy timothy an individual reindeer developed a large rumen (Olsen et al, 1995).Rumen fluid from this animal demonstrated a delayed cellulolysis in vitro compared to animals that did not develop a large rumen (Olsen et al, 1995).
The composition of the cellulolytic bacterial population adherent to the rumen particles in reindeer fed grass has not been examined earlier.The cellulolytic bacterial population in rumen contents from semi-domesticated reindeer in the Scottish highland ranged from 0.05-2.0% of the total viable population (Hobson et al, 1976), resembling that of domestic ruminants like sheep and cow (van Gylswyk & Schwartz, 1984).Orpin et al. (1985) found the cellulolytic bacterial population in the rumen fluid of the high-Arctic Svalbard reindeer (Rangifer t. platyrhynchus) to constitute as much as 14.6 and 35.0% of the total viable population in summer and winter, respectively.The four major cellulolytic bacterial species Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes and B. fibrisolvens were all isolated from Svalbard reindeer on natural pastures (Orpin et al, 1985).The same isolation technique was used in the present study, but bacterial isolations were made using an acid swollen cellulose medium and only CMC-hydrolysing bacteria were characterised.Numbers of bacteria adherent to the rumen solids obtained from the individual animals were generally higher on the M8SC medium which was incubated for 6-7 days, compared to the numbers on M8V medium incubated for 48 h (Table 2).The high total concentration of carbohydrates in the M8V medium allowed a very rapid growth of some of the bacterial strains, resulting in very large colonies after a 48 h incubation period, and the slow-growing strains may therefore have escaped counting on this medium.None of the CMC hydrolysing bacteria isolated from the silagefed reindeer were found to be cellulolytic, contrasting the fact that cellulolytic B. fibrisolvens were obtained from all the hay-fed calves (Table 3).B. fibrisolvens is generally not considered to be a rapid cellulose digester compared to the other cellulolytic bacterial strains (Hungate, 1966;van Gylswyk & Labuschagne, 1971), but is found to be the most numerous strain in the rumen of the Svalbard reindeer (Orpin et al, 1985).A cellulolytic Clostridium sp. was also isolated from one of the hay-fed animals (Table 3).Cellulolytic members of the genus Clostridium have occasionally been isolated from the bovine rumen, but they appear to be of less importance (Hungate, 1957;Shane, 1969).F. suainogenes does not grow well in cellulose agar medium (Stewart et al., 1981), and since isolations of CMChydrolysing bacteria from reindeer rumen feed particles were made from acid swollen cellulose agar medium, this might have excluded possible strains of this species present.
Growth of pure cultures of cellulolytic strains are inhibited at low pH values, the most resistant strain, B. fibrisolvens, is inhibited at pH 6.1 5 (Russel & Dombrowski, 1980).Re-feeding starved reindeer may induce ruminal acidosis (Bøe et al., 1982;Sletten & Hove, 1990).Water soluble carbohydrates (WSC) may be present in high concentrations in timothy silage, and silage may therefore cause an initial drop in ruminal pH when given to reindeer due to rapid fermentation of the WSC.Such a drop in pH might cause changes in the ruminal cellulolytic bacterial population.The pH recorded in rumen fluid obtained from two adult female reindeer instrumented with rumen fistulas during the initial 47 hours of ad lib.access to leafy timothy silage with (on DM basis) 30% WSC, following a 7 weeks period of ad lib.access to lichen and 4 days of starvation, revealed no dramatic drop in pH, the lowest recorded pH being 6.90 and 6.45 in the two animals, respectively (M. A. Olsen & T. H. Aagnes, unpubl. data).Hence, reindeer seem to increase the intake of timothy silage, in such a way that rumen acidosis is prevented duting the initial feeding period.Compared to pelleted rations, structural grass will probably stimulate rumination and hence salivation, buffering the microbial VFA produced in the rumen.The differences in the composition of the adherent bacterial population in reindeer fed silage and hay is therefore unlikely to be explained by ruminal acidosis, more likely the chemical composition of the two different substrates.Addition of energy in the form of molasses, increasing the contents of WSC to 16% DM in the timothy silage, increased DM food intake significantly compared to when fed just a pure timothy silage diet (Moen et al., 1998).Easily fermentable carbohydrates support microbial growth in the rumen and may therefore also increase cell wall digestion.A low content of WSC in the timothy silage (Table 1) may therefore have led to energy deficiency for the rumen microorganisms in these animals.The hay was rich 62 in true protein compared to the timothy silage (Table 1).NH4-N available for microbial synthesis was higher in hay-fed than in silage-fed reindeer (Moen et al.. 1998: Table 4).It is therefore concluded that low ruminal levels of N in reindeer calves fed timothy silage, compared to animals fed hay, seems to suppress ruminal microbial synthesis of cellulolytic bacteria, in particular when intake of easily available energy is low.

Table 2 .
Numbers of viable adherent anaerobic bacteria growing on M8V and M8SC medium and the proportion hydrolysing CMC and cellulose from the rumen of reindeer fed lichen (Cladonia stellaris), timothy (Pbleum pratense) hav or silage.