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Amino acid and fatty acid composition of microorganisms passing into the abomasum of sheep
Livestock Production Science, 3(1976)57--63 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
57
AMINO ACID AND FATTY ACID COMPOSITION OF MICROORGANISMS PASSING INTO THE ABOMASUM OF SHEEP
N.V. KURILOV, N.A. SEVASTIANOVA, V.N. KC RSHUNOV, N.D. MYSNIK and A~E. PODSHIBYAKIN All-Union Scientific Research Institute of Farm Animal Physiology, Biochemistry and Nutrition, Borovsk, Kaluga Region (U.S.S.R.)
(Received June 13th, 1974)
ABSTRACT Kurilov, N.V., Sevastianova, N.A., Korshunov, V.N., Mysnik, N.D. and Podshibyakin, A.E., 1976. Amino acid and fatty acid composition of microorganisms passing into the abomasum of sheep. Livest. Prod. Sci., 3: 57--63. The bacterial and protozoal fractions were isolated by a differential centrifugation method from daily samples of chyme passing into the abomasum from the forestomach of sheep. The mean values for the nitrogen content (percentage of dry matter) were 8.3 + 0.2% for the bacterial fraction and 5.3 ± 0.2% for the protozoal fraction. They were not significantly affected by the different products, methionine, lysine or starch, added to the basal ration. The amino acid composition of the bacterial and protozoal proteins was relatively constant, though some slight variations in certain amino acids were observed in relation to the nature of the supplements. The total lipid content ranged from 5.42 to 8.85% of the dry matter for the bacterial fraction. Its mean value was 9.63% for the protozoal fraction. It was not affected significantly by the supplements. Free fatty acids dominate the lipid composition of bacteria and phospholipids dominate in that of protozoa. The principal fatty acids of protozoal lipids were palmitic acid (41.32%) and oleic acid (19.33%). INTRODUCTION K n o w l e d g e o f t h e c h e m i c a l c o m p o s i t i o n o f t h e m i c r o b i a l m a s s passing i n t o t h e a b o m a s u m is o f n u t r i t i o n a l i m p o r t a n c e . A n a l y s e s o f r u m e n b a c t e r i a a n d p r o t o z o a f o r p r o t e i n ( S m i t h a n d B a k e r , 1 9 4 4 ; M c N a u g h t et al., 1 9 5 0 ; Weller, 1 9 5 7 ) , a m i n o acid (Weller, 1 9 5 7 ; Bergen e t al., 1 9 6 8 ) , lipids ( G a r t o n a n d Oxf o r d , 1 9 5 5 ) a n d f a t t y acids (Williams a n d D i n u s s o n , 1 9 7 3 ) h a v e b e e n reported. T h e p u r p o s e o f o u r w o r k was t o d e t e r m i n e t h e p r o t e i n , a m i n o acid a n d lipid c o m p o s i t i o n o f b a c t e r i a l a n d p r o t o z o a l f r a c t i o n s passing o u t o f t h e f o r e s t o m a c h i n t o t h e a b o m a s u m o f s h e e p a n d t o s t u d y t h e e f f e c t o f various diets o n t h a t c o m p o s i t i o n .
58
MATERIALS AND METHODS
The experiments were carried out on five Cigay sheep. Each animal was fitted with a chronic rumen fistula and external anastomosis between the omasum and the abomasum (Kurilov et al., 1973). Each experimental period lasted for 25--30 days. During the first period, all the sheep were given a hay concentrate ration, balanced as regards the main nutrients, and were provided with water ad libitum. This basal ration consisted of 800 g clover/timothy hay, 400 g all-mash, 500 g fodder beet. During the other experimental periods the basal ration was supplemented with either 2 g d/-methionine, or 3 g /-lysine, or 50 g corn starch, or 20 g starch + 2 g methionine. Rumen content samples were collected regularly 3 h after feeding. At the end of the experimental period, daily total chyme entering the abomasum was determined during two diurnal tests. Every hour, chyme samples were taken (20--50 ml, constituting 1/10 of chyme passing in 1 h). For the purpose of isolation of the protozoal and bacterial fractions, a differential centrifugation method was applied to the daily chyme sample which had been preliminarily filtered through four layers of cheesecloth. Then 500 ml of the strained chyme was mixed with an equal volume of a fixing fluid that consisted of 0.2% Tween-80 dissolved in an 8% formalin solution. The mixture obtained was stored at room temperature for 48 h with periodical mixing to allow better isolation of microbial cells from food debris. For isolation of the protozoa, the mixture was centrifuged at 1 000 rpm for 3--5 rain. The sediment was mixed with 0.9 N NaC1 solution and re-centrifuged. Bacteria were isolated by centrifuging the supernatant fluid at 15 000 rpm for 15 min. The sediment was mixed with 0.9 N NaC1 solution and centrifuged ag~n. The protozoal and bacterial preparations were repeatedly washed and centrifuged until pure microbial cells were observed by microscopical examination. They were air dried to constant weight at 65°C. Total nitrogen was determined according to Kjeldahl (1970), total lipids according to Folch (1970), and the lipid fractions separated by chemical methods (Pokrovsky, 1969). Amino acids were determined in acid (with 6 N HC1); hydrolysates of bacterial and protozoal biomass, with a Hd-1200 E automatic analyser (U.S.S.R. product). Fatty acids were only determined in total lipid samples of rumen protozoa by a method of gas--liquid chromatography using a "Chrom-4" model chromatograph. RESULTS On average, the mean nitrogen content was 8.3+ 0.2% of dry matter for the bacterial mass, and 5.3-+ 0.2% for the protozoal mass (Table I). It was not significantly modified by the different supplements added to the basal ration. The slight decrease for the protozoa found in sheep with the starch supplements could be explained by the incorporation of starch granules within the cells.
59
TABLE I Nitrogen and protein c o n t e n t of bacterial and protozoal mass, isolated from the c h y m e ,
leaving the sheep f o r e s t o m a c h u n d e r v a r i o u s diets. D i f f e r e n c e s at e x p e r i m e n t a l s t a g e s in all cases are n o n - s i g n i f i c a n t (P > 0.1). Ration Total nitrogen
Hay concentrate r a t i o n ( B R ) ( n = 5) B R + s t a r c h (n = 5) B R + m e t h i o n i n e (n = 4) B R + l y s i n e (n = 5) BR + methionine + s t a r c h (n -- 4)
Protein (nitrogen × 6.25)
Bacteria
Protozoa
Bacteria
8.34 8.22 8.40 8.10
5.34 5.00 5.47 5.43
52.12 51.38 52.50 50.62
± 0.28 +- 0 . 1 1 -+ 0 . 2 9 +- 0 . 1 8
8.30 ± 0.12
± 0.19 ± 0.27 ± 0.37 ± 0.19
4 . 8 8 ± 0.21
± ± ± ±
Protozoa
1.75 0.69 1.81 1.12
51.88 ± 0.75
33.38 31.25 34.19 33.94
± ± ± ±
1.18 1.69 2.31 1.19
3 0 . 5 0 ± 1.31
When the sheep were fed the basal hay concentrate ration, mean amounts entering the abomasum per day were 33.3 g of bacterial dry matter, 48.2 g of protozoal dry matter and 32.5 g o f microbial protein. These amounts were increased b y the addition of 2 g dl-methionine to the basal ration: 43.8 g bacteria, 56.6 g p r o t o z o a and 41.88 g microbial protein. Approximately the same a m o u n t of microbial protein (41.25 g) entered the abomasum when 3 g l-lysine was added. The addition of these amino acids resulted in an increase in the contribution of bacterial protein to the total microbial protein (59.1% with the lysine supplement). Amino acid composition of bacteria and protozoa is relatively stable b u t in some cases there were significant differences in the c o n t e n t of certain amino acids according to the experimental period (Table II). Thus, the addition of methionine to the basal ration brought a b o u t a higher c o n t e n t of arginine, aspartic acid and serine in bacterial protein, and a slight decrease in isoleucine and leucine contents. On the contrary, the c o n t e n t of leucine and isoleucine increased in the protozoal protein b u t the serine and threonine levels decreased. These modifications in amino acid composition may be explained by the modifications in species composition of the population. So the addition of dl-methionine p r o m o t e d an increase in the number of amylolytic, lactate fermenting and urea assimilating bacteria. Protozoal species composition became more variable (Kolenjko et al., 1973). The amounts of total lipids entering the abomasum ranged from 40.3 to 81.5 g per day depending u p o n the ration. The total lipid c o n t e n t of the bacteria ranged from 5.42 to 8.85%. The value for the protozoa was less variable (mean: 9.63%). However, a significant decrease from 10.2+ 0.81 to 6.9+ 0.47% (P < 0.05) was observed when 50 g starch was added to the basal ration. Table III shows the percentage composition of bacterial and protozoal lipids when the sheep were given the basal hay-concentrate ration. Phospholipids are the major lipid fraction in the p r o t o z o a (mean: 51.78+ 0.92; maximum: 58.18%), and free fatty acids (41.32 + 9.5%) in the bacteria. The
60
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T A B L E III Lipid class c o m p o s i t i o n o f b a c t e r i a l a n d p r o t o z o a l mass. Values are e x p r e s s e d as a p e r c e n t age o f t o t a l lipids Lipid classes
Protozoa
Bacteria
Phospholipids Triglycerides Free f a t t y acids Cholesterol Others
51.78 10.83 13.28 3.24 20.87
28.09 3.59 41.32 4.30 22.70
± 0.92 ± 0.71 +- 0.59 ± 0.11
± 3.52 ± 0.77 -+ 9 . 5 0 ± 0.31
proportion of triglycerides is higher in the protozoa than in the bacteria. 30 fatty acids, of chain lengths ranging from 8 to 22 carbon atoms, were identified in the protozoa (Table IV). Saturated fatty acids constitute 66.5% of the total. Palmitic acid is the major fatty acid (41.32%). The presence of branched fatty acids (4.37% of the total) is also typical of protozoal lipids. TABLE IV F a t t y acid c o m p o s i t i o n o f lipids in o v i n e m i x e d r u m e n p r o t o z o a . Values are e x p r e s s e d as a p e r c e n t a g e o f t o t a l f a t t y acids c a l c u l a t e d using p e a k h e i g h t s a n d r e t e n t i o n t i m e . T h e n u m b e r f o l l o w i n g a semi c o l o n i n d i c a t e s t h e n u m b e r o f d o u b l e b o n d s . F a t t y Acids
M ± m
Cs--C12 Cl~:o Cl3:o b r C~4:o Cl4:o b r C~s:o Cis:o b r C1,:o C,6:obr C16:, C1~:o C1~:o b r C,8:o Cls: o b r Cle:l C1s:2 C1s:3 Ci9:0
0.37 0 . 2 2 ± 0.04 0 . 1 2 ± 0.03 0.8 +- 0 . 0 6 0.20 ± 0.05 1.67 ± 0 . 1 0 1.26 +- 0.07 4 1 . 3 2 ± 1.60 1.30 -+ 0.20 0 . 8 6 +- 0 . 1 5 0 . 2 9 ± 0.04 1 . 0 2 -+ 0 . 0 6 11.50 ± 0.93 0.47 1 9 . 3 3 ± 0.89 9 . 0 9 -+ 0 . 9 0 1.34 ± 0 . 2 0 0.43 5.50+- 1 . 1 0 2.89
C22:o Other
S a t u r a t e d acids 66.51 U n s a t u r a t e d acids 33.49 T o t a l i s o m e r s o f C13, Cl4, C~s, Cl,, C1~, C18, 4.37 br -- branched
62 REFERENCES Bergen, W.G., Purser, D.B. and Ceine, J.N., 1968. Determination of limiting amino acids of rumen isolated microbial proteins fed to rats. J. Dairy Sci., 51(10): 1698--1700. Garton, G.A. and Oxford, A.E., 1955. The nature bacterial lipids in the rumen o f hay-fed sheep. J. Sci. Prod. Agric., 6: 142. Hungate, R.E., 1966. The Rumen and Its Microbes. Academic Press, New York, N.Y. Kolenjko, E.I., Tarakanov, B.V., Guschin, N.N., Dolgov, I.A. and Shavyrina, T.A., 1973. Forestomach microflora of sheep fed routine diets with DL-methionine and L-Lysine. Nauchn. Tr. Vses. Nauchno-Issled. Inst. F.Bi. Promsti. Borovsk, XII: 243--249 (in Russian). Kurilov, N.V., Sevastianova, N.A., Mysnik, N.D., Korshunov, V.N. and Agafonova, T.A., 1973. Effect of various types of feeding on the synthesis of bacterial protein, amino acids and lipids in sheep rumen. Nauchn. Tr. Vses. Nauchno-Issled. Inst. Promsti, Borovsk, XII: 142--149 (in Russian). McNaught, M.L., Smith, J.A.B., Henry, K.M. and Kon, S.K., 1950. The utilization of nonprotein nitrogen in bovine rumen. 5. The isolation and nutritive value of preparation of dried tureen bactei-ia. Biochem. J., 46:32. Pokrovsky, A.A., 1969. Biokhimicheskie metodi issledovaniya v klinike. M., "Meditsina." Smith, J.A. and Baker, F., 1944. The utilization of urea in the bovine rumen. IV. The isolation of the synthesized material and the correlation between protein synthesis and microbial activity. Biochem. J., 38: 496. Williams, P.P. and Dinusson, W.E., 1973. Amino acid and fatty acid composition of bovine ruminal bacteria and protozoa. J. Anita. Sci., 36(I): 151--155.
RESUME Kurilov, N.V., Sevastianova, N.A., Korshunov, V.N., Mysnik, N.D. et Podshibyakin, A.E., 1976. Les teneurs en acides amines et acides gras des micro-organismes passant dans la caillette du' mouton. Livest. Prod. Sci., 3 : 5 7 - - 6 3 (en anglais). Les bact~ries et les protozoaires du contenu digestif passant du feuillet dans la caillette du m o u t o n ont ~t~ isol~s par une m~thode de centrifugation diff~rentielle. Les teneurs en azote o n t ~t~ de 8.3 -+ 0.2% (de la mati~re s~che) pour les bact~ries et de 5.3 +- 0.2% pour les protozoaires. Elles n ' o n t pas ~t~ modifi~es de fa~on significative par les diff~rents compigments, m~thionine, lysine ou amidon, qui ont ~t~ ajout~ ~ la ration de base. La composition en acides amines des prot~ines, des bact~ries et des protozoaires a ~t6 relativement constante; la p r o p o r t i o n de certains acides amines a cependant pr~sent~ des variations limitfies selon la nature des compl~ments. La teneur en lipides t o t a u x a vari~ de 5.42 ~ 8.85% de la mati~re s ~ h e pour les bact~ries et elle a ~t~ en moyenne de 9.63% pour les protozoaires. Elle n'a pas ~t~ modifi~e significativement par les compl~ments. Les acides gras libres sont les constituants majeurs des lipides bact~riens et les phospholipides de ceux des lipides des protozoaires. La composition en acides gras de ces derniers est caract~ris~e par une proportion ~l~v~e d'acide palmitique (41.32%) et d'acide ol~ique (19.33%). KURZFASSUNG Kurilov, N.V., Sevastianova, N.A., Korshunov, V°N., Mysnik, N.D. and Podshibyakin, A.E., 1976. Zusammensetzung yon Aminos~uren und Fetts~iuren von Mikroorganismen, die in den Labmagen yon Schafen iibertreten. Livest. Prod. Sci., 3 : 5 7 - - 6 3 (in Englisch). Die Bakterien- und Protozoenfraktionen wurden mit Hilfe einer Differentialzentrifugenmethode aus einer t/iglichen Durchschnittsprobe des Chymus isoliert, der aus dem Vormagen
63
des Schafes in den Labmagen iibertritt. Die Durchschnittswerte fiir den Stickstoffgehalt (% der Trockenmasse) betrugen 8.3 +- 0.2% ftir die Bakterienfraktion sowie 5.3 +- 0.2% fiir die Protozoenfraktion. Sie wurden dutch Zusatz verschiedener 8toffe zur Grundration, wie Methionin, Lysin oder St~rke, nicht signifikant beeinflusst. Die Amino~urenzusammensetzung des Bakterien- und Protozoeneiweiss blieb relativ konstant, obgleich einige geringe Variationen bei bestimrnten Aminos~iuren in Abh~/ngigkeit yon der Art der Zusatzstoffe beobachtet wurden. Der Gesamtgehalt an Lipoiden schwankte zwischen 5.42 und 8.85% des Trockengehaltes der Bakterienfraktion. Der durchschnittliche Gehalt betrug 9.63% fiir die Protozoenfraktion. Er wurde dutch Zusatzstoffe nicht signifikant beeinflusst. In der Lipoidzusammensetzung der Bakterien dominieren die freien Fetts~uren, w~/hrend die Phosphorlipoide bei den Protozoen dominieren. Die bedeutendsten Fett~uren der Protozoenlipoide waren Palmitins~ure (41.32%), Ols~ure (19.33%), Stearins~iure (11.50%) und Linol~ure (9.09%). PE3©ME
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57
AMINO ACID AND FATTY ACID COMPOSITION OF MICROORGANISMS PASSING INTO THE ABOMASUM OF SHEEP
N.V. KURILOV, N.A. SEVASTIANOVA, V.N. KC RSHUNOV, N.D. MYSNIK and A~E. PODSHIBYAKIN All-Union Scientific Research Institute of Farm Animal Physiology, Biochemistry and Nutrition, Borovsk, Kaluga Region (U.S.S.R.)
(Received June 13th, 1974)
ABSTRACT Kurilov, N.V., Sevastianova, N.A., Korshunov, V.N., Mysnik, N.D. and Podshibyakin, A.E., 1976. Amino acid and fatty acid composition of microorganisms passing into the abomasum of sheep. Livest. Prod. Sci., 3: 57--63. The bacterial and protozoal fractions were isolated by a differential centrifugation method from daily samples of chyme passing into the abomasum from the forestomach of sheep. The mean values for the nitrogen content (percentage of dry matter) were 8.3 + 0.2% for the bacterial fraction and 5.3 ± 0.2% for the protozoal fraction. They were not significantly affected by the different products, methionine, lysine or starch, added to the basal ration. The amino acid composition of the bacterial and protozoal proteins was relatively constant, though some slight variations in certain amino acids were observed in relation to the nature of the supplements. The total lipid content ranged from 5.42 to 8.85% of the dry matter for the bacterial fraction. Its mean value was 9.63% for the protozoal fraction. It was not affected significantly by the supplements. Free fatty acids dominate the lipid composition of bacteria and phospholipids dominate in that of protozoa. The principal fatty acids of protozoal lipids were palmitic acid (41.32%) and oleic acid (19.33%). INTRODUCTION K n o w l e d g e o f t h e c h e m i c a l c o m p o s i t i o n o f t h e m i c r o b i a l m a s s passing i n t o t h e a b o m a s u m is o f n u t r i t i o n a l i m p o r t a n c e . A n a l y s e s o f r u m e n b a c t e r i a a n d p r o t o z o a f o r p r o t e i n ( S m i t h a n d B a k e r , 1 9 4 4 ; M c N a u g h t et al., 1 9 5 0 ; Weller, 1 9 5 7 ) , a m i n o acid (Weller, 1 9 5 7 ; Bergen e t al., 1 9 6 8 ) , lipids ( G a r t o n a n d Oxf o r d , 1 9 5 5 ) a n d f a t t y acids (Williams a n d D i n u s s o n , 1 9 7 3 ) h a v e b e e n reported. T h e p u r p o s e o f o u r w o r k was t o d e t e r m i n e t h e p r o t e i n , a m i n o acid a n d lipid c o m p o s i t i o n o f b a c t e r i a l a n d p r o t o z o a l f r a c t i o n s passing o u t o f t h e f o r e s t o m a c h i n t o t h e a b o m a s u m o f s h e e p a n d t o s t u d y t h e e f f e c t o f various diets o n t h a t c o m p o s i t i o n .
58
MATERIALS AND METHODS
The experiments were carried out on five Cigay sheep. Each animal was fitted with a chronic rumen fistula and external anastomosis between the omasum and the abomasum (Kurilov et al., 1973). Each experimental period lasted for 25--30 days. During the first period, all the sheep were given a hay concentrate ration, balanced as regards the main nutrients, and were provided with water ad libitum. This basal ration consisted of 800 g clover/timothy hay, 400 g all-mash, 500 g fodder beet. During the other experimental periods the basal ration was supplemented with either 2 g d/-methionine, or 3 g /-lysine, or 50 g corn starch, or 20 g starch + 2 g methionine. Rumen content samples were collected regularly 3 h after feeding. At the end of the experimental period, daily total chyme entering the abomasum was determined during two diurnal tests. Every hour, chyme samples were taken (20--50 ml, constituting 1/10 of chyme passing in 1 h). For the purpose of isolation of the protozoal and bacterial fractions, a differential centrifugation method was applied to the daily chyme sample which had been preliminarily filtered through four layers of cheesecloth. Then 500 ml of the strained chyme was mixed with an equal volume of a fixing fluid that consisted of 0.2% Tween-80 dissolved in an 8% formalin solution. The mixture obtained was stored at room temperature for 48 h with periodical mixing to allow better isolation of microbial cells from food debris. For isolation of the protozoa, the mixture was centrifuged at 1 000 rpm for 3--5 rain. The sediment was mixed with 0.9 N NaC1 solution and re-centrifuged. Bacteria were isolated by centrifuging the supernatant fluid at 15 000 rpm for 15 min. The sediment was mixed with 0.9 N NaC1 solution and centrifuged ag~n. The protozoal and bacterial preparations were repeatedly washed and centrifuged until pure microbial cells were observed by microscopical examination. They were air dried to constant weight at 65°C. Total nitrogen was determined according to Kjeldahl (1970), total lipids according to Folch (1970), and the lipid fractions separated by chemical methods (Pokrovsky, 1969). Amino acids were determined in acid (with 6 N HC1); hydrolysates of bacterial and protozoal biomass, with a Hd-1200 E automatic analyser (U.S.S.R. product). Fatty acids were only determined in total lipid samples of rumen protozoa by a method of gas--liquid chromatography using a "Chrom-4" model chromatograph. RESULTS On average, the mean nitrogen content was 8.3+ 0.2% of dry matter for the bacterial mass, and 5.3-+ 0.2% for the protozoal mass (Table I). It was not significantly modified by the different supplements added to the basal ration. The slight decrease for the protozoa found in sheep with the starch supplements could be explained by the incorporation of starch granules within the cells.
59
TABLE I Nitrogen and protein c o n t e n t of bacterial and protozoal mass, isolated from the c h y m e ,
leaving the sheep f o r e s t o m a c h u n d e r v a r i o u s diets. D i f f e r e n c e s at e x p e r i m e n t a l s t a g e s in all cases are n o n - s i g n i f i c a n t (P > 0.1). Ration Total nitrogen
Hay concentrate r a t i o n ( B R ) ( n = 5) B R + s t a r c h (n = 5) B R + m e t h i o n i n e (n = 4) B R + l y s i n e (n = 5) BR + methionine + s t a r c h (n -- 4)
Protein (nitrogen × 6.25)
Bacteria
Protozoa
Bacteria
8.34 8.22 8.40 8.10
5.34 5.00 5.47 5.43
52.12 51.38 52.50 50.62
± 0.28 +- 0 . 1 1 -+ 0 . 2 9 +- 0 . 1 8
8.30 ± 0.12
± 0.19 ± 0.27 ± 0.37 ± 0.19
4 . 8 8 ± 0.21
± ± ± ±
Protozoa
1.75 0.69 1.81 1.12
51.88 ± 0.75
33.38 31.25 34.19 33.94
± ± ± ±
1.18 1.69 2.31 1.19
3 0 . 5 0 ± 1.31
When the sheep were fed the basal hay concentrate ration, mean amounts entering the abomasum per day were 33.3 g of bacterial dry matter, 48.2 g of protozoal dry matter and 32.5 g o f microbial protein. These amounts were increased b y the addition of 2 g dl-methionine to the basal ration: 43.8 g bacteria, 56.6 g p r o t o z o a and 41.88 g microbial protein. Approximately the same a m o u n t of microbial protein (41.25 g) entered the abomasum when 3 g l-lysine was added. The addition of these amino acids resulted in an increase in the contribution of bacterial protein to the total microbial protein (59.1% with the lysine supplement). Amino acid composition of bacteria and protozoa is relatively stable b u t in some cases there were significant differences in the c o n t e n t of certain amino acids according to the experimental period (Table II). Thus, the addition of methionine to the basal ration brought a b o u t a higher c o n t e n t of arginine, aspartic acid and serine in bacterial protein, and a slight decrease in isoleucine and leucine contents. On the contrary, the c o n t e n t of leucine and isoleucine increased in the protozoal protein b u t the serine and threonine levels decreased. These modifications in amino acid composition may be explained by the modifications in species composition of the population. So the addition of dl-methionine p r o m o t e d an increase in the number of amylolytic, lactate fermenting and urea assimilating bacteria. Protozoal species composition became more variable (Kolenjko et al., 1973). The amounts of total lipids entering the abomasum ranged from 40.3 to 81.5 g per day depending u p o n the ration. The total lipid c o n t e n t of the bacteria ranged from 5.42 to 8.85%. The value for the protozoa was less variable (mean: 9.63%). However, a significant decrease from 10.2+ 0.81 to 6.9+ 0.47% (P < 0.05) was observed when 50 g starch was added to the basal ration. Table III shows the percentage composition of bacterial and protozoal lipids when the sheep were given the basal hay-concentrate ration. Phospholipids are the major lipid fraction in the p r o t o z o a (mean: 51.78+ 0.92; maximum: 58.18%), and free fatty acids (41.32 + 9.5%) in the bacteria. The
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61
T A B L E III Lipid class c o m p o s i t i o n o f b a c t e r i a l a n d p r o t o z o a l mass. Values are e x p r e s s e d as a p e r c e n t age o f t o t a l lipids Lipid classes
Protozoa
Bacteria
Phospholipids Triglycerides Free f a t t y acids Cholesterol Others
51.78 10.83 13.28 3.24 20.87
28.09 3.59 41.32 4.30 22.70
± 0.92 ± 0.71 +- 0.59 ± 0.11
± 3.52 ± 0.77 -+ 9 . 5 0 ± 0.31
proportion of triglycerides is higher in the protozoa than in the bacteria. 30 fatty acids, of chain lengths ranging from 8 to 22 carbon atoms, were identified in the protozoa (Table IV). Saturated fatty acids constitute 66.5% of the total. Palmitic acid is the major fatty acid (41.32%). The presence of branched fatty acids (4.37% of the total) is also typical of protozoal lipids. TABLE IV F a t t y acid c o m p o s i t i o n o f lipids in o v i n e m i x e d r u m e n p r o t o z o a . Values are e x p r e s s e d as a p e r c e n t a g e o f t o t a l f a t t y acids c a l c u l a t e d using p e a k h e i g h t s a n d r e t e n t i o n t i m e . T h e n u m b e r f o l l o w i n g a semi c o l o n i n d i c a t e s t h e n u m b e r o f d o u b l e b o n d s . F a t t y Acids
M ± m
Cs--C12 Cl~:o Cl3:o b r C~4:o Cl4:o b r C~s:o Cis:o b r C1,:o C,6:obr C16:, C1~:o C1~:o b r C,8:o Cls: o b r Cle:l C1s:2 C1s:3 Ci9:0
0.37 0 . 2 2 ± 0.04 0 . 1 2 ± 0.03 0.8 +- 0 . 0 6 0.20 ± 0.05 1.67 ± 0 . 1 0 1.26 +- 0.07 4 1 . 3 2 ± 1.60 1.30 -+ 0.20 0 . 8 6 +- 0 . 1 5 0 . 2 9 ± 0.04 1 . 0 2 -+ 0 . 0 6 11.50 ± 0.93 0.47 1 9 . 3 3 ± 0.89 9 . 0 9 -+ 0 . 9 0 1.34 ± 0 . 2 0 0.43 5.50+- 1 . 1 0 2.89
C22:o Other
S a t u r a t e d acids 66.51 U n s a t u r a t e d acids 33.49 T o t a l i s o m e r s o f C13, Cl4, C~s, Cl,, C1~, C18, 4.37 br -- branched
62 REFERENCES Bergen, W.G., Purser, D.B. and Ceine, J.N., 1968. Determination of limiting amino acids of rumen isolated microbial proteins fed to rats. J. Dairy Sci., 51(10): 1698--1700. Garton, G.A. and Oxford, A.E., 1955. The nature bacterial lipids in the rumen o f hay-fed sheep. J. Sci. Prod. Agric., 6: 142. Hungate, R.E., 1966. The Rumen and Its Microbes. Academic Press, New York, N.Y. Kolenjko, E.I., Tarakanov, B.V., Guschin, N.N., Dolgov, I.A. and Shavyrina, T.A., 1973. Forestomach microflora of sheep fed routine diets with DL-methionine and L-Lysine. Nauchn. Tr. Vses. Nauchno-Issled. Inst. F.Bi. Promsti. Borovsk, XII: 243--249 (in Russian). Kurilov, N.V., Sevastianova, N.A., Mysnik, N.D., Korshunov, V.N. and Agafonova, T.A., 1973. Effect of various types of feeding on the synthesis of bacterial protein, amino acids and lipids in sheep rumen. Nauchn. Tr. Vses. Nauchno-Issled. Inst. Promsti, Borovsk, XII: 142--149 (in Russian). McNaught, M.L., Smith, J.A.B., Henry, K.M. and Kon, S.K., 1950. The utilization of nonprotein nitrogen in bovine rumen. 5. The isolation and nutritive value of preparation of dried tureen bactei-ia. Biochem. J., 46:32. Pokrovsky, A.A., 1969. Biokhimicheskie metodi issledovaniya v klinike. M., "Meditsina." Smith, J.A. and Baker, F., 1944. The utilization of urea in the bovine rumen. IV. The isolation of the synthesized material and the correlation between protein synthesis and microbial activity. Biochem. J., 38: 496. Williams, P.P. and Dinusson, W.E., 1973. Amino acid and fatty acid composition of bovine ruminal bacteria and protozoa. J. Anita. Sci., 36(I): 151--155.
RESUME Kurilov, N.V., Sevastianova, N.A., Korshunov, V.N., Mysnik, N.D. et Podshibyakin, A.E., 1976. Les teneurs en acides amines et acides gras des micro-organismes passant dans la caillette du' mouton. Livest. Prod. Sci., 3 : 5 7 - - 6 3 (en anglais). Les bact~ries et les protozoaires du contenu digestif passant du feuillet dans la caillette du m o u t o n ont ~t~ isol~s par une m~thode de centrifugation diff~rentielle. Les teneurs en azote o n t ~t~ de 8.3 -+ 0.2% (de la mati~re s~che) pour les bact~ries et de 5.3 +- 0.2% pour les protozoaires. Elles n ' o n t pas ~t~ modifi~es de fa~on significative par les diff~rents compigments, m~thionine, lysine ou amidon, qui ont ~t~ ajout~ ~ la ration de base. La composition en acides amines des prot~ines, des bact~ries et des protozoaires a ~t6 relativement constante; la p r o p o r t i o n de certains acides amines a cependant pr~sent~ des variations limitfies selon la nature des compl~ments. La teneur en lipides t o t a u x a vari~ de 5.42 ~ 8.85% de la mati~re s ~ h e pour les bact~ries et elle a ~t~ en moyenne de 9.63% pour les protozoaires. Elle n'a pas ~t~ modifi~e significativement par les compl~ments. Les acides gras libres sont les constituants majeurs des lipides bact~riens et les phospholipides de ceux des lipides des protozoaires. La composition en acides gras de ces derniers est caract~ris~e par une proportion ~l~v~e d'acide palmitique (41.32%) et d'acide ol~ique (19.33%). KURZFASSUNG Kurilov, N.V., Sevastianova, N.A., Korshunov, V°N., Mysnik, N.D. and Podshibyakin, A.E., 1976. Zusammensetzung yon Aminos~uren und Fetts~iuren von Mikroorganismen, die in den Labmagen yon Schafen iibertreten. Livest. Prod. Sci., 3 : 5 7 - - 6 3 (in Englisch). Die Bakterien- und Protozoenfraktionen wurden mit Hilfe einer Differentialzentrifugenmethode aus einer t/iglichen Durchschnittsprobe des Chymus isoliert, der aus dem Vormagen
63
des Schafes in den Labmagen iibertritt. Die Durchschnittswerte fiir den Stickstoffgehalt (% der Trockenmasse) betrugen 8.3 +- 0.2% ftir die Bakterienfraktion sowie 5.3 +- 0.2% fiir die Protozoenfraktion. Sie wurden dutch Zusatz verschiedener 8toffe zur Grundration, wie Methionin, Lysin oder St~rke, nicht signifikant beeinflusst. Die Amino~urenzusammensetzung des Bakterien- und Protozoeneiweiss blieb relativ konstant, obgleich einige geringe Variationen bei bestimrnten Aminos~iuren in Abh~/ngigkeit yon der Art der Zusatzstoffe beobachtet wurden. Der Gesamtgehalt an Lipoiden schwankte zwischen 5.42 und 8.85% des Trockengehaltes der Bakterienfraktion. Der durchschnittliche Gehalt betrug 9.63% fiir die Protozoenfraktion. Er wurde dutch Zusatzstoffe nicht signifikant beeinflusst. In der Lipoidzusammensetzung der Bakterien dominieren die freien Fetts~uren, w~/hrend die Phosphorlipoide bei den Protozoen dominieren. Die bedeutendsten Fett~uren der Protozoenlipoide waren Palmitins~ure (41.32%), Ols~ure (19.33%), Stearins~iure (11.50%) und Linol~ure (9.09%). PE3©ME
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