A proposed simple method for determining the outflow of ciliates from the reticulo-rumen

Two adult male sheep were used to determine the outflow of protozoa from the reticulo-rumen. The outflow of ciliates was calculated on the basis of their concentration in reticular digesta and volume of effluent leaving the reticulo-rumen. The concentration of ciliates from the genus Entodinium and Diplodinium in reticulum was by 33-63 % and 32-73% lower than in the rumen. The concentration of Holotricha varied between 55.7 and 126.8% of the ruminal density. No differences were found between the concentration of protozoa in reticular digesta taken at the contraction and resting phases of the reticulum. The number of ciliates leaving the reticulo-rumen per day equaled 66-177 % of their number in the rumen, and the apparent residence time of protozoa varied between 13.8 and 36.6 h. It was longest in the case of ciliates from the genus Dipolodinium and the shortest in the case of Isotricha. A technique for the collection of reticular digesta at the contraction and resting phases of the reticulum is described.


INTRODUCTION
The forestomachs of ruminants are inhabited by numerous bacteria, fungi and protozoa.The protozoa are selectively retained in the rumen (Michalowski et al., 1986) but part of their population passes down from the reticulo-rumen to the omasum and then to the abomasum and duodenum.The outflow of protozoa from the rumen has been measured indirectly, i.e as flow of protozoal protein at the duodenum (Harrison et al., 1979;Steinhour et al., 1982;Whitelaw et al., 1984;Cockburn and Williams, 1984;John and Ulyatt, 1984;Meyer et al., 1986) and by a more direct method, i.e. by counting ciliates in omasal liquid taken from omasum (Weller and Pilgrim, 1974;Punia and Leibholz, 1994) or in omasal effluent (Michalowski et al., 1986).No satisfactory method has, however, been developed yet.
On the other hand it has been shown that the concentration of ciliates in omasal influent and in reticular digesta are almost the same (Harmeyer and Michalowski, 1991).It was also found in the same study that only reticular contents passed down to the omasum and that inflow to omasum was observed only during the second phase of contraction of the reticulum.If this is the case the concentration of ciliates in the reticulum could be used for calculation the number of protozoa leaving the reticulo-rumen over the day.The data cited suggest also that the samples taken at the contraction of reticulum would be the best for this purpose.
The aim of the present study was to determine the daily outflow of protozoa from the reticulo rumen on the basis of their concentration in reticular digesta and compare the obtained results with the data of other authors who measured the outflow of ciliates to lower gut of ruminants.

Animals and feeds
Two adult male sheep (L and R) weighing 77 and 85 kg and fitted with 100 mm permanent rumen cannulas were used.The animals were kept in separate pens and given 300 g pelleted concentrate (15.5 % protein) and 700 g hay at 8 a.m. and 4 p.m. Water was available all the time.

Experimental procedure
A 2 weeks adaptation period of animals to the food and feeding frequency was allowed before any collection of the samples of rumen and reticular digesta was started.
The samples of reticular digesta were taken via rumen cannula with the use of plastic tube of 10 mm ID and 500 mm in length.The tube was fitted with a tight rubber piston (Figure 1).A small balloon of an apparatus for recording contraction of the reticulum was fixed to the tube near its end and introduced into the reticulum during the sampling.The samples of reticular digesta were taken when the second phase of contraction of the reticulum wall had begun or during the resting phase.The samples were taken by suction made by pulling the piston to the opposite end of the collecting tube.The tube was then taken out and the sampled digesta was poured to a beaker.It was subsampled for protozoa counts (5 g) and for dry matter (DM) determination (the remaining part of the sampled material).
Rumen content (about 500 g) was taken from different places in the rumen.The sampled material was thoroughly mixed, subsampled for protozoa counts (2 x 5 g) and returned immediately to the rumen.The collection of rumen content was made immediately after sampling of reticular digesta.Both types of the material was collected at 2, 4, 6 and 8 h after morning feeding.The sampling was repeated four times on four different days.
The pool size of digesta was measured by removing and weighing all the rumen contents.The removed digesta was thoroughly mixed, subsampled for DM determination (20-30 g) and returned immediately to the rumen.The procedure was repeated four times, but no more than 2 times a week.
The volume of liquid leaving the reticulo-rumen was measured with the use of CrEDTA.A solution of marker (60 ml) containing 750 mg Cr was introduced into the rumen at 8 a.m. and samples of rumen fluid (20 ml) were taken at, 1,2,3,4,6,8,10,14,20 and 24 h thereafter.The collected samples were centrifuged at 20.000 g for 15 min and supernatant was used for Cr determination.
The experiment was repeated three times.

Analyses
Samples for protozoa counting, fixed in 4% formaldehyde solution (1:1 w/v) were analyzed under light microscope.Entodinia were counted in a Fuchs-Rosenthal chamber while other ciliates by the method described earlier (Michalowski, 1975).The protozoa were identified according to Dogiel (1927) and Grain (1966).Dry matter was determined by drying the samples at 95°C for 48 h.Cr was estimated by atomic absorption spectrometry as described by Michalowski et al. (1986).

Calculations
Apparent residence time of protozoa in the rumen was calculated from the equation T = V:L x 24 h; where T = apparent residence time (h), V = rumen content volume x protozoa concentration in rumen digesta, L = effluent volume x protozoa concentration in reticular digesta.The volume of effluent leaving the reticulo-rumen per day was calculated according to Hyden (1961) To obtain the daily flow of reticular digesta to the omasum the calculated liquid volume was corrected for DM content of reticular digesta.
All of the statistical calculations were made according to Ruszczyc (1970).

RESULTS
The weight of the digesta in the rumen of sheep varied between 12.1 and 17.1 kg and the volume of the effluent leaving the reticulo-rumen was 20.2-33.41/d.Mean values are given in Table 1.The concentration of ciliates in the rumen and reticulum of sheep "L" and "R" is presented in Tables 2 and 3, respectively.The ^Entodinium group" composed of many species of which the most numerous were Entodinium simplex, Entodinium caudatum and Entodinium longinucleatum.Anoplodinium denticulatum, Diploplastron affine, Polyplastron multivesiculatum and Ostracodinium obtusum formed the Diplodinium group, while Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium the holotricha group".Very large variations in the number of Diplodinium and Holotricha were observed during the sampling period.No Diplodinia other than Polyplastron multivesiculatum were found in the rumen of sheep "L" on the first sampling day and no more than 160 cells/g during the second collection.The concentration of ciliates from the genus Diplodinium in the rumen of sheep "R" increased during the collection period from 1400 to over 28000 per gram.On the other hand, the number of Holotricha not exceeded 500 cells/g during the second collection day while the highest concentra-  tion was over 22000/g.Due to the mentioned variations the standard deviations were commensurately high.Regardless of the day to day variations, the concentrations of Entodinium and Diplodinium in the reticulum were by about 33-63% and 32-73% lower, respectively as compared with their densities in the rumen.The higher concentration of Holotricha in the rumen was noted only 2 and 4 (sheep "L") or 2 and 8 (sheep "R") h after morning feeding.No significant differences were found between the concentration of ciliates in the reticulum contents taken at the contraction and resting phase.The densities of particular populations tended, however, to be, in majority of cases, higher in the samples taken at contraction.Of the Diplodinium and Holotricha the ciliates Polyplastron multivesiculatum and Dasytricha ruminantium were counted separately.The concentration of Polyplastron multivesiculatum in the rumen of sheep "L" varied from 360 to 3400 and in the reticulum from 160 to 2080/g.The respective concentration in sheep "R" was 800-4480 and 240-2160/g.The population density of Dasytricha ruminantium in the rumen and reticulum of sheep "L" and "R" were 1700-20500 and 1800-10800, and 1700-17100 and 1400-12200/g, respectively.
Total number of ciliates in the rumen of sheep "L" as well as outflow rate and apparent residence time of the different groups of protozoa are presented in Table 4.The number of ciliates leaving the reticulo-rumen during a day was equal to 66-174.2 % of their number in the rumen.According to this the apparent residence time varied from 14.7 to 36.6 h.The residence time of Entodinium was similar regardless of the calculation basis.The residence time of other populations tended to be longer when it was calculated on the basis of concentration of protozoa in the samples taken at the resting phase of the reticulum.
A significant difference, however, was found in the case of "other Diplodinia" and "Dasytricha ruminantium" only.
The number of ciliates passing down from the reticulum of sheep "R" varied from 77 to over 177% of their number in the rumen.The smallest outflow was noted in the case of Polyplastron multivesiculatum while the largest in the case of Isotricha sp.All the results are presented in Table 5.

DISCUSSION
The results obtained provide direct evidence for reduced concentration of ciliates from the genus Entodinium and Diplodinium in reticular digesta compared with rumen contents of sheep.The observed reduction can result from selective retention of ciliates in the rumen due to attachment of these organisms to particulate fractions of rumen contents and their sequestration between large particles of food forming a sort of filter on the way from the rumen to reticulum.A similar phenomenon was already observed in earlier investigations (Michalowski, 1990).Conversely no sequestration seems occur in reticulum as the reticular digesta is composed of the liquid phase and very small food particles (unpublished).Due to this no differences were observed between the concentration of ciliates in reticular content and omasal influent (Harmeyer and Michalowski, 1991).
In contrast to Entodinium and Diplodinium, the concentration of Isotricha sp. and Dasytricha ruminantium in the rumen and the reticular digesta were sametimes similar.Holotrichs, however, seem to belong to organisms of the liquid fraction of rumen contents.Moreover the cuticula of these ciliates is not very rigid as of entodiniomorphs and, especially, of large Diplodinium.Due to this the cells of Holotricha are very flexible and this property could make any passage of these ciliates through the filter mentioned above much easier.Noteworthy is also fact that these protozoa temporarily attach to the reticulum wall (Abe et al., 1981).Thus their detachment could raise the concentration in reticular digesta.
The apparent residence time of protozoa in the rumen varied from about 13 to over 36 h.The residence time of Polyplastron multivesiculatum and "other Diplod-inia" in the both sheep and Entodinium in sheep "R", were similar while that of Entodinium in sheep "L" was something shorter as compared with the results obtained by Michalowski et al. (1986).The residence time, however, was calculated there on the basis of concentration of ciliates in the omasal effluent thus any sequestration and/or lysis some of protozoa within the omasum can not be precluded and such a possibility has been suggested by the authors.The residence time of the both holotricha sp" and Dasytricha ruminantium were by two time shorter than calculated by cited authors.These protozoa, however, are very mobile and due to this they could escape of the passage to the omasum.The lysis of holotrichs within the omasum can not be, also precluded.Thus this phenomenon needs experimental explanation.
The aim of this investigation was to verify a suggestion that the concentration of ciliates in reticular digesta could be suitable to calculate the outflow of protozoa down the omasum.The basis for this assumption was similar concentration of ciliates in reticular digesta and in omasal influent (Harmeyer and Michalowski, 1991).Unfortunately there did not exist any possibility for simultaneous collection of the digesta from the reticulum and omasum.Thus it is only possible to compare the obtained results with the data of other authors.The mean concentration of ciliates from the genus Diplodinium in reticulum equaled 43-52% of their density in the rumen.The respective values fox Holotricha were 75-95%).Similar differences were found between the concentration of holotrichs and large and medium entodiniomorphs in the ruminal and omasal digesta (Punia et al. 1992;Punia and Leibholz, 1994).Thus the cited findings suggest that outflow of Diplodinium and Holotricha could be calculated on the basis of their concentration in reticulum.The concentration of Entodinium in reticulum of sheep was by about 31-53%) lower than in the rumen.The concentration of small ciliates (presumably, mainly Entodinium) in the omasal digesta account only for 26-45%) of the density in the rumen (Punia et al., 1992; Punia and Leibholz, 1994).This suggest a possibility of some overestimation when the concentration of Entodinium in the reticulum would be the basis for calculation of the outflow of these ciliates down the omasum.On the other hand, however, no differences were found between the concentration of ciliates in the reticular digesta and omasal influent (Harmeyer and Michalowski, 1991).In fact, these last data concern only to the total number of protozoa but Entodinium accounted there for over 90%o of the total count.Thus further investigation are necessary to explain this discrepancy.
The calculated daily outflow of protozoa equaled 66-180%o of their number in whole rumen contents per day.These values are much higher in comparison with the findings of Weller and Pilgrim (1974) who calculated the outflow of ciliates on the basis of their concentration in the liquid taken from the omasal canal.It should be emphasized, however, that the outflow of protozoa described by the mentioned authors seems to be exceptionally low.It was not confirmed by other investigators either by indirect or by more direct methods (Harrison et al., 1979;Steinhour et al., 1981;Michalowski et al., 1986;Punia et al. 1992;Punia and Leibholz, 1994).The outflow rate of ciliates in this experiment varied in high ranges in relation to the group of protozoa.The outflow of Polyplastron multivesiculatum and" other Diplodinia" was within the range calculated on the basis of concentration of ciliates in omasal effluent (Michalowski et al., 1986).The outflow rate, however, of Entodinium and especially Holotricha was faster.It is possible that these ciliates, having more delicate cuticlula, are more susceptible to disintegration in the omasum.Thus their inflow to and outflow from the omasum may not be equal.Such a possibility is suggested by low proportion of protozoal N in the microbial N leaving the omasum of the sheep (Michalowski, 1990).Protozoal N was calculated in that study on the basis of N content in single cells of ciliates and their number leaving the omasum.The obtained results are distinctly lower than those calculated from concentration of different markers or the amino acid profile (Harrison et al., 1979;Steinhour et al., 1982;Cockburn and Williams, 1984;Meyer et al., 1986) as well as on the basis of protozoa concentration in the omasal digesta (Punia et al., 1992).
The presented results and the data of other authors, cited above, seem to suggest that perhaps the concentration of ciliates in the reticulm could be the basis for calculation of the outflow of ciliates from the reticulo-rumen down the lower digestive tract.However, further investigations are necessary to confirm this hypothesis, and especially the experiments enabling simultaneous sampling of the reticular and omasal digesta from the same animals are necessary to confirm this hypothesis.A collection of numerous samples during each day of experiment seem to be also necessary to minimize any possibility of overestimation.
The concentration of ciliates in digesta taken from the reticulum at the contraction and resting phases were similar.This suggests that recording the motility of reticulum can be omitted in the proposed sampling technique.On the other hand, however, recording by the method described in this paper precludes any uptake of a sample of digesta from another compartment in the reticulo-rumen.

Figure I .
Figure I. Device construction for collection of the reticular digesta via rumen cannula, a -collecting tube; b -rubber piston; c -ballon; d -connecting tube; e-recording apparatus The concentration of ciliates in the rumen (x 10 3 /g) and reticular digesta (% ruminal concentration) of sheep"L" during the contraction and resting phases of reticulum and at different time after feeding (h)

TABLE 3
Total number (x lOVrumen), outflow rate (% mean ruminal number/d) and apparent residence time (h) of ciliates in reticulo-rumen of sheep "R".Outflow rate and residence time were calculated on the basis of ciliate number in the reticular digesta during contraction (A) and resting (B) phases of reticulum