The effects of different silage additives on rumen protozoan number and volatile fatty acid concentration in sheep fed corn silage

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1 Small Ruminant Research 48 (2003) Technical note The effects of different silage additives on rumen protozoan number and volatile fatty acid concentration in sheep fed corn silage N. Dönmez a,, M.A. Karslı b, A. Çınar a, T. Aksu b, E. Baytok b a Department of Physiology, Faculty of Veterinary Medicine, University of Yüzüncü Yıl, Van, Turkey b Department of Animal Nutrition, Faculty of Veterinary Medicine, University of Yüzüncü Yıl, Van, Turkey Accepted 16 December 2002 Abstract The main objective of this study was to determine the effects of different silage additives on protozoan population, genera and total volatile fatty acid (VFA) concentrations and percentage of VFAs in corn silage. Four ruminally fistulated Morkaraman Kıvırcık lambs were used in a 4 4 Latin square design with 14-day adaptation and 1-day sampling periods. The animals were offered 20% cottonseed meal and 80% corn silage with or without treatment with silage additives, ad libitum intake. Silages used in the experiment were corn silage without treatment, treated with 5% molasses, 0.05% formic acid, and 10 g/t enzyme. Total protozoan number was significantly different among treatments (P <0.05). It was the highest in sheep fed silage treated with molasses ( ml 1 ) and the lowest in sheep fed silage treated with formic acid ( ml 1 ). Entodinium, Epidinium, Isotricha, Dasytricha, Diplodinium, and Osphyroxscolex types were observed in all treatments, but major protozoan genera were Entodinium, comprising 61 69% of total protozoan population. Total VFA concentration was significantly less in sheep fed enzyme-treated silage compared with other treatments. Percentage of acetic acid was significantly lower (P <0.05) in sheep fed silage treated with molasses compared with other treatments, but percentages of propionic acid, and butyric acid were similar among treatments. Ruminal ammonia-n concentrations ranged from 7.71 to mg/dl and were lowest in sheep fed enzyme-treated silage among treatments (P <0.05). In conclusion, the highest protozoan counts were observed in the rumen of sheep fed corn silage treated with molasses Elsevier Science B.V. All rights reserved. Keywords: Corn silage; Rumen protozoa; Rumen volatile fatty acids; Silage additives; Sheep 1. Introduction Ruminants can better utilize forages compared with mono-gastric animals because they posses microorganism in their rumen, which release enzyme to digest forages (Jounay, 1989). These microorganisms in- Corresponding author. Tel.: /1506; fax: address: donmez68@hotmail.com (N. Dönmez). clude bacteria, protozoan, yeast, and fungi (Bölükbaşı, 1989). Because of greater ability to utilize forages, ruminant diets contain substantial amount of forage. Silage has been gaining more acceptances among dairy farmers. Lactic acid is desired in silage-making, thus many different silage additives have been developed to improve conservation of silage (Rotz and Muck, 1994). Rumen protozoa have an important role on nutrition of ruminants (Hristov et al., 2001). It is known /03/$ see front matter 2003 Elsevier Science B.V. All rights reserved. doi: /s (03)

2 228 N. Dönmez et al. / Small Ruminant Research 48 (2003) that type of diet has a great impact on the number and type of protozoa (Kocabatmaz et al., 1992; Williams and Coleman, 1992; Hristov et al., 2001), consequently, on concentrations of volatile fatty acids (VFAs) in the rumen fluid, because each rumen protozoan species produces different volatile fatty acid in the rumen (Orskov and Ryle, 1990). In the literature, number of rumen protozoan ranged from to ml 1 in animals fed different diets (Sulu et al., 1988; Jounay, 1989; Jounay et al., 1992). Even though there are many studies on the effects of different diets on rumen protozoan count, there is a lack of information regarding the use of different silage additives on rumen protozoan count. Therefore, the objective of this study was to determine the effects of different silage additives on rumen protozoan count and consequently, rumen volatile fatty acid concentrations. 2. Materials and methods Four rumen fistulated Morkaraman Kıvırcık lambs, weighing 35 ± 1.2 kg, were used. The experiment was carried out using 4 4 Latin square designs with 14-day adaptation and 1-day sampling periods. The animals were offered 20% cottonseed meal and 80% corn silage with or without treatment with silage additives, ad libitum intake. Silage treatments included control (no additives) 5% molasses, 0.05% formic acid, and 10 g/t enzyme (maize-all obtained from Alltech). As recommend by manufacturer, inoculant was added at cfu/g of fresh forage. The chemical compositions of diets are presented in Table 1. Table 1 Chemical composition, ph and lactic acid content of corn silage treated with different silage additives (percentage of DM) DM b b b a 1.26 NDF a ab a b 5.06 ADF a ab a b 3.23 CP 6.84 b 7.17 b 6.63 b 9.26 a 1.18 ph Lactic acid 1.73 b 3.60 a 2.60 b 4.39 a 0.51 Means in same row with different letters (a and b) statistically differ (P <0.05). During the experiment, all animals were housed in metabolism cages and fed twice daily at 08:00 and 20:00 h. Drinking water and Vitamin mineral block were always available. Forty-milliliter samples of rumen fluid were removed at 2 h after morning feeding via the rumen cannula by suction pump. Twenty milliliters of rumen fluid was used for protozoan identification just after sampling without any additive. The remaining 20 ml was analyzed for volatile fatty acid concentrations using gas chromatography (Shimadzu, GC-14B) as described by Leventini et al. (1990). Protozoa were counted using McMaster counting chamber as described by Boyne et al. (1957) and protozoa genera were identified according to the criteria described by Boyne et al. (1957) and Ogimoto and Imai (1981). The ph of each sample was determined in triplicate using approximately 25 g wet ensilage added to 100 ml of distilled water. After hydration for 10 min using blender, the ph was determined using digital ph meter (Polan et al., 1998). The filtrate were filtered through filter paper, centrifuged and stored for organic acid analysis. Lactic acid analysis were accomplished by using gas chromatograph (Shimadzu, GC-14B) as described by Leventini et al. (1990). DM, ash, and CP contents of feed were determined following the procedure of Association of Official Analytical Chemists (1980). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed according to the method of Van Soet and Robertson (1979) Statistical analysis of data All data were subjected to analysis of variance using General Linear Model procedure of SAS (1985). Mean treatment differences were determined by Duncan s multiple range tests with a level of statistical significance of 5% (Steel and Torrie, 1980). 3. Results The chemical composition, ph, and lactic acid content of silages are shown in Table 1. Addition of molasses into corn silage significantly increased DM, CP, and lactic acid content, and decreased the concentrations of NDF and ADF (P <0.05) because molasses

3 N. Dönmez et al. / Small Ruminant Research 48 (2003) Table 2 Ruminal protozoon count ( 10 3 ml 1 ) and genera of sheep fed corn silage treated with different silage additives Total protozoa bc b c a Entodinium minimum Entodinium caudatum 1.5 b 5 a 5.75 a 1.75 b 0.89 Entodinium longinucleatum 4.75 a 2.25 b 2 b 2.75 b 0.89 Isotricha intestinalis 0.25 b 3 a 2.75 ab 4.25 a 1.76 Isotricha prostama Diplodinium ruminantium Epidinium ecaudatum 1.75 b 4.5 ab 8.25 a 1.75 b 3.67 Epidinium tricaudatum Diplodinium 26.5 a c 25.5 ab 22 bc 2.67 Osphyroxscolex caudatum Means in same row with different letters (a, b and c) statistically differ (P <0.05). contained considerably higher DM and CP and lower NDF and ADF compared with corn silage. Enzyme treatment also significantly increased lactic acid content (P <0.05), but tended to decrease NDF and ADF content of silage. Silage ph were similar among treatments (P >0.05). Protozoan numbers and genera are presented in Table 2. Total protozoan number was significantly different among treatments (P < 0.05). It was the highest in sheep fed silage treated with molasses ( ml 1 ) and the lowest in sheep fed silage treated with formic acid ( ml 1 ). Entodinium, Epidinium, Isotricha, Dasytricha, Diplodinium, and Osphyroxscolex types were observed in all treatments, but major protozoan genera were Entodinium, comprising 61 69% of total protozoan population in all treatments. The concentrations and percentages of VFAs are shown in Table 3. Total VFA concentration was significantly less in sheep fed enzyme-treated silage compared with other treatments. Acetic acid was significantly lower (P <0.05) in sheep fed silage treated with molasses compared with other treatments, but propionic acid, and butyric acid concentrations were similar among treatments and ranged from 60 to 67% for acetic acid, and 16 20% for propionic, and butyric acids. Ruminal ammonia-n concentrations ranged from 7.71 to mg/dl and were lowest in sheep fed enzyme-treated silage among treatments (P <0.05). Table 3 Ruminal VFAs and ammonia-n contents of sheep fed corn silage treated with different silage additives VFAs (mmol/l) Total ab b a a Acetic acid bc c a ab 8.41 Propionic acid a a a a 5.77 Butyric acid a a a 27.7 a 6.49 Proportions of VFAs (percentage of total VFA) Acetic acid a ab ab b 4.09 Propionic acid Butyric acid Ammonia-N (mg/dl) a 7.71 b ab a 2.92 Means in same row with different letters (a, b and c) statistically differ (P <0.05).

4 230 N. Dönmez et al. / Small Ruminant Research 48 (2003) Discussion The main objective of this study was to determine the effects of different silage additives on protozoan population, genera and total VFA concentrations and proportion of VFAs in corn silage. Total protozoan number and genera are associated with diet type and changes with ruminal proteolytic activities (Veira, 1986). Total protozoan numbers observed in the study were similar to those reported in Angora goat (Kocabatmaz et al., 1988) and in sheep (Jounay et al., 1992). In contrast, Ivan et al. (1991) reported that casein and soybean meal supplementation decreased total protozoan number in corn silage, for which total protozoan numbers were less compared with the current study. It is well known that total protozoan number can easily be affected by diet type, feeding frequency, geography where animals live, and ruminal ph (Veira, 1986; Leek, 1993). This difference on total protozoan number may explain the differences among different studies. The differences on total protozoan number were mainly results of treatment because animals were kept under same conditions. The major protozoan genera in the current study were Entodinium, comprising 61 69% of total protozoan population in all treatments. This finding is an agreement with the results of Ivan et al. (2000). Ruminants mainly depends on fermentation product produced in the rumen for energy, namely, VFAs. Even though the concentration of VFAs highly differs among diets, It generally ranges from 60 to 120 mmol/l (Bölükbaşı, 1989; Leek, 1993; Houtert, 1993). Total VFAs ranged from to mmol/l in the current study, which are in agreement with values reported in the literature (Strokes et al., 1991; Hristov et al., 2001). Among VFAs, while acetic acid has an important role in meeting animal s metabolic energy requirement and milk fat synthesis, butyric acid supplies energy to ruminal epithelial cells. Approximately, 60% of animal s glucose requirement is provided by propionic acid. Thus, the percentages of VFAs have a very important role on animal production. The percentage of VFAs is mainly associated with the composition of diet. As sugar and starch content increase in diet, percentage of acetic acid decreases, but propionic acid content increases (Leek, 1993; Houtert, 1993). Similarly, acetic acid percentage was significantly different among treatments, but the percentages of propionic and butyric acids were numerically greater in sheep fed silage treated with molasses compared with sheep fed control diet in this study (Table 3). Percentage of acetic acid also seemed to be related to total protozoan count. As percentage of acetic acid increased, total protozoan count decreased. Even though ruminal ammonia-n concentrations were lowest in sheep fed enzyme-treated silage among treatments, all ruminal ammonia-n concentrations were above the critical level for optimal microbial growth in the rumen reported by Hespell and Bryant (1979). 5. Conclusion In conclusion, the highest protozoan count was observed in the rumen of sheep fed corn silage treated with molasses. Protozoan count seemed to be related to percentage of ruminal acetic acid in this study. Thus, protozoan number may affect composition of milk, especially percentage of milk fat in ruminant animals. References AOAC, Official Methods of Analysis, 13th ed. Association of Official Analytical Chemists, Washington, DC. Bölükbaşı, F., In: Cilt, I. (Ed.), Textbook of Physiology: Body Heat and Digestion System. A. Ü. Basımevi, Ankara. Boyne, A.W., Eadie, J.M., Raitt, K., The development and testing of a method of counting rumen ciliate protozoa. J. Gen. Microbiol. 17, Hespell, R.B., Bryant, M.P., Efficiency of rumen microbial growth: influence of some theoretical and experimental factors on YATP. J. Anim. Sci. 49, Houtert, M.F.J., The production and metabolism of volatile fatty acids by ruminants fed roughages: a review. Anim. Feed Sci. Technol. 43, Hristov, A.N., Ivan, M., Rode, L.M., McAllister, T.A., Fermentation characteristics and ruminal ciliate protozoal populations in cattle fed medium- or high concentrate barley-based diets. J. Anim. Sci. 79, Ivan, M., Charmley, L.L., Neill, L., Hıdıroglou, M., Metabolic changes in the rumen following protozoal inoculation of fauna-free sheep fed a corn silage diet supplemented with casein or soybean meal. Ann. Rec. Vet. 22 (2), Ivan, M., Neill, L., Forster, R., Alimon, R., Rode, L.M., Entz, T., Effects of Isotricha, Dasytricha, Entodinium and total fauna on ruminal fermentation and duodenal flow in wethers fed different diets. J. Dairy Sci. 83 (4), Jounay, J.P., Effects of diet on populations of rumen protozoa in relation to fibre digestion. In: Nolan, J.V., Leng, R.A., Demeyer, D.I. (Eds.), The Roles of Protozoa and Fungi

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