The effect of supplementing cows with live yeast , Saccharomyces cerevisiae , on ciliate fauna and ruminal fermentation

The infl uence of a live culture of Saccharomyces cerevisiae on the number of ciliates, activity of carboxymethylcellulose(CMC), xylanand starch-degrading enzymes, as well as the concentration of VFA in the rumen of two cows was examined. The animals were fed either hay and concentrate alone or supplemented with live yeast given at the rate of 5 g/d. It was found that the response of rumen fauna to yeast supplementation was related to the animal. The total protozoa and Entodinia counts increased signifi cantly only in one cow (P≤0.05). The number of protozoa from the genus Diplodinium remained unchanged (cow 1) or decreased signifi cantly (cow 2). Epidinia decreased in the rumen of both animals, while Isotrichidae did not change signifi cantly (P>0.05).The activity of CMC-ase and xylan-degrading enzymes varied between 7.2-9.4 and 108.3-130.0 μM released reducing sugars/g DM/h, respectively. The effect of yeast on the activity of fi brolytic enzymes was related to the host animal. CMC-ase decreased signifi cantly only in cow 2, while xylanase, in cow 1. The activities of starch-degrading enzymes as well as pH of rumen fl uid and volatile fatty acids were not affected by supplementation of cows with yeast.


INTRODUCTION
Live yeast have been fed to ruminants for more than 70 years.Nonetheless, their effect on rumen microbiota, especially on protozoa inhabiting in this chamber, is still not well known.The response of rumen fauna and some of the morphological groups of rumen protozoa was studied by Doreau and Jouany, (1998), Jouany et al. (1998), Mwenya et al., (2005) and Galip (2006), but the results were inconsistent 158 S. CEREVISIAE, ON CILIATE FAUNA AND RUMINAL FERMENTATION despite the nutritional and environmental requirements of the examined groups of rumen ciliates differing signifi cantly (Williams and Coleman, 1992).
The effect of yeast on carbohydrate digestion and fermentation in the rumen also has not been well defi ned yet.It has been shown that the supplementation of feed with yeast increased the digestibility of NDF and ADF (Newbold et al., 1995).However, neither Kmet' et al. (1992) nor Brossard et al. (2006) were able to show a positive effect of live yeast on cellululoytic and amylolytic activity in the rumen content.In addition, Koul et al. (1998) and Doležal et al. (2005), observed an increase in the concentration of VFA and in the proportion of acetate.In contrast, Chaucheyras et al. (1996) and Brossard et al. (2006) showed that neither total VFA nor acetate and propionate were infl uenced by enrichment of the diet with yeast.
The objective of this study was to compare the response of ciliates inhabiting the rumen of two cows to the supplementation of the hosts with a live culture of S. cerevisiae.The effect of yeast on the activity of fi brolytic and amylolytic enzymes as well as on pH and concentration of volatile fatty acids in the rumen fl uid of both animals was also examined.

MATERIAL AND METHODS
The experiment was carried out on two rumen-fi stulated Black-and-White Lowland cows.The animals were fed a diet (7.2 kg DM/d) consisting of 87% meadow hay and 13% concentrate.The concentrate was composed of, %: crushed barley 80, rapeseed oilmeal 10, and soyabean oilmeal 10.The supplemental dose of a vitamin-mineral mixture was 50 g/d.The daily ration was divided into two equal parts and fed at 8.00 and 16.00 h.A dose of 5 g of live Saccharomyces cerevisiae CNCM I-1077 (1·10 10 CFU/g), when given, was introduced into the rumen of animals at 8.00 h via the fi stula.Drinking water was available ad libitum.
The period of preliminary feeding of cows lasted for four weeks and was followed by the sampling period.The samples of fl uid and digesta were taken from the rumen just before the morning feeding and at 2, 4 and 8 h thereafter.The sampling was repeated on three consecutive days.The samples of rumen content for enzymatic examination (20 g) were frozen and kept at -20°C, whereas these for protozoa counts (5 g) were fi xed with aqueous formaldehyde (4% W/V).Samples of rumen fl uid for determination of volatile fatty acids (5 ml) were preserved with 0.5 ml of formic acid.To obtain representative samples, the content and fl uid were taken from different places in the rumen.The withdrawn material was thoroughly mixed and the appropriate samples were collected while the remaining material was immediately replaced in the rumen.
The samples of rumen content for enzymatic studies were treated with CCl 4 according to Huhtanen and Khalili (1992) and the obtained preparation was used to determine the activity of carboxymethylcellulose-(CMC), xylan-and starchdegrading enzymes.The reaction mixture consisted of 750 μl of 2% (W/V) substrate dissolved in McIlvaine buffer (pH 6.0), 50 μl of enzyme preparation and 450 ml of the same buffer.It was incubated for 1 h at 40°C and the released reducing sugars were measured using the dinitrosalicilic acid reagent (Miller et al., 1960) and a Beckman DU-64 Spectrophotometer.The concentration of VFA in rumen fl uid was determined by gas chromatography according to Ziołecki and Kwiatkowska (1973) using a Hewlett Packard 5890 apparatus.The pH of rumen fl uid was measured using a Beckman pH-meter.The protozoa were identifi ed and classifi ed according to Dogiel (1927) and counted under a light microscope according to Michałowski et al. (2001).
The statistical calculations were carried by the tests using the Statistica 5.1.software package.The normality of the distribution of variables was tested by the Shapiro-Wilk test.Mean values were calculated and compared by the pairedsamples Student-t test for normal distribution.Variables without normal distribution were elaborated by the Wilcoxon test.

Rumen ciliates
The ciliate fauna of the cows was composed of twenty one species belonging to four genera of the family Ophryoscolecidae as well as three species from the family Isotrichidae.The concentration of ciliates in the rumen of both cows is presented in Table 1.In cow No. 1, the total number of protozoa and Entodinium count were significantly higher when this animal was fed the experimental diet compared with the control.The concentration of ciliates from the remaining groups was not affected by the diet.The total concentration of protozoa as well as the number of Entodinium and Isotrichidae in cow No. 2 remained unchanged when yeast was added to the control diet composed of hay and concentrate.In contrast, the number of Diplodinium and Epidinium decreased signifi cantly.

Carbohydrate digestion
The activity of enzymes involved in the digestion of structural carbohydrates and starch is presented in Table 2.The activity of xylan degrading enzymes was higher when cow No. 1 was fed the control diet compared with the yeast-supplemented diet.The activity of CMC and starch degrading enzymes remained unchanged.The activity of CMC-ase in the rumen of cow No. 2 was signifi cantly lower when this animal was fed the diet supplemented with yeast than when control feed was given.The xylan-and starch-degrading enzymes were not affected by the diet.

Fermentation products
No signifi cant differences were found between pH or between the concentration of total VFA and particular acids in the rumen fl uid of cows fed control and experimental diets (Table 3).

DISCUSSION
Twenty-one species comprised the rumen ciliate fauna of cows.The population density of particular species was not determined because the identifi cation of particular species after feeding the host animals was nearly impossible.This concerns especially the small Entodinia, as numerous individuals from this group were fi lled with starch granules and this precluded their true identifi cation.
The response of rumen ciliates to the supplementation of cows with Saccharomyces cervisiae varied in relation to the host and group of microorganisms.The effect of this additive on the total number of protozoa found in our experiment was consistent with the relationships described earlier by Doreau and Jouany, (1998) and Mwenya et al. (2005) if their results are considered together, as the cited authors observed either a positive reaction or no effect.In contrast to the results obtained in our study and in the experiments cited above, Newbold et al. (1995) observed a negative effect of yeast NCYC 240, NCYC 694, and Yea-Sacc on the population density of rumen ciliates.These studies were, however, performed under in vitro conditions using a Rusitec system, which provides growth conditions differing considerably from those in the rumen.It is also noteworthy that changes in the total number of protozoa always refl ect changes in the most numerous group of ciliates and Entodinia have most often been such a population (Williams and Coleman, 1992).This was also the case with the cows used in the reported studies.Galip (2006) reported that the number of ciliates from the genus Diplodinium decreased when rams were fed a diet supplemented with yeast, while Doreau and Jouany (1998) did not observe changes in the number of Diploplastron affi ne, which often are an important representative of the genus Diplodinium.In general, our results are in accordance with the fi ndings of the cited authors since Diplodi-nia either decreased (cow 2) or remained unchanged (cow 1) when live cultures of Saccharomyces cerevisiae were given to the animals.In contrast to Diplodinia, ciliates from the genus Epidinium reacted negatively to the supplementation of cows with yeast and such a response was observed in the case of both cows.Unfortunately, no relevant data were found in available literature and due to this no comparison was possible.
The ciliates from the family Isotrichidae were the only protozoa that did not change when live cultures of Saccacharomyces cerevisiae were supplemented to the rumen of cows.Thus, the fi ndings obtained in our experiment are in accordance with the results of Doreau and Jouany (1998) and Galip (2006) who supplemented sheep with yeast.Such a result can suggest that these protozoa were either less sensitive than ophryoscolecids to the changes in the rumen ecosystem caused by the additives or that yeast did not infl uence the growth conditions required by the rumen holotrichs.
The supplementation of the diet with yeast lowered the activity of CMC-ase in cow No. 2 and xylan-degrading enzymes in cow No. 1.This suggests that live cultures of Saccacharomyces cerevisiae negatively infl uenced some of the microorganisms that are able to synthesize fi brolytic enzymes.According to Dehority (1993), the most cellulolytic species of rumen ciliates are Eudiplodinium maggii and Ostracodinium obtusum, which belong to the genus Diplodinium.Both of them were present in the rumen of cows.On the other hand, the ciliates Epidinium ecaudatum caudatum are also characterized by high fi brolytic activity (Takenaka et al., 1999, Michałowski et al., 2001, Wereszka et al., 2004).All of the protozoa and Anoplodinium denticulatum also exhibited the ability to digest xylan (Stan et al., 2006).The comparison of the obtained data suggest a relationship between the changes in the number fi brolytic ciliates and in fi brolytic activity.Unfortunately, the only relationship that was found here was the positive correlation between the number of Dilodiunium and the activity of the mentioned enzymes in the rumen of cow No. 2. No such relationship was found in relation to ciliates from the genus Epidinium.On the other hand, the concentration of these ciliates in the rumen of both cows was presumably too low to have a signifi cant impact on the total activity of enzymes involved in the degradation of dietary polysaccharides In contrast to the fi ndings of Kmet' et al. (1992), yeast had no effect on the activity of enzymes involved in starch digestion in the rumen of cows.Such fi ndings may suggest that either the impact of protozoa on the total activity of amylolytic enzymes was not very important or that the changes in this activity resulting from the changes in the ciliate count were compensated by appropriate changes in amylolytic bacteria.This last suggestion is supported by the negative correlation between ciliates and amylolytic bacteria in the rumen (Ushida et al., 1991).
The results of the measurements of pH and quantifi cation of VFA showed that supplementation of cows with yeast did not change the fermentation pattern in the rumen of these animals.Thus they are consistent with the data of the in vitro and in vivo studies of Newbold et al. (1995) and Marrero et al. (2006), respectively, but contradictory to the fi ndings of Strzetelski et al. (1995) and Doležal and Doležal (2005), who reported that the addition of live S. cerevisiae to the diet increased the concentration of VFA in the rumen fl uid of ruminants.They are also inconsistent with the results of Zeleňák et al. (1994), Lynch and Martin (2002) and Mwenya et al. (2005) who reported that not only the total concentration, but also the molar proportion of particular acids were infl uenced by yeast supplementation.The fermentation pattern in the rumen of cows was characterized by a very high proportion of acetate, which contributed up to 80 % of total VFA.This may suggest that the microorganisms involved in acetate production predominated in the rumen of the examined animals.On the other hand, the absence of changes in VFA concentration and in molar proportions of particular acids suggest that yeast given to cows did not infl uence the population of the microorganisms mentioned above.Further inspection of the results also suggests that the impact of protozoa on carbohydrate metabolism was rather small because the concentration of butyrate was much lower than propionate, and such a proportion is not typical of these organisms (Michałowski, 1987).

CONCLUSIONS
The non-uniform reaction of ciliates to supplementation of the host with yeast resulted presumably from differences in the sensitivity of particular groups of protozoa to the changes in the rumen environment caused by the additive.On the other hand, the non-uniform response of similar groups of ciliates inhabiting in the rumen of two different hosts resulted presumably from differences in the reaction of the compared rumen ecosystems to the same doses of yeast.Despite the drop in the activity of enzymes involved in fi bre degradation, neither the quantity of carbohydrates fermented in the rumen nor the microorganisms responsible for the fermentation pattern were probably signifi cantly infl uenced by yeast in the offered dose.

Table 1 .
The concentration of ciliates (×10 3 /g digesta) in the rumen of cows fed hay and concentrate diet alone (control) or supplemented with live yeast (cxperimental)

Table 2 .
The activity of CMC-ase, xylan and starch degrading enzymes (μM released reducing sugars/g DM/min) in the rumen content of cows fed hay and concentrate alone (control) or diet supplemented with yeast (experimental)

Table 3 .
Effect of live yeast on pH and concentration of total and particular acids in the rumen of cows (mmol/l) fed diet without (control) and with yeast supplement (experimental)