RUMEN FAUNA IN CALVES RAISED IN ISOLATION FROM THE DAMS

Crha J., A. Holub: Rumen Fauna in CalfJes Raised in Isolation from the Dams. Acta vet. Bmo, 47, 1978: 127-135. Six Slovak Spotted calves raised in isolation form their dams were examined for the presence of rumen protozoa from birth to 29 weeks of age. Entodinia were first observed 19 weeks after the animals had been fed roughage for 8 weeks. Eremoplastron spp. and Epidinium spp. occurred from the 25th week and Eudoplodimum spp. from the 27th week after birth. In three calves left with their dams for 20 days after birth entodinia were observed as early as 13 weeks after birth and epidinia and isotricha were present from the 18th week after birth. Rumen microorganisms, protozoa, Entodiniomorpha, Holotricha. There are conflicting reports as to the time at which protozoa first appear in the rumen of calves but it is, no doubt, rather late after birth. Although they may make their appearance in the first or second week after birth, provided that ruminal pH is higher than 6.0 they were generally demonstrated from the third week after birth (Lengemann and Allen 1959). According to other reports they do not appear until calves are 8 weeks old and then only in 15 per cent of animals (Latteur 1953), or even as late as 16 weeks after birth (Conrad et al. 1958). These discrepancies are not surprising when one reftects that the time at which protozoa possibly with the exception of ftagellates (Eadie 1962), appear in the rumen depends on contact of the calves with older, protozoa-harbouring animals (Strelkov et a1. 1933; Pounden and Hibbs 1950; Bryant et al. 1958; Eadie 1962, Borhami et al. 1967). It is therefore conceivable that the methods of rearing particularly under intensive husbandry conditions inftuence faunation of the calves to a considerable extent. Since early establishment of ciliates in the rumen is important to the development and growth of calves, all practices that advance or delay the onset of rumen faunation, thus affecting the progress of transformation of a suckling into a ruminant, are of great importance and may have far-reaching consequences. Material and Methods Animals The experimental animals were Slovak Spotted calves. They were separated from the dams immediately after birth before being licked by the cows. They were transferred first to a calf house where they were given a shower-bath, were brushed and then individually penned in cages bedded with straw. They were fed a milk diet four times a day for the first four days and three times a day for the next 15 days. At 20 days of age they were shifted to granulated alfalfa meal. At one month of age they were changed to clover hay. After being penned in cages up to 11 weeks of age, the animals were transferred to another calf house, about 200 m distant from the previous one, where they were loose-housed. One week later they were transferted to a third calf house, about 200 m distant fromthe previous one, where they were housed about twenty to a pen and fed alfalfa and clover hay ad libitum, received barley-and-maize meal three times a day and were provided with water. Afterwards they were transferted to a fourth calf house, about 800 m distant from the previous one, where they were loose-housed and were fed on maize silage; once a week they received fresh green clover. The four calf houses were under the charge of different attendants.

There are conflicting reports as to the time at which protozoa first appear in the rumen of calves but it is, no doubt, rather late after birth.Although they may make their appearance in the first or second week after birth, provided that ruminal pH is higher than 6.0 they were generally demonstrated from the third week after birth (Lengemann and Allen 1959).According to other reports they do not appear until calves are 8 weeks old and then only in 15 per cent of animals (Latteur 1953), or even as late as 16 weeks after birth (Conrad et al. 1958).
These discrepancies are not surprising when one reftects that the time at which protozoa possibly with the exception of ftagellates (Eadie 1962), appear in the rumen depends on contact of the calves with older, protozoa-harbouring animals (Strelkov et a1. 1933;Pounden and Hibbs 1950;Bryant et al. 1958;Eadie 1962, Borhami et al. 1967).
It is therefore conceivable that the methods of rearing particularly under intensive husbandry conditions inftuence faunation of the calves to a considerable extent.Since early establishment of ciliates in the rumen is important to the development and growth of calves, all practices that advance or delay the onset of rumen faunation, thus affecting the progress of transformation of a suckling into a ruminant, are of great importance and may have far-reaching consequences.

Animals
The experimental animals were Slovak Spotted calves.They were separated from the dams immediately after birth before being licked by the cows.They were transferred first to a calf house where they were given a shower-bath, were brushed and then individually penned in cages bedded with straw.They were fed a milk diet four times a day for the first four days and three times a day for the next 15 days.At 20 days of age they were shifted to granulated alfalfa meal.At one month of age they were changed to clover hay.After being penned in cages up to 11 weeks of age, the animals were transferred to another calf house, about 200 m distant from the previous one, where they were loose-housed.One week later they were transferted to a third calf house, about 200 m distant fromthe previous one, where they were housed about twenty to a pen and fed alfalfa and clover hay ad libitum, received barley-and-maize meal three times a day and were provided with water.Afterwards they were transferted to a fourth calf house, about 800 m distant from the previous one, where they were loose-housed and were fed on maize silage; once a week they received fresh green clover.The four calf houses were under the charge of different attendants.
Six calves born during one week and raised as described above were included in the experiment.An additional three calves were left with their dams for 20 days after birth and then transferred to cages and raised in the same way as the calves separated from the dams immediately after birth.

Collection of Samples
Rumen liquor was collected by means of an oesophageal tube at 14-day intervals, invariably between 10 and 12 a.m., from the first week after birth.The liquor was strained through a sieve and diluted 1 : 1 (2 ml + 2 ml) with 10 per cent formol.

Microscopical Examination
The sediment of each sample was examined for the presence of ciliates.If they were present the sample was shaken and a portion of it was transferred to a Fuchs-Rosenthal chamber.The ciliates in each sample were counted four times; the results were averaged and were expressed per 1 ml.For species identification, the ciliates were stained with carmine dye (nuclei) and with modified Lugol's solution (skeleton plates) (Dogiel 1927, Kofoid and Mac Lennan 1930, 1932, 1933;Wertheim 1935;Lubinsky 1957;Noirot -Timothee 1960;Latteur 1966Latteur , 1968Latteur , 1970)) In isolated calves rumen ciliates were first observed 19 weeks after birth and were consistently found in all subsequent rumen liquor samples till the end of the experiment.The only genus represented up to 23 weeks after birth was Ento- First appearance of ciliated protozoa in the rumen liquor of isolated calves (Group 2) and control calves kept with dams after birth (Group 1) Numbers of ciliated protozoa (thousands/ml) in the rumen liquor of isolated ea1ves and control animals left with th~ d"ml after birth   1 and 2).The first species to appear was Entodinium simplex followed by Entodinium bursa, Entodinium augustatum, Entodinium longinucleatum, Eremoplastron hovis, Epidinium ecaudatum, Entodinium bifidum, Eudiplodinium maggii, Entodiniium caudatum, Eremoplastron rostratum and Entodinium dilobum (Table 3).Large specimens of Entodinium bursa and Entodonium longinucleatum were to contain ciliates of the species Entodinium simplex.
In calves left with their dams after birth ciliates were first observed at 13 weeks of age, i. e. as early as the second week after the shift to roughage (Fig. 1, Tables 1 and 2).The first species to appear were Entodinium simplex and Entodinium bursa followed by Epidinium ecaudarum, Entodinium caudatum, Entodinium minimum and Isotricha prostoma.

Discussion
As might be expected, rumen ciliates appeared earlier in calves left with their dams than in calves strictly isolated from the dams immediately after birth.Oxford (1955) reported sequential establishment of entodinia before diplodinia before holotrichs.Bryant et al. (1958) found, similarly to our results, no proto- zoa in three isolated calves during 13 weeks after birth; entodinia were first observed in these animals at 17 weeks of age after the calves were placed in contact with adult cattle and the sequence of establishment of entodinia, diplodinia and holotrichs was the same as reported by Oxford (1955).Naga et al. (1969) reported that Entodinium followed by Eudiplodinium were the first two genera to appear in the rumen of buffalo calves where ciliated protozoa were seen earlier than in cow calves in which isotrichs and entodinia occurred almost simultaneously.
Entodinia.are therefore the first ciliates to appear in the rumen of ruminants.They are, with few exceptions, small in size and morphologically primitive and ingest starch grains and bacteria that are present in ruminants at this time.In our isolated calves as well as in the controls left with the dams the appearance of primitive entodinia was followed by morphologically more complicated ciliates such as epidinia which were not observed in cow and water buffalo calves in Egypt (Naga et al. 1969).
Rumen ciliates that are larger in size and have thicker skeletal plates (eudiplodinia, epidinia, ostracodinia) feed, for the most part, on grass scraps.However, diplodini~ ~;nd Entodinium bursa (syn.Entodinium vorax) (DogieI1927), the largest species of primitive entodinia, are predacious.In our study we found predacious Entodinium bursa specimens containing specimens of Entodinium simplex.Moreover, we found large specimens of Entodinium longinucleatum with swallowed Entodinium simplex specimens.Also in the higher members of the family predatory habits were found only in species where large specimens occur (Lubinsky 1957).
A number of investigators have studied the presence of ciliates in young animals in relation to growth rate and health status.Belenkij (1953) reported that calves with gastric or intestinal disease had no ciliates in the rumen.Artifical faunation protected calves from coliparatyphoid and colibacillosis.Similarly, Williams and Dinusson (1972) reported that two of three isolated calves developed scours, which is in keeping with our observations.Borhami et al. (1967) found a more rapid growth rate in inoculated than in uninoculated calves.Bryant and Small (1960) on the other hand, reported that the lack of a normal microbiological population in calves did not have any drastic effect on the animals' growth or health up to 17 weeks of age.According to Minakov (1967) rumen ciliates appear earlier in calves inoculated with rumen contents from adult cattle than in uninoculated calves.A milk diet did not promote the establishment of ciliates, but ciliates inoculated into milk-fed calves did not die.Williams and Dinusson (1972) described the case of an isolated female calf that was free of protozoa till she was artifically inseminated.The establishment of ciliated protozoa in the rumen of isolated calves can be hastened by artifical faunation, i. e. by inoculation with rumen contents from healthy adult animals.Faunation stimulates digestion in the ontogenesis of the calf.The processes of digestive function in young animals are associated with the formation of reflex response to food during the differentiation of the central nervous system (c.N. S.).The sooner the relation between digestive receptors and the C. N. S. is established, the better the process of digestion and, in consequence, the more rapid growth rate of the young (Be~ lenkij 1953).
Natural transfaunation has been explained since the observations of Strelkov (1933) as a result of direct contact of young ruminants with adult ones through licking or the cud.The question, however, arises why ciliates do not appear in the rumen of calves immediately after birth and why only entodinia become established at first although one might expect that other protozoal species occurring in large numbers in adult cattle (Dogiel 1927;Wertheim 1934;Minakov 1967;Crha 1969;Vasily and Mitchel 1974;a. 0.) would be transferred through the cud or licking at the same time.Rumen ciliates are anaerobes and are very sensitive to oxygen.In vitro, they survive only in a culture medium containing 0.7 mg oxygen per ml (Gaumont and Grain 1967).The least air sensitive ciliates are entodinia and isotrichs (Sedloev 1973).On the other hand, rumen ciliates endure extremely well thermal shocks which they survive for several hours and continue to develop after being reexposed to their optimum growth temperature, i. e. 38°C (Florentin 1964(Florentin , 1965)).The replication of ciliates has been related among other things, to the intake of roughage.Bazadov and Prokudin (1959) reported that the appearance of rumen ciliates does not invariably coincide with the start of roughage feeding, but is often delayed, which is in keeping with our observations.Large quantities of rumen ciliates appear in calves after the animals are placed on fresh green forage; this was also the case in our experiments.There is also in vitro evidence to indicate that entodinia survive in rumen fluid much longer if fresh clover leaves are added (Kandacu and T akachasi 1956).Hungate (1966) found entodinia and diplodinia on sheep pasture plants, but neither cysts nor pseudocysts of these ciliates were detected as was demonstrated more recently by Nouzarede (1965).

Table 2
Perce:atatre proportioaa of protozoal pner8 in the rumen Uquor of Isolated calves aacl control lUll ...... left with the clams after birth dinium.Samples collected from the 25th week of age onwards contained in addition Eremoplastron spp., Epidinium spp.andEudiplodinium spp.In other words, ciliates first appeared after the calves were fed dry roughage for 8 weeks and the genera represented increased in number only after another 5 weeks in association with the shift to another diet(Fig 1, Tables

Table 3
Percentage proportions of protozoal species in the rumen liquor of isolated calves and control animals left with the dam.after birth