Rumen Ciliated Protozoa of the Free-Living European Bison (Bison bonasus, Linnaeus)

This study aims to perform population analysis of the rumen ciliated protozoa of the free-living European bison (wisent, Bison bonasus, Linnaeus). The samples of the rumen fluid from the 18 bison subjected to the controlled culls within the free-ranging population in the Bialowieza primeval forest in Poland were collected and examined. The examined ciliates population consisted of the species of the families Isotrichidae and Ophryoscolecidae. There were 12 genera (Isotricha, Dasytricha, Diplodinium, Elytroplastron, Entodinium, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, Ophryoscolex, and Ostracodinium) and 32 morphospecies of the ciliates. We observed the prevalence of a type B protozoan population (56% animals) with the typical Epidinium and Eudiplodinium genera members. Other examined animals possessed the mixed A–B population with Ophryoscolex genus, distinct for type A ciliate population. The average total ciliates count was 2.77 ± 1.03 × 105/ml (mean ± SD). The most abundant genera were Entodinium, 83%, and Dasytricha, 14%. The abundance of other genera was <1% of the total count. Within the 16 Entodinium species determined, the most abundant species was Entodinium nanellum (16.3% of total ciliates count). The average Shannon–Wiener diversity index was 2.1 ± 0.39, evenness was 0.7 ± 0.11, and species richness was 24 ± 3.0 (mean ± SD). Our study is the first report on the population composition and diversity of rumen ciliates of European bison. The composition and counts of ciliate genera and species were similar to the composition and counts of the rumen ciliated protozoa of American bison and many other kinds of free-living and domestic ruminants. Our European bison ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior.


INTRODUCTION
The European bison (wisent, Bison bonasus L.) is the largest terrestrial mammal in Europe. The European bison has been successfully restored after the extinction in the wild at the beginning of the twentieth century. The total area of European bison habitat covers about 130,000 ha in Poland. Due to herd management of growing free-ranging wisent population in the Bialowieza Forest (northeast Poland), the annual culls have been conducted since 1971 (Krasińska and Krasiński, 2013). They aim to reduce the population on average by 11%. The most frequent reasons for bison selections were various injuries, entering fields, aggression toward people, and, after 1980, changes in the genitourinary system caused by posthitis/balanoposthitis illness (Krasińska and Krasiński, 2013). The free-living ruminants are interesting from a scientific perspective because few studies describe their complex rumen microbial ecosystems (Ishaq et al., 2015). The complex rumen microbial population comprises prokaryotes (eubacteria and archaea) and eukaryotes (fungi and protozoa). The dominant component of rumen protozoa is ciliated protozoa (Williams and Coleman, 1992), which contribute to the breakdown of plant and microbial carbohydrates and proteins. Protozoa influence the rumen microbial population by predation on other protozoa, prokaryotes, and fungi, suggesting that protozoa are still attractive from a scientific point of view (Williams et al., 2020). In general, the number of rumen ciliates species in different host animals is about 45 or less, influenced mainly by host feeding behavior and seasonal variation in the diet (Dehority, 2004). The studies on the rumen ciliates population revealed two main types, types A and B. Type A population is characterized by the presence of genera of Polyplastron and Ophryoscolex (Eadie, 1962(Eadie, , 1967. Type B population is characterized by the presence of genera of Epidinium and Eudiplodinium. On the other hand, some other genera are commonly present in most ruminants (Entodinium spp. and Isotrichids). Rumen ciliates were described in many ruminants' species; however, their description of the genus Bison is limited only to American bison (A. bison, Bison bison). All protozoan species found in A. bison have also been reported in domestic livestock. The type B population predominated in free-living A. bison without contacts with cattle. The mixed A-B population occurred in bison in areas inhabited by domestic livestock (Towne et al., 1988b). The percentage distribution of rumen ciliate species among A. bison varies among the geographical regions, depending on the type and quantity of consumed feed and on the contact with other animals in the group. The present study aimed to examine and describe the ciliate population of the rumen fluid of European bison obtained from culled animals of the free-ranging population in Bialowieza. The description of rumen ciliate protozoa of the European bison contributes to our knowledge of the ecology and diversity of rumen ciliates of the free-living European ruminants.

MATERIALS AND METHODS
The material was obtained from culled European bison originating from the free-ranging population in the Bialowieza Forest (northeast Poland; longitude between 23 • 31 ′ and 24 • 21 ′ E and latitude 52 • 29 ′ and 52 • 57 ′ N). Samples were collected immediately after death (1-3 h) from 18 animals of both sexes of various ages during February and March 2007 (Table 1). For the microscopic counts, about 10 g/animal of the rumen contents were preserved with an equal amount of 8% formaldehyde solution (w/w), strained through four layers of cheesecloths into 10 ml polypropylene tubes with screw cups and stored at 8 • C in a refrigerator until analysis. Ciliates were counted microscopically in an aliquot of the suitably diluted sample (Williams and Coleman, 1992). At least four replicates were counted per sample and per ciliate species. The protozoan genera and species were identified according to the size and the shape of cells, skeletal plates (if present), macronucleus, and ciliature arrangement (Dogiel, 1927;Ogimoto and Imai, 1981;Williams and Coleman, 1992;Ito and Imai, 1998;Ito et al., 2001;Cedrola et al., 2017aCedrola et al., ,b, 2018. Different staining procedures were used to stain skeletal plates (iodine solution), nuclei (methyl greenformalin-saline and chrome-alum-carmine), and infraciliature (pyridinated silver carbonate method) (Ogimoto and Imai, 1981). The pictures of ciliates were taken under bright field illumination by Moticam Pro CCD Camera (Motic Incorporation Ltd., Hong Kong) mounted on a BA400 microscope (Motic Incorporation Ltd., Hong Kong). The images were processed and analyzed using ImageJ software according to ImageJ software documentation (Abramoff et al., 2004;Siritantikorn et al., 2012;Choudhry, 2016). Morphometric measurements were performed by image analysis of at least 20 cells. Only ciliates of the families Isotrichidae and Ophryoscolecidae were present in the samples. Differentiated count of species of genus Entodinium was performed in 13 samples (animals). Direct bacterial count estimated the total bacteria count through image analysis of pictures taken under bright field illumination of dried smears of formaldehyde-fixed samples (Siritantikorn et al., 2012;Choudhry, 2016). Two smears stained with methylene blue and known dimensions and known volumes per sample (animal) were prepared according to the Breed method (Horáková, 1988). Twenty randomly selected pictures per smear were taken at an objective magnification of ×100 by the Moticam Pro CCD Camera (Motic Incorporation Ltd., Hong Kong) mounted on a BA400 microscope (Motic Incorporation Ltd., Hong Kong). The images were processed and analyzed using ImageJ software (Selinummi et al., 2005). Ciliates and bacteria counts per milliliter were expressed as geometric means of log natural transformed values ± geometric standard deviation and arithmetic means ± standard deviations. We evaluated bison age and sex effects on the total count of bacteria and ciliates by nonparametric Kruskal-Wallis test (GraphPad Prism, GraphPad Software, Inc., San Diego, CA, USA). The biodiversity indices (Shannon-Wiener diversity index, evenness, and species richness) (Spellerberg and Fedor, 2003;Jost, 2006) were calculated with an Excel calculator (Microsoft Office Professional, 2007, Microsoft, Redmond, WA, USA). The Shannon-Wiener (SW) diversity index was computed to explain the entropy, taking into account the species richness and evenness of the community. The species richness was evaluated by counting the number of taxa per sample (animal). Correlation analysis on counts of ciliates genera was calculated

DISCUSSION
The rumen protozoal population of free-living ruminants is influenced mainly by host feeding behavior and seasonal variation in the diet (Kamler, 1999;Dehority and Odenyo, 2003;Booyse and Dehority, 2011;Clauss et al., 2011;Obidziński et al., 2017). European bison can be considered mixed feeders with 68-97% of herbaceous plants in their natural diet (Gebczyńska et al., 1991;Kowalczyk et al., 2019). In some other references, the European bison are considered as grazers with 68% of grass in their natural diet (Pucek et al., 2002;Clauss et al., 2006;Przybyło et al., 2019). The population of rumen ciliated protozoa of grazers (e.g., cattle) is generally more diverse in comparison with typical browsers, selectors (e.g., roe deer and blue duiker), but this is not a rule (Kofoid and Christianson, 1934;Sládeček, 1946;Prins and Geelen, 1971;Giesecke and Gylswyk, 1975;Imai, 1988;Williams and Coleman, 1992;Dehority, 1994;Robbins et al., 1995;Dehority and Odenyo, 2003;Clauss et al., 2011). More generically diverse ciliate populations of grass and roughage eaters may result from a slower passage rate and higher rumen pH (Dehority and Odenyo, 2003). However, in the Bialowieza forest, other food (hay) is available to bison during winter (Gebczyńska et al., 1991;Pucek et al., 2002;Kowalczyk et al., 2011). Therefore, the differences in both summer and winter rumen ciliate populations are likely small. To our knowledge, this is the first study describing the population of protozoa in the rumen of European bison of the Białowieza region. Generally, the species composition of rumen ciliates of European bison was similar to the composition of rumen ciliates populations of many Frontiers in Microbiology | www.frontiersin.org other species of free-living and domesticated ruminants (Kofoid and Christianson, 1934;Sládeček, 1946;Crha, 1972;Towne et al., 1988a,b;Ito et al., 1994;Moon-van der Staay et al., 2014). Recent research pointed to the existence of a core rumen microbiome across a wide geographical range, which is modified by the diet and the host (O'Kelly and Spiers, 1992;Guan et al., 2008;Shi et al., 2008;Moon-van der Staay et al., 2014;Henderson et al., 2015;Ishaq et al., 2015;Tapio et al., 2017;Reis et al., 2019;Furman et al., 2020;Xue et al., 2020). These studies indicate that dietary and animal feeding strategies dominate over host species. In the study of Henderson et al. (2015), the variability of protozoa between and within animal groups was much greater than that of bacteria and archaea. Analyses of different 18S ribosomal RNA (rRNA) genes showed extremely complex but related ciliate communities, which occur in the rumen of cattle, sheep, goats, and red deer (Moon-van der Staay et al., 2014). Although individual host genetic characteristics might influence the composition of the rumen prokaryotes (Shi et al., 2008;Xue et al., 2020), it seems that ciliates' feed preferences and relationship within ciliates contribute remarkably to their composition in the rumen (Eadie, 1967;Dehority, 1998;Martinele and D'Agosto, 2008).
In the type B population, the predatory behavior of E. bubali on Epidinium, Enoploplastron, and Entodinium was observed in sheep rumen (Martinele and D'Agosto, 2008). However, the predatory activity of E. bubali in our bison samples was low.
Our correlation analysis revealed the prevalence of the positive correlations among the individual rumen protozoal genera counts. Most numerous positive correlations were observed on Isotricha spp. (with total counts, Entodinium, Eremoplastron, Eudiplodinium, Elytroplastron, and Ostracodinium counts). We can speculate that the growth of Isotricha can be promoted by soluble metabolic products of hydrolytic and proteolytic activities of Entodinium, Eremoplastron, Eudiplodinium, Elytroplastron, and Ostracodinium species. Isotricha is known to prefer soluble substrates (Williams, 1986). On the other hand, Entodiniomorphid ciliates prefer solid substrates (plant and bacterial particles) (Williams and Coleman, 1992). On the other hand, we have observed no correlations of Dasytricha and Metadinium with other ciliate genera. Differences in metabolic activities were observed between Isotricha and Dasytricha. They remarkably differ in carbohydrate fermentation (Howard, 1959). Dasytricha has greater metabolic versatility than Isotricha. Therefore, Dasytricha is probably less dependent on intermediate metabolic products of other members of the ciliate population. We can speculate on similar features of Metadinium species. For example, Metadinium medium could degrade starch, amylose, amylopectin, and hemicellulose (Naga and El-Shazly, 1968).The Eudipl. Elytropl.
Diplod. The first value is correlation coefficient r, and a number in parentheses is probability value P. The significant values are highlighted.

Gender
Frontiers in Microbiology | www.frontiersin.org comparison with other studies is difficult because of different analysis methods, different animal diets, and different ciliate population structures observed. In Tan et al. (2020), the correlation of molecular data revealed the positive association of the Metadinium with Eudiplodinium, Isotricha with Dasytricha, and Polyplastron with Ostracodinium and Ophryoscolex. In our study, those associations were not observed. The influence of the season may also contribute to these differences. Our samples were taken in the winter when the animals eat a predominantly fibrous diet with a lack of green fodder rich in soluble nutrients. Our ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior. We observed a similar total number of rumen ciliates of European bison (277.10 3 /ml) and American bison (328.10 3 /ml) (Towne et al., 1988a,b). A similar ciliate density was also observed in domestic ruminants (cattle and goats) (Imai, 1988;Ito et al., 1994Ito et al., , 1995de la Fuente et al., 2009;Mishima et al., 2009). On the other hand, the domestic ruminants have the lower average number of ciliate species per host (8-18; Imai, 1988;Ito et al., 1994Ito et al., , 1995de la Fuente et al., 2009;Mishima et al., 2009). It is considered that species evenness decreases in the ruminants on high concentrate feed (Ito et al., 1994). On the other hand, when ruminants are fed on high forage, the number of species per host increases to more than 30 (Mishima et al., 2009). There were considerable variations in the counts of all examined ciliates species (genera). The animal-to-animal variations in both the differential counts of ciliate species and total counts were also observed in A. bison and other ruminants (Towne et al., 1988a,b;Kittelmann and Janssen, 2011). Purser and Moir (Purser and Moir, 1966a,b) showed that rumen volume could be a factor involved in individual animal differences in rumen parameters of sheep fed the same diet. The significant differences in the total ciliates counts were removed after the counts' adjustment for rumen volume (Dehority, 1978). The same author observed decreased rumen volume of sheep fed concentrate diet than the forage (alfalfa) diet. The changes in the rumen volume regarding the feed changes were also observed in other ruminants (Kamler et al., 2003). Some studies also suggest the effects of animal age, sex, and weight on the ciliate population (Clauss et al., 2011;Duarte et al., 2018). Our correlation analysis revealed no effects of animal sex on ciliates counts. However, sex in our bison collection was not evenly represented among age groups. No effects of host age and sex on rumen protozoa were observed on Spanish ibex and domestic goats (de la Fuente et al., 2009). We have observed a positive correlation of Ophryoscolex counts with animal age and a negative correlation of Epidinium counts with host weight. Those relationships' physiological backgrounds are unclear, as analysis revealed the positive correlation of animal age and weight. It can point to the possible antagonistic relationship between O. purkyniei and E. parvicaudatum. This phenomenon needs more not only microscopic but also molecular and in vitro physiological studies.

CONCLUSION
Our study is the first report on the population composition and diversity of rumen ciliates of European bison. The population structure and counts of ciliate genera and species of European bison were similar to the composition and counts of the rumen ciliated protozoa of American bison and many other kinds of free-living and domestic ruminants. Our European bison ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethical Committee of the Institute of Animal Physiology of Centre of Biosciences of SAS approved the experimental protocol (resolution number Ro-3355/16-221).

AUTHOR CONTRIBUTIONS
SK: project administration, investigation and methodology, validation, samples analysis and evaluation, data curation, manuscript writing, review, and editing. DD and KM: samples analysis and evaluation. ZV: investigation and methodology and manuscript review. JP: samples collection. MS-S: funding acquisition and supervision and manuscript review. AC: conceptualization, funding acquisition and supervision, and manuscript review. ZG: conceptualization, samples collection, and manuscript review. All authors read and approved the final manuscript.

FUNDING
This study was supported by the Grant Scientific Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences (VEGA 2/0009/08) and by the Faculty of Veterinary Medicine and Animal Science, Poznan University of Life Sciences, Poland, from the Department of Animal Nutrition (no. 506.533.04.00).