Activity of cellulolytic enzymes in the contents of reticulorumen and caecocolon of roe deer (Capreolus capreolus)

Comparative Biochemistry and Physiology Part A 119 (1998) 925 – 930 Activity of cellulolytic enzymes in the contents of reticulorumen and caecocolon of roe deer (Capreolus capreolus) Anke Deutsch a,*, Matthias Lechner-Doll a, Gerhard A. Wolf b b a Institute for Zoo Biology and Wildlife Research (IZW), P.O. Box 601103, 10252 Berlin, Germany Institute for Plant Pathology and Plant Protection, Uni6ersity of Göttingen, Griesebachstr. 6, 37077 Göttingen, Germany Received 3 June 1997; received in revised form 18 November 1997; accepted 3 December 1997 Abstract Selective ruminants, which prefer easily digestible plants, cannot digest fibrous forage as well as grass eaters. Low enzyme activity or short retention time of ingesta particles in fermentation chambers appeared to be responsible for reduced cellulose breakdown. Seasonal activity of cellulolytic enzymes, cellulose concentration and protozoa population in reticulorumen (RR) and caecocolon (CC) of roe deer as a typical concentrate selector were investigated. Cellulase activities were lowest in winter when cellulose concentration in RR contents were highest. Highest enzyme activities and lowest cellulose concentration were measured in early spring. Cellulolytic activities were significantly correlated with the number of protozoa in RR. Only one entodinomorphic genus was identified in the RR. The enzyme activities in CC were far lower compared with those in RR. Low cellulose digestion in the RR cannot be compensated for by cellulose breakdown in the CC. The reduced cellulose digestion of roe deer may be attributed to the short retention time of food particles in spring and summer, whereas decreased colonisation of microorganisms in the rumen may be the main reason for low cellulose breakdown in winter. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Caecocolon; Cellulase activity; Cellulose; Morphophysiological adaptation; Protozoa; Reticulorumen; Roe deer; Seasonality 1. Introduction Roe deer (Capreolus capreolus) is one of the smallest wild ruminants in the temperate climate zone. Distribution extends from Western Europe over Western Asia to East and Southeast China [22,23]. Roe deer, like other concentrate selectors, are equipped with a digestive system far less suited to optimise plant fibre digestion compared with grass and roughage eaters [13]. They are adapted to processing easily digestible forage. Thereby they avoid cellulose and choose a diet which is rich in plant cell contents e.g. forbs, flowers and leaves [24]. In roughage eaters like sheep or cattle, digestion of cellulose from their grass-based diets is a main source of energy. An effi* Corresponding author. Tel.: + 49 30 5168732; fax: 49 30 5126104. 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)00004-X cient cellulose breakdown can be achieved by high microbial cellulolytic activities and a long particle retention time in fermentation chambers. The aim of the present study was to examine cellulolytic activities in the roe deer digestive system. Is the microbial cellulase activity influenced by the seasonal or the feeding situation, respectively, and is it possible that reduced cellulose digestion in the reticulorumen (RR, rumen and reticulum) is compensated for by cellulose breakdown in the caecocolon (CC, caecum and ansa proximalis coli) as observed in the Greater Kudu [4]? 2. Materials and methods Seasonal changes of cellulose concentration and cellulase activity in the ingesta of RR and CC of roe deer were investigated and protozoa population in the RR 926 A. Deutsch et al. / Comparati6e Biochemistry and Physiology, Part A 119 (1998) 925–930 was characterised. Samples from domestic sheep were obtained for comparative reasons. 2.1. Collection and preparation of samples Whole ingesta of RR (n =37) and CC (n =28) were obtained from 38 male roe deer (yearling and adult) rapidly after being shot in Schleswig-Holstein and Brandenburg (Germany) between February and August. RR ingesta samples were collected from two cannulated sheep kept on a standard hay-concentrate diet in April and May. Every sample was mixed to obtain representative subsamples. About 5 g of fresh RR contents (n =7) were put into methyl green formalin (1000 ml 10% formalin, 0.6 g methyl green, 8 g NaCl) in relation 1:1 for protozoa identification [17]. About 100 g of the remaining samples were stored frozen (−20°C) prior to analysis of cellulose concentration and cellulase activity within 1 month. 2.2. Determination of cell wall constituents and identification of protozoa About 30 g of frozen RR and CC ingesta samples were dried at 80°C to a constant weight, ground in a willey mill to pass a 1 mm screen and subsampled for determination of neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) according to the technique of van Soest [25]. Cellulose contents can be determined with the equation: cellulose =ADF −ADL [20]. RR ingesta samples in methyl green formalin were mixed, diluted with distilled water 1:10 and filtered through a sieve (1-mm pore size) to separate coarse constituents [17]. About six drops of well-mixed filtrate were filled into a special counting chamber (Nageotte, vol.: 50 mm3). Protozoa were identified and counted directly under the light microscope using a magnification 1:1000. vals (70% treatment time). Finally, the samples were filtered through a 200-mm mesh (Faust, Berlin, Germany) and centrifuged at 26000×g and 4°C for 20 min (Beckmann J2-MC, Germany) (Fig. 1). Dye-labelled substrate (50 ml, Carboxymethyl-Cellulose-Remazol Brilliant Clue, 4 mg/ml) and buffer (100 ml, 0.2 M sodium acetate – acetic acid buffer, pH 5.5) were preincubated on microtitre plates (350-ml cavities) in a water bath (SBD 50 Heto-Holten, Wettenberg, Germany) at 40°C for 5 min. After the addition of cellulase extract (50 ml), the plates were sealed with a low evaporation lid and incubated for 60 min. The reaction was stopped and the non-degraded high-polymeric substrates were precipitated by adding 50 ml of 2 N HCl and subsequent cooling on ice. Blanks were prepared in a similar way but without the addition of enzyme extract during incubation. Subsequently, the plates were centrifuged (1050× g, 10 min, Hettich Rotana/TRC 4401, Tuttlingen, Germany). Supernatants (150 ml), containing soluble dye-labelled degradation products were transferred to a new microtitre plate (350-ml cavities) and measured spectrophotometrically (Spectra Shell, SLT Labinstruments, Germany) at 600 nm against blanks. All data on cellulase activity are given as extinction which indicates relative enzyme activity. 2.4. Statistics The influence of the season on cellulose concentration and enzyme activity was tested with the Kruskal– Wallis non-parametric ANOVA Test. Changes of cellulose concentration and cellulase activity between seasons February/March (S1), April/May (S2) and June to August (S3) were tested with Dunn’s multiple comparisons test. Correlations between the values of RR and CC were determined with Spearman correlation coefficients. 2.3. Assay of cellulolytic acti6ity A new method as described by Wolf and Wirth [27] was modified to quantify cellulase activity in digesta samples. In principle the colorimetric assay of cellulase activity is based on enzymic degradation of soluble polysaccharide derivates, labelled covalently with dye. About 10 g of each frozen sample were homogenised in 40 ml cold (4°C) sodium acetate−acetic acid buffer (pH 5.5, 0.2 M) by a blender (Alaska STM 130, Germany) for 1 min at highest speed. Cellulolytic enzymes were solubilised by ultrasonic treatment on ice at a frequency of 20 kHz using an ultrasonic receiver (Sonoplus HD70 of Bandelin electronic, Berlin, Germany). Ultrasonic treatment times were 4 min at inter- Fig. 1. Extraction of cellulolytic enzymes from digesta samples. A. Deutsch et al. / Comparati6e Biochemistry and Physiology, Part A 119 (1998) 925–930 927 Fig. 2. Cellulose content in RR of roe deer (February, n = 2; March, n = 2; April, n = 2; May, n = 10; June, n= 12; July, n = 3; August, n = 5). Comparisons between roe deer and domestic sheep were carried out using the Mann – Whitney U-test. 3. Results 3.1. Seasonal changes of cellulose concentration in reticulorumen and caecocolon of roe deer Cellulose concentration in RR was 21% of dry matter (DM) in February and March. In early spring the concentration decreased to 10% of DM and rose steadily to 15% of DM in late summer (Fig. 2). The influence of the season on cellulose concentration in RR was highly significant (P= 0.0031). The cellulose concentration of RR was significantly higher in February and March (S1) than in both April/May (S2) (P B 0.01), and June to August (S3) (P B0.05), whereas no significant differences between cellulose concentration in S2 and in S3 were observed. The cellulose concentration in CC of roe deer (Fig. 3) was significantly (P B0.0001) correlated with cellulose concentration in RR. The concentration of cellulose in Fig. 4. Cellulase activity in RR and CC of roe deer from February to August (test: pH 5.5, 40°C, incubation time 60 min). CC was significantly higher in S1 than in S2 (PB 0.05) and S3 (P B0.05) like in RR. 3.2. Seasonal changes of cellulase acti6ity in reticulorumen and caecocolon of roe deer In February the activities of cellulolytic enzymes in RR were 0.26290.084 (mean9SD). The activities reached a peak at 0.509 9 0.004 in April and decreased steadily thereafter to 0.2419 0.086 in August (Fig. 4). The enzyme activities in CC were on average 63% lower compared with the cellulolytic activities in RR. The statistical comparison of cellulase activities in both RR and CC indicated a significant influence of the season on enzyme activities (RR, P= 0.0007; CC, P= 0.0021). The cellulolytic activity in RR contents was significantly higher in S2 than in S1 (P B0.05) and S3 (P B0.001), whereas cellulase activity in CC contents was significantly higher in S2 than in S1 (P B0.01). 3.3. Cellulose concentration and cellulase acti6ity in reticulorumen of roe deer compared with domestic sheep Cellulose concentration and cellulase activity in RR were compared between roe deer (n =12) and domestic sheep (n =2; nine measurements) in April and May, showed in Table 1. The percentage concentration of Table 1 Average cellulose concentration and cellulase activity in RR of roe deer (n= 9) and sheep (n = 2 with six repeats) in April and May Roe deer Fig. 3. Cellulose content in CC of roe deer (February, n = 1; March, n = 2; April, n = 2; May, n = 9; June, n = 8; July, n = 3; August, n = 3). Cellulose concentration (%) 10.62 9 2.72 Cellulase activity (extinction 600 0.448 9 0.074 nm) Sheep 19.19 9 3.51 0.278 9 0.051 928 A. Deutsch et al. / Comparati6e Biochemistry and Physiology, Part A 119 (1998) 925–930 Fig. 5. Correlation between cellulase activity and protozoa concentration in RR of roe deer. cellulose was significantly lower in RR contents of roe deer than in RR contents of sheep (P= 0.0009), whereas the cellulase activity in RR contents of both species were similar in this season. 3.4. Identification of protozoa in reticulorumen contents of roe deer Only one entodinomorphic genus was identified in the RR of roe deer. No protozoa were found in one of seven samples investigated. The quantity of ciliates in RR varied seasonally from (82.9914.8) ×103 to (2476.395.8) × 103/g fresh RR contents. The highest density of entodinia was observed at the beginning of the vegetation period (April), whereas the number of protozoa decreased in March and August. Cellulolytic activity correlated significantly with the number of ciliates in RR content (F= 45.372, P = 0.0067) as shown in Fig. 5. 4. Discussion In the present work, the cellulose content, cellulase activity and protozoa concentration in RR and CC of male roe deer were studied from February to August. The data from male roe deer can be considered representative of the species as long as diet selection is concerned. Feed intake may be higher in females in late pregnancy (March and April) and during lactation (May – July), with unknown consequences for cellulose digestion. A new method by Wolf and Wirth [27] was employed the first time to determine cellulase activity in RR and CC of roe deer by a special cellulose derivate. Anaerobic cultures of microbes are not necessary. The use of soluble chromogenic substrates offers a direct and more specific, sensitive and rapid detection of endo-acting polysaccharide hydrolases [2,3,27]. Endoglucanases initiate the cellulose breakdown and transform great insol- uble polysaccharides to small soluble units. Endoglucanase activity is the rate-limiting step in cellulose breakdown. Activities of exoglucanases and b-glucosidases are not rate-limiting and thus cannot be determined with this method [18,19,26]. The colorimetrical method used in the present study is less complex yet more sensitive and reliable than viscosimetry, turbidimetry or methods employing insoluble or even chromogenic substrates. The solubility of the CM-cellulose leads to a better distribution of substrate in the solution and to a quick breakdown. Short incubation periods prevent losses of enzyme activities caused by denaturation of protein. Behavioural and morphophysiological adaptation of roe deer to seasonal changes of the forage quality and availability may be summarised as follows. Several structures of its digestive system e.g. large salivary glands, relative small rumen, and large reticulo-omasal ostium, are the base of a specific adaptation to a low-fibre forage rich in plant cell contents [12]. In spring and summer the nutritional strategy of roe deer is orientated towards high and frequent intake of readily digestible forages. Feed intake of roe deer in enclosures as estimated by Barth [10] increased sharply by 50% in April after a low intake during winter. The quantity of food consumed by roe deer is directly proportional to its level of assimilation and protein content, and inversely related to its cellulose content [25]. The largest quantity of plant matter is consumed during spring when assimilation is highest [5]. High feed intake and large salivary glands of roe deer, which supply more diluting liquid, reduce retention time and allow rapid rumen turnover [16]. At the end of autumn and in winter, on the other hand, as a consequence of low forage quality, limited diversity and decreased availability, roe deer reduce forage intake and metabolism. Reduced food intake results in a structural transformation of the digestive system over a period of 10– 20 days [10,11]. The main factor is the reduction of the ruminal mucosal surface. This results from a reduction of papillary number and length, caused by reduced blood flow due to decreased fermentation and volatile fatty acids concentrations [14]. Fewer intake periods and a smaller reticulo-omasal ostium in roe deer restrict passage rate of slowly digestible fibrous forage and increase the ratio of rumen content to rumen capacity [15]. This may result in increased rumen fill, and decreased rumen turnover in order to make cell wall digestion and absorption more complete [1]. Longer retention times in winter were measured by Holand [16]. The adaptation processes are cyclic and occur again when highly digestible forage becomes available during March/April. The bucks reduce forage intake during the rut in August like in winter. A. Deutsch et al. / Comparati6e Biochemistry and Physiology, Part A 119 (1998) 925–930 In our study, lower cellulose concentrations were observed both in the RR and CC contents of roe deer during spring and summer than in winter. These results indicate a seasonal shift in the quality and availability of forages consumed by roe deer. During spring, the main food of roe deer consists of young leaves, forbs and the buds and shoots of woody browse [9], whereas in winter only slowly digestible fibrous forage is available. Digestibility of plants decreases with increasing cellulose content in the cell wall. Increased proportions of lignin in cell walls lead to a maximum of fibre content of forage plants in winter. Seasonal changes of cell wall concentration in the rumen contents has been also reported by Holand [15]. Seasonal changes of cellulase activity in RR and CC of roe deer were observed in the present study. During winter, when feed intake of roe deer is greatly reduced and retention time of feed particles in the RR increases, which theoretically should result in an improvement of cellulose digestion, cellulase activity is very low. As a consequence, cellulose breakdown remains low even in winter, although the proportion of cellulose in the diet is much higher compared with the other seasons. Our results point to a significant increase of cellulase activity in April. Activity of enzymes depends on substrate concentration and availability for the microbes as well as physicochemical conditions in the RR (shortchain fatty-acid concentration, pH). The high consumption of easily digestible forage rich in accessible plant cell contents (soluble carbohydrates, protein) and nutrients leads to an improvement of the growing conditions for microorganisms due to a substrate change in the rumen. Increasing proliferation results in a higher density of microorganisms including cellulolytes adhering to forage particles in the rumen. Increasing density of microorganisms and consequently a higher concentration of cellulolytic enzymes, but not a higher cellulose content of forage, appear to be reasons for higher cellulase activity in RR of roe deer in April. The cellulase activity is an indicator for the colonisation of feed particles by cellulolytic microorganisms in the rumen [18]. Cellulase activity decreased from May to August depending on forage quality and intake of roe deer and reached a minimum during the rutting season (August) of roe bucks, whose intake then is very low. Cellulolytic activity in CC is only about 37% of that in the RR on average in all seasons. As a consequence, reduced cellulose digestion in the rumen cannot be compensated for by cellulose breakdown in the CC. The distal fermentation chambers (caecum and ansa proximalis coli) are particularly voluminous and are mainly used for hemicellulose breakdown [8]. Cellulolytic fermentation in CC, however, is supplementing the energy balance of roe deer only negligibly [6]. 929 The cellulase activity in the forestomach of roe deer under optimal feeding conditions (April/May) is not different from sheep although roe deer select and ingest plant material which contains very little cellulose in spring time. Hence it may be concluded that reduced cellulose breakdown in the rumen of roe deer depends slightly on enzymic activity of microorganisms in spring. The relatively small and simple rumen, inducing frequent feeding bouts and with wide ostia for rapid passage of ingesta may be an important reason for slow cellulose fermentation mainly in the vegetation period. Large salivary glands of roe deer supply more diluting liquid, which also reduce retention time [13]. In contrast, a large subdivided rumen, narrow ostia and small salivary glands of sheep prevent quick outflow of ingesta promoting a high cellulose digestion. In the rumen of domestic ruminants more species of ciliata are found than in rumen of wild ruminants [21]. Only one genus (entodinomorphic genus) was identified in ingesta of RR of roe deer in our study and similarly by Drescher-Kaden [7]. The concentration of protozoa was higher in rumen of roe deer than in rumen of chamois and red deer as compared with results of Drescher-Kaden [7]. Frequent feeding rhythm and consumption of easily digestible nutritious forage may also cause a high density of protozoa in the rumen. When ingesting low quality feed in winter, decreasing concentrations of ciliata down to 50% were observed in both roe deer and other wild ruminants [7]. During the rutting season of roe bucks the protozoa density is decreasing even below the concentration of protozoa in winter due to reduced feed intake and high water content of the ingesta. Lower substrata availability for the microbes results in declining proliferation of ciliata. Furthermore water intake and salivation leads to diluted ingesta. A positive correlation between protozoa concentration and cellulase activity was observed. Protozoa density increased with increasing cellulolytic activity in rumen of roe deer. This may be a hint that protozoa concentration is an indicator for microbial colonisation in the rumen. The proliferation of cellulolytic bacteria increases concurrently with better growing conditions for ciliata. 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