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Effect of lactate on the in vitro digestion of Agropyron elongatum by rumen microorganisms
Animal Feed Science and Technology, 16 (1986) 161--167
161
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
EFFECT OF LACTATE ON THE IN VITRO DIGESTION OF A G R O P Y R O N ELONGATUM BY RUMEN MICROORGANISMS
J.P. F A Y and F.M.A. O V E J E R O
Department of Animal Production, AgriculturalResearch Station, National Instituteof Agricultural Technology (INTA ), 7620 Balcarce (Argentina) (Received 14 May 1985; accepted for publication24 April 1986)
ABSTRACT Fay, J.P, and Ovejero, F.M.A., 1986. Effect of lactate on the in vitro digestion of Agropyron elongatum by rumen microorganisms. Anita. Feed Sci. Technol., 16: 161--167. Sodium lactate concentrations higher than 20 mM increasingly inhibited the in vitro digestion of Agropyron elongatum by rumen microorganisms. The highest concentration tested (200 mM) reduced forage dry matter loss due to microbial degradation by 65%, and completely prevented the digestion of forage fiber. A role for .lactate in the depression of fiber digestion in grain supplemented ruminants is suggested. INTRODUCTION
The addition of grains or starch (main carbohydrate in grains) to forage usually results in decreased cellulose and dry matter digestion by ruminants (Burroughs et al., 1949; Torres and Boelcke, 1976; van der Linden et al., 1984). A similar effect was found when in vitro cellulose digestion by rumen microbes was studied in the presence of glucose or cellobiose (Hiltner and Dehority, 1983) or barley (Stewart, 1977). Lactic acid concentrations in rumen liquor are usually very low, but they may rise considerably when grains or easily fermentable carbohydrates are supplied {Stewart, 1977; Counotte et al., 1983; Mackie et al., 1984). Volatile and other fatty acids may inhibit growth of several rumen bacteria (Henderson, 1973; Stewart, 1975; Prins and Clarke, 1980) and lactic acid has been shown to prevent the anaerobic growth of some non-tureen bacterial species (Grau, 1981). However, the Dotential of lactic acid as an inhibitor for rurnen microorganisms has not been tested. Since both an increase in lactic acid concentration and a pH drop occur simultaneously when a rapidly fermentable carbohydrate source is present (Malestein et al., 1981; Counotte et al., 1983), any possible inhibition of rumen microbial activity by lactic acid would be confounded with the inhibition due to a low pH in the medium (Stewart, 1977; Mertens, 1979; Hiltner and Dehority, 1983). To minimize an effect of pH, we undertook to
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© 1986 Elsevier Science Publishers B.V.
162
determine the effect of different concentrations of NaLac on the in vitro ruminal digestion of Agropyron elongatum [Eritrigia pontica (Podp) Holub], a forage grass which is likely to be supplemented with grain as a diet for cattle in Argentina (Tortes and Boelcke, 1976; Rosso et al., 1980}. MATERIALS AND METHODS Grass was cut at a height of 8--10 cm from early tillering of vigorous plants in the field, dried at 60°C for 24 h, mixed and ground in a Willey mill to pass a l-ram screen. The ground forage sample was kept at room temperature in the dark, and every 2 weeks 0.5-g sub-samples were placed into 80-ml digestion tubes for the dry and organic matter digestibility assays according to Tilley and Terry (1963). To calculate ADF (acid detergent fibre} digestibility, ADF was determined in the forage and in the residues recovered after a 48-h incubation with rumen inocula. Prior to the addition of rumen inocula, adequate amounts of NaLac (DL-lactic acid sodium salt) or NaC1 were added to the tubes in 10 ml of distilled water to obtain the final concentrations of Tables I and II. Inocula (50 ml) were made by squeezing the rumen contents from a fistulated steer kept on an alfalfa hay diet through 4 layers of cheesecloth and mixing (1:4, v/v) the rumen liquor with McDougall's artificial saliva (McDougall, 1949) containing 0.85 g 1-1 of urea. Three types of control were included in each assay: (1} tubes in which NaLac or NaC1 solutions were replaced by 10 ml of distilled water (inoculated controls, 0% inhibition of microbial digestion assumed); (2) tubes in which rumen inoculum was replaced by an equal volume of artificial saliva plus 10% (v/v) of a saturated HgC12 solution or 1.6 mg m1-1 of streptomycin as microbial inhibitors (non-inoculated controls, 100% inhibition of microbial digestion assumed); (3) tubes in which NaC1 200 mM was added instead of NaLac (to control effects due to increased Na concentrations and osmolality in the medium). pH was measured in all the tubes immediately before and after the 48-h incubation with rumen inocula. ADF, NDF (neutral detergent fibre} and permanganate lignin were determined as described by Goering and Van Soest (1970), and crude protein (N × 6.25) was estimated by a modified Kjeldahl procedure. All chemicals used were reagent grade. Forage material recovered after Tilley and Terry's procedure (Tilley and Terry, 1963) was dried at 100°C for 24 h and then ashed at 500°C for 24 h to calculate total dry (DMD) and organic (OMD) matter disappearances, respectively. Percentage inhibition by salts (NaLac or NaC1} of dry (MDMD) and organic (MOMD) matter, and of ADF (MADFD) disappearances due only to microbial digestion were calculated as follows: % Inhibition of MDMD (or MOMD or MADFD) = [DMD (or OMD or ADFD) in inoculated control -- DMD (or OMD or ADFD) when salt added] / [DMD (or OMD or ADFD) in inoculated control - DMD (or OMD or ADFD) in non-inoculated control] × 100.
67.3 67.4 67.7 67.2 66.3 65.1 60.5 56.0 51.6 46.0 44.1 41.8 53.6
27,5 ± 0.9 b
0 (inoculated control) NaLac 5 10 20 40 60 80 100 125 150 175 200 NaCl 200
0 (Non-inoculated control) ~
100 b
0a --0.3 --1.0 0.1 3.5 6.4 17.8 28.8 41.4 54.5 59.9 64.4 34.8 ± + + + + ± ± + + ± ± ±
1.0 a 1.9 a 1.3 a 1.6 a 2.9 a 4.1 ab 3.7 ab 5.4 ab 3.5 ab 2.4 ab 1.4 ab 2.9 ab
Inhibition of MDMD3 (%) -+ 1.1 a ± 1.1 a + 1.0 a ± 0.9 a ± 1.2 a + 1.7 a -+ 2.1 ab ± 2.0 ab + 2.7 ab -+ 1.9 ab + 1.5 ab ± 1.0 ab ± 1.7 ab
23.5 ± 0.9 b
69.8 69.8 69.8 69.6 68.6 67.2 62.2 56.6 51.5 45.2 43.0 40.0 53.9
OMD 4 (%)
± 0.8 a -+ 1.3 a ± 1.0 a ± 1.3 a 2 2.7 a ± 4.0 ab ± 3.7 ab + 5.3 ab + 3.4 ab ± 2.4 ab ± 1.7 ab ± 2.9 ab 100 b
0a --0.1 --0.2 0.3 3.5 6.5 17.5 28.8 41.4 53.9 59.6 64.6 34.5
Inhibition of MOMD5 (%)
6.89
6.97 6.97 6.97 6.97 6.97 6.98 6.98 7.03 7.01 7.09 7.02 7.05 6.91
pH 6 Initial
6.87
6.90 6.91 6.91 6.94 6.93 6.97 6.98 6.97 6.95 6.97 6.97 6.90 6.79
Final
Agropyron elongatum d r y a n d o r g a n i c m a t t e r b y t u r e e n m i c r o o r g a n -
1 M e t h o d o f Tilley a n d T e r r y ( 1 9 6 3 ) . R u m e n i n o c u l u m a d d e d t o all t h e t u b e s except to the non-inoculated control. Values (means +_ SEM) o b t a i n e d f r o m 8 - - 1 2 d u p l i c a t e assays. 2T o t a l d r y m a t t e r d i s a p p e a r a n c e . 3M D M D , d r y m a t t e r d i s a p p e a r a n c e d u e o n l y t o m i c r o b i a l d i g e s t i o n . T o t a l Organic m a t t e r d i s a p p e a r a n c e . s MOMD, organic m a t t e r d i s a p p e a r a n c e d u e o n l y t o m i c r o b i a l d i g e s t i o n . 6 M e a s u r e d at t h e b e g i n n i n g (initial) a n d a t t h e e n d (final) o f t h e 48-h i n c u b a t i o n w i t h t u r e e n m i c r o b e s ( m e a n o f 3 d e t e r m i n a t i o n s ) . 10% (v/v) o f a s a t u r a t e d Hg Cl~ s o l u t i o n a d d e d . aDiffer f r o m n o n - i n o c u l a t e d c o n t r o l ( P ~ 0.05), b D i f f e r f r o m i n o c u l a t e d c o n t r o l (P ~ 0.05).
+- 0.8 a + 0.9 a + 0.8 a -+ 0.7 a + 1.0 a + 1.5 a + 1.8 ab + 1.6 ab + 2.3 ab ± 1.6 ab +- 1.3 ab ± 0.8 ab ± 1.4 ab
DMD 2 (%)
Salt a d d e d (raM)
isms ~
E f f e c t ' o f s o d i u m lactate ( N a L a c ) o n t h e in v i t r o d i g e s t i b i l i t y o f
TABLE I
t.a
164 TABLE H Effect of sodium lactate (NaLac) on the in vitro digestibility of Agropyron elongatum acid detergent fiber by tureen microorganisms ~ Salt added (raM)
0 (Inoculated Control) NaLac 80 200 NaC1 200 0 (Non-inoculated control) ~
ADFD 2 (%)
42.8 38.4 --0.6 20.9 0.2
+ 1.2 a + 1.3 a + 2.0 b + 2.6 ab + 1.0 b
Inhibition of MADFD 3 (%) 0a 10.0 -+ 3.5 ab 102.5 + 5.8 b 51.3 + 5.4 ab 100 b
pH 4 Initial
Final
7.00 7.01 7.02 6.91 6.98
6.92 + 0.04 a 7.07 + 0.02 b 7.05 + 0.05 6.86 + 0.03 a 7.17 + 0.03 b
+- 0.03 + 0.03 + 0.03 + 0.04 + 0.02
Rumen inoculum was added to all the tubes except to the non-inoculated control. After 48 h of incubation ADF was determined according to Goering and Van Soest (1970), Values (means +- SEM) obtained from 6 duplicate assays. Total ADF disappearance. 3MADFD, ADF disappearance due only to microbial digestion. 4Measured at the beginning (initial) and at the end (final) of the 48-h incubation. 5 Streptomycin (1.6 mg ml -~) added. aDiffer from non-inoculated control (P ~ 0.05}. bDiffer from inoculated control (P ~ 0.05}. D a t a w e r e a n a l y z e d b y analysis o f variance a n d m e a n s w e r e c o m p a r e d using D u n n e t ' s (Steel a n d Torrie, 1960} a n d S t u d e n t ' s t tests. RESULTS T h e forage used in this w o r k h a d t h e f o l l o w i n g p e r c e n t a g e c o m p o s i t i o n ( w t / w t ) : c r u d e p r o t e i n , 21.0; N D F , 48.0; A D F , 33.1; lignin, 3.6; t o t a l ash, 7.2. T h e in vitro digestibility o f A. elongatum was significantly (P < 0 . 0 5 ) d e c r e a s e d b y c o n c e n t r a t i o n s o f 80 m M o r g r e a t e r o f N a L a c a n d b y 200 m M NaC1 ( T a b l e I). C o n c e n t r a t i o n s as l o w as 20 m M N a L a c also s h o w e d a n inh i b i t i o n t r e n d . A l t h o u g h t h e smallest c o n c e n t r a t i o n s t e s t e d (5 and 10 m M ) a p p e a r e d to slightly s t i m u l a t e digestibility, d i f f e r e n c e s w e r e n o t significant
{P > 0.05). When D M D values w e r e c o m p a r e d w i t h t h e i r c o r r e s p o n d i n g O M D values n o d i f f e r e n c e s w e r e d e t e c t e d (P > 0.05). A t c o n c e n t r a t i o n s higher t h a n 20 m M t h e e x t e n t o f i n h i b i t i o n increased w i t h increasing c o n c e n t r a t i o n s o f N a L a c . A s e x p e c t e d , D M D and O M D in t h e a b s e n c e o f r u m e n m i c r o o r g a n i s m s (leaching o f forage soluble c o m p o n e n t s in artificial saliva} was l o w e r t h a n in i n o c u l a t e d t u b e s (leaching + m i c r o b i a l digestion o f f o r a g e components). Values o f % i n h i b i t i o n o f M D M D a n d M O M D s h o w e d t h a t t h e m i c r o b i a l c o n t r i b u t i o n t o t h e digestion o f forage was a f f e c t e d b y b o t h N a L a c a n d
165
NaC1 (Table I). Inhibition of microbial digesting activity increased between 20 and 200 mM NaLac. Concentrations below 20 mM appeared to slightly increase both MDMD and MOMD, but differences were not significant (P > 0.05). Almost 65% of microbial digestion of forage was inhibited by 200 mM NaLac vs. only 35% by the same concentration of NaC1. Medium pH was not substantially altered by the additions of either NaC1 or NaLac. Forage ADF digestibility was also impaired by both NaLac and NaC1 (Table II). Complete inhibition of MADFO was obtained with 200 mM NaLac, but the inhibition provoked by 200 mM NaC1 was considerably lower. Significant differences in medium pH were detected only at the end of the incubation. NaLac 80 mM was the only addition that modified pH with respect to the values of the inoculated control. Percentage inhibitions of both MDMD and MOMD were positively correlated with NaLac concentrations as follows: % Inhibition of MDMD = --7.6 + 0.37 (raM NaLac), (r 2 = 0.86, P ~< 0.05) % Inhibition of MOMD = --7.3 + 0.37 (raM NaLac), (r: = 0.87, P ~< 0.05) DISCUSSION According to the preceding equations, the minimum concentration of NaLac concentration of about 2.7 mM will elicit a 1% depression of both plete inhibition of microbial digestibility would be obtained with NaLac concentrations greater than 290 raM. Above 20 mM NaLac, every increase in NaLac concentration of about 2.7 mM will elicit a 1% depression of both MDMD and MOMD. Fiber digesting activity of rumen microbes seems to be more sensitive to high NaLac concentrations since complete inhibition of ADF digestion was obtained with only 200 mM of the salt. The inclusion of gains or other high-energy feeds in the diet usually provokes a decrease in tureen pH as more VFA and lactic acid are produced by microbial fermentation (Counotte et al., 1983; Mackie et al., 1984). This pH drop has been suggested to be the main cause for the observed decrease in fiber digestion after grain supplementation because ceUuloytic activity of rumen microbes is maximal at pH 6.8--7.1 and markedly decreases below these values (Mertens, 1979). In the present study, lactic acid was added to the medium as its sodium salt and during the assays pH remained within the reported optimum range for ceUulolysis; a depression of digestion due to a pH change, therefore, can be ruled out. Lactate concentrations that originate in the rumen when gains or other concentrates are included in the diet normally do not exceed 20 mM (Malestein et al., 1981; Mackie et al., 1984). According to our results, these concentrations would not be high enough to depress forage digestibility provided that pH is kept above 6.8 (Mertens, 1979). However, Grau (1981) showed that the inhibitory effect of lactic acid on growth of some nontureen bacteria increases when pH decreases from 6.1 to 5.5, and we can speculate that the inhibition of rumen microbes by lactate may also be
166
potentiated when pH falls as a consequence of grain supplementation. Furthermore, a pH drop may result in higher ruminal levels of lactic acid due to reduced microbial degradation of this compound below pH 6 (Malenstein et al., 1981) compared to degradation at higher pH values. When grains are supplied in excessive amounts, either ground or without a previdus adaptation of the animals to these feeds, ruminal concentrations of lactic acid rapidly increase and can reach levels well over 20 mM (Counotte and Prins, 1981; Muir et al., 1981). According to results of this study, these high concentrations of lactate will be inhibitory for the microbial digesting activity even at pH values within the normal range in the tureen. Increased osmolality reduces cellulose digestibility by rumen microbes (Bergen, 1972) and in this work increasing concentrations of NaLac induced increasing inhibitions of A. elongatum digestion in vitro. Nevertheless, the inhibition of MDMD, MOMD and MADFD by 200 mM NaC1 was only half that caused by an equivalent concentration of NaLac which clearly indicates that lactate per se depress forage digestion by rumen microbes. The mechanisms of the inhibition of forage digestion by lactate could be similar to those proposed by E1-Shazly et al. (1961) and by Mertens (1979) to explain the depression of cellulose digestion by starch. In addition, the possibility that lactate itself may be toxic for fiber digesting rumen microbes cannot be ruled out since this compound has been shown to be toxic to some microorganisms (Grau, 1981). We conclude that lactate is a potential inhibitor of forage digestion by rumen microbes and, therefore, it may contribute to the reported decrease of fiber digestion in grain-supplemented ruminants. ACKNOWLEDGEMENTS
Thanks are due to Dr. H. Fernandez for his help with the statistical analysis, and to M. Patriarca, C. Bastarrechea and M.E. Giuliano for their technical assistance. This work was supported in part by a grant from the Consejo Nacional de Investigaciones Cientlficas y T~cnicas de la Repfiblica Argentina (CONtCET). J.P. Fay is a Career Investigator of the CONICET. REFERENCES Bergen, W.G., 1972. Role of osmolality in feed intake control in sheep. J. Anita. Sci., 34: 1054--1060. Burroughs, W., Gerlaugh, P., Edgington, B.H. and Bethke, R.M., 1949. The influence of corn starch upon roughage digestion in cattle. J. Anita. Sci., 8: 271--278. Counotte, G.H.M. and Prins, R.A., 1981. Regulation of lactate metabolism in the rumen. Vet. Res. Commun., 5: 101--115. Counotte, G.H.M., Lankhorst, A. and Prins, R.A., 1983. Role of DL-Lactic acid as an intermediate in rumen metabolism of dairy cows. J. Anita. Sci., 56: 1222--1235. EI-Shazly, K., Dehority, B.A. and Johnson, R.R., 1961. Effect of starch on the digestion of cellulose in vitro and in vivo by tureen microorganisms. J. Anita. Sci., 20 : 268--273.
167 Goering, H.K. and Van Soest, P.J., 1970. Forage fibre analyses (apparatus, reagents, procedures and some applications). Agric. Handbook No. 379. ARS, USDA, Washington DC, pp. 1--20. Grau, F.H., 1981. Role o f pH, lactate and anaerobiosis in controlling the growth of some fermentative gram negative bacteria on beef. Appl. Environ. Microbiol., 42: 1043-1050. Henderson, C., 1973. The effects of fatty acids on pure cultures of rumen bacteria. J. Agric. Sci., 81: 107--112. Hiltner, P. and Dehority, B.A., 1983. Effect o f soluble carbohydrates on digestion of eellulose b y pure cultures of rumen bacteria. Appl. Environ. Microbiol., 46: 642--648. Mackie, R.I., Gilchrist, F.M.C. and Heath, S., 1984. An in vivo study of ruminal microorganisms influencing lactate turnover and its contribution to volatile fatty acid production. J. Agric. Sci., 103: 37--51. Malestein, A., van 't Klooster, A.T., Counotte, G.H.M. and Prins, R.A., 1981. Concentrate feeding and ruminal fermentation. 1. Influence of the frequency of feeding concentrates on rumen acid composition, feed intake and milk production. Neth. J. Agric. Sci., 29: 239--248. McDougall, E.I., 1949. Studies on ruminant saliva. I. The composition and output of sheep's saliva. Biochem. J., 43: 99--109. Mertens, D.R., 1979. Effects of buffers upon fiber digestion. In: E.W.H. Hale and P. Meinhardt (Editors), Regulation of Acid-Base Balance. Church and Dwight, Piscataway, NJ, pp. 65--80. Muir, L.A., Rickes, E.L., Duquette, P.F. and Smith, G.E., 1981. Prevention of induced lactic acidosis in cattle by thiopeptin. J. Anita. Sci., 52 : 635--643. Prins, R.A. and Clarke, R.T.J., 1980. Microbial ecology of the rumen. In: Y. Ruckebusch and P. Thivend (Editors), Digestive Physiology and Metabolism in Ruminants. MTP Press, Lancaster, pp. 179w204. Rosso, O.R., Gomez, P.O. and Verde, S.C., 1980. Supplementation with corn grain and nitrogen sources of steers fed grass hays. In: L.S. Verde and A. Fernandez (Editors), Proc. IV World Conf. Anim. Prod. Asociaci6n Argentina de Producci6n Animal, Buenos Aires, pp. 128--137 (in Spanish). Steel, R.G. and Torrie, J.H., 1960. Principles and procedures of statistics. McGraw Hill, New York, pp. 111--112. Stewart, C.S., 1975. Some effects o f phosphate and volatile fatty acid salts on the growth o f rumen bacteria. J. Gen. Microbiol., 89: 319--326. Stewart, C.S., 1977. Factors affecting the ceUulolytic activity of rumen contents. Appl. Environ. Microbiol., 33: 497--502. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104--111. Torres, F. and Boelcke, C., 1976. Grain supplementation of low quality grasses, In: P.V. Fonnesbeck, L.E. Harris and L.C. Kearl (Editors), First International Symposium, Feed Composition, Animal Nutrient Requirements and Computerization of Diets. Utah Agric. Exp. Stn., Utah State University, Logan, UT, pp. 408--414. Van Der Linden, Y., Van Glyswyk, N.O. and Schwartz, H.M., 1984. Influence of supplementation of corn stover with corn grain on the fibrolytic bacteria in the rumen of sheep and their relation to the intake and digestion o f fiber. J. Anim. Sci., 59: 772-783.
161
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
EFFECT OF LACTATE ON THE IN VITRO DIGESTION OF A G R O P Y R O N ELONGATUM BY RUMEN MICROORGANISMS
J.P. F A Y and F.M.A. O V E J E R O
Department of Animal Production, AgriculturalResearch Station, National Instituteof Agricultural Technology (INTA ), 7620 Balcarce (Argentina) (Received 14 May 1985; accepted for publication24 April 1986)
ABSTRACT Fay, J.P, and Ovejero, F.M.A., 1986. Effect of lactate on the in vitro digestion of Agropyron elongatum by rumen microorganisms. Anita. Feed Sci. Technol., 16: 161--167. Sodium lactate concentrations higher than 20 mM increasingly inhibited the in vitro digestion of Agropyron elongatum by rumen microorganisms. The highest concentration tested (200 mM) reduced forage dry matter loss due to microbial degradation by 65%, and completely prevented the digestion of forage fiber. A role for .lactate in the depression of fiber digestion in grain supplemented ruminants is suggested. INTRODUCTION
The addition of grains or starch (main carbohydrate in grains) to forage usually results in decreased cellulose and dry matter digestion by ruminants (Burroughs et al., 1949; Torres and Boelcke, 1976; van der Linden et al., 1984). A similar effect was found when in vitro cellulose digestion by rumen microbes was studied in the presence of glucose or cellobiose (Hiltner and Dehority, 1983) or barley (Stewart, 1977). Lactic acid concentrations in rumen liquor are usually very low, but they may rise considerably when grains or easily fermentable carbohydrates are supplied {Stewart, 1977; Counotte et al., 1983; Mackie et al., 1984). Volatile and other fatty acids may inhibit growth of several rumen bacteria (Henderson, 1973; Stewart, 1975; Prins and Clarke, 1980) and lactic acid has been shown to prevent the anaerobic growth of some non-tureen bacterial species (Grau, 1981). However, the Dotential of lactic acid as an inhibitor for rurnen microorganisms has not been tested. Since both an increase in lactic acid concentration and a pH drop occur simultaneously when a rapidly fermentable carbohydrate source is present (Malestein et al., 1981; Counotte et al., 1983), any possible inhibition of rumen microbial activity by lactic acid would be confounded with the inhibition due to a low pH in the medium (Stewart, 1977; Mertens, 1979; Hiltner and Dehority, 1983). To minimize an effect of pH, we undertook to
0377-8401/86/$03.50
© 1986 Elsevier Science Publishers B.V.
162
determine the effect of different concentrations of NaLac on the in vitro ruminal digestion of Agropyron elongatum [Eritrigia pontica (Podp) Holub], a forage grass which is likely to be supplemented with grain as a diet for cattle in Argentina (Tortes and Boelcke, 1976; Rosso et al., 1980}. MATERIALS AND METHODS Grass was cut at a height of 8--10 cm from early tillering of vigorous plants in the field, dried at 60°C for 24 h, mixed and ground in a Willey mill to pass a l-ram screen. The ground forage sample was kept at room temperature in the dark, and every 2 weeks 0.5-g sub-samples were placed into 80-ml digestion tubes for the dry and organic matter digestibility assays according to Tilley and Terry (1963). To calculate ADF (acid detergent fibre} digestibility, ADF was determined in the forage and in the residues recovered after a 48-h incubation with rumen inocula. Prior to the addition of rumen inocula, adequate amounts of NaLac (DL-lactic acid sodium salt) or NaC1 were added to the tubes in 10 ml of distilled water to obtain the final concentrations of Tables I and II. Inocula (50 ml) were made by squeezing the rumen contents from a fistulated steer kept on an alfalfa hay diet through 4 layers of cheesecloth and mixing (1:4, v/v) the rumen liquor with McDougall's artificial saliva (McDougall, 1949) containing 0.85 g 1-1 of urea. Three types of control were included in each assay: (1} tubes in which NaLac or NaC1 solutions were replaced by 10 ml of distilled water (inoculated controls, 0% inhibition of microbial digestion assumed); (2) tubes in which rumen inoculum was replaced by an equal volume of artificial saliva plus 10% (v/v) of a saturated HgC12 solution or 1.6 mg m1-1 of streptomycin as microbial inhibitors (non-inoculated controls, 100% inhibition of microbial digestion assumed); (3) tubes in which NaC1 200 mM was added instead of NaLac (to control effects due to increased Na concentrations and osmolality in the medium). pH was measured in all the tubes immediately before and after the 48-h incubation with rumen inocula. ADF, NDF (neutral detergent fibre} and permanganate lignin were determined as described by Goering and Van Soest (1970), and crude protein (N × 6.25) was estimated by a modified Kjeldahl procedure. All chemicals used were reagent grade. Forage material recovered after Tilley and Terry's procedure (Tilley and Terry, 1963) was dried at 100°C for 24 h and then ashed at 500°C for 24 h to calculate total dry (DMD) and organic (OMD) matter disappearances, respectively. Percentage inhibition by salts (NaLac or NaC1} of dry (MDMD) and organic (MOMD) matter, and of ADF (MADFD) disappearances due only to microbial digestion were calculated as follows: % Inhibition of MDMD (or MOMD or MADFD) = [DMD (or OMD or ADFD) in inoculated control -- DMD (or OMD or ADFD) when salt added] / [DMD (or OMD or ADFD) in inoculated control - DMD (or OMD or ADFD) in non-inoculated control] × 100.
67.3 67.4 67.7 67.2 66.3 65.1 60.5 56.0 51.6 46.0 44.1 41.8 53.6
27,5 ± 0.9 b
0 (inoculated control) NaLac 5 10 20 40 60 80 100 125 150 175 200 NaCl 200
0 (Non-inoculated control) ~
100 b
0a --0.3 --1.0 0.1 3.5 6.4 17.8 28.8 41.4 54.5 59.9 64.4 34.8 ± + + + + ± ± + + ± ± ±
1.0 a 1.9 a 1.3 a 1.6 a 2.9 a 4.1 ab 3.7 ab 5.4 ab 3.5 ab 2.4 ab 1.4 ab 2.9 ab
Inhibition of MDMD3 (%) -+ 1.1 a ± 1.1 a + 1.0 a ± 0.9 a ± 1.2 a + 1.7 a -+ 2.1 ab ± 2.0 ab + 2.7 ab -+ 1.9 ab + 1.5 ab ± 1.0 ab ± 1.7 ab
23.5 ± 0.9 b
69.8 69.8 69.8 69.6 68.6 67.2 62.2 56.6 51.5 45.2 43.0 40.0 53.9
OMD 4 (%)
± 0.8 a -+ 1.3 a ± 1.0 a ± 1.3 a 2 2.7 a ± 4.0 ab ± 3.7 ab + 5.3 ab + 3.4 ab ± 2.4 ab ± 1.7 ab ± 2.9 ab 100 b
0a --0.1 --0.2 0.3 3.5 6.5 17.5 28.8 41.4 53.9 59.6 64.6 34.5
Inhibition of MOMD5 (%)
6.89
6.97 6.97 6.97 6.97 6.97 6.98 6.98 7.03 7.01 7.09 7.02 7.05 6.91
pH 6 Initial
6.87
6.90 6.91 6.91 6.94 6.93 6.97 6.98 6.97 6.95 6.97 6.97 6.90 6.79
Final
Agropyron elongatum d r y a n d o r g a n i c m a t t e r b y t u r e e n m i c r o o r g a n -
1 M e t h o d o f Tilley a n d T e r r y ( 1 9 6 3 ) . R u m e n i n o c u l u m a d d e d t o all t h e t u b e s except to the non-inoculated control. Values (means +_ SEM) o b t a i n e d f r o m 8 - - 1 2 d u p l i c a t e assays. 2T o t a l d r y m a t t e r d i s a p p e a r a n c e . 3M D M D , d r y m a t t e r d i s a p p e a r a n c e d u e o n l y t o m i c r o b i a l d i g e s t i o n . T o t a l Organic m a t t e r d i s a p p e a r a n c e . s MOMD, organic m a t t e r d i s a p p e a r a n c e d u e o n l y t o m i c r o b i a l d i g e s t i o n . 6 M e a s u r e d at t h e b e g i n n i n g (initial) a n d a t t h e e n d (final) o f t h e 48-h i n c u b a t i o n w i t h t u r e e n m i c r o b e s ( m e a n o f 3 d e t e r m i n a t i o n s ) . 10% (v/v) o f a s a t u r a t e d Hg Cl~ s o l u t i o n a d d e d . aDiffer f r o m n o n - i n o c u l a t e d c o n t r o l ( P ~ 0.05), b D i f f e r f r o m i n o c u l a t e d c o n t r o l (P ~ 0.05).
+- 0.8 a + 0.9 a + 0.8 a -+ 0.7 a + 1.0 a + 1.5 a + 1.8 ab + 1.6 ab + 2.3 ab ± 1.6 ab +- 1.3 ab ± 0.8 ab ± 1.4 ab
DMD 2 (%)
Salt a d d e d (raM)
isms ~
E f f e c t ' o f s o d i u m lactate ( N a L a c ) o n t h e in v i t r o d i g e s t i b i l i t y o f
TABLE I
t.a
164 TABLE H Effect of sodium lactate (NaLac) on the in vitro digestibility of Agropyron elongatum acid detergent fiber by tureen microorganisms ~ Salt added (raM)
0 (Inoculated Control) NaLac 80 200 NaC1 200 0 (Non-inoculated control) ~
ADFD 2 (%)
42.8 38.4 --0.6 20.9 0.2
+ 1.2 a + 1.3 a + 2.0 b + 2.6 ab + 1.0 b
Inhibition of MADFD 3 (%) 0a 10.0 -+ 3.5 ab 102.5 + 5.8 b 51.3 + 5.4 ab 100 b
pH 4 Initial
Final
7.00 7.01 7.02 6.91 6.98
6.92 + 0.04 a 7.07 + 0.02 b 7.05 + 0.05 6.86 + 0.03 a 7.17 + 0.03 b
+- 0.03 + 0.03 + 0.03 + 0.04 + 0.02
Rumen inoculum was added to all the tubes except to the non-inoculated control. After 48 h of incubation ADF was determined according to Goering and Van Soest (1970), Values (means +- SEM) obtained from 6 duplicate assays. Total ADF disappearance. 3MADFD, ADF disappearance due only to microbial digestion. 4Measured at the beginning (initial) and at the end (final) of the 48-h incubation. 5 Streptomycin (1.6 mg ml -~) added. aDiffer from non-inoculated control (P ~ 0.05}. bDiffer from inoculated control (P ~ 0.05}. D a t a w e r e a n a l y z e d b y analysis o f variance a n d m e a n s w e r e c o m p a r e d using D u n n e t ' s (Steel a n d Torrie, 1960} a n d S t u d e n t ' s t tests. RESULTS T h e forage used in this w o r k h a d t h e f o l l o w i n g p e r c e n t a g e c o m p o s i t i o n ( w t / w t ) : c r u d e p r o t e i n , 21.0; N D F , 48.0; A D F , 33.1; lignin, 3.6; t o t a l ash, 7.2. T h e in vitro digestibility o f A. elongatum was significantly (P < 0 . 0 5 ) d e c r e a s e d b y c o n c e n t r a t i o n s o f 80 m M o r g r e a t e r o f N a L a c a n d b y 200 m M NaC1 ( T a b l e I). C o n c e n t r a t i o n s as l o w as 20 m M N a L a c also s h o w e d a n inh i b i t i o n t r e n d . A l t h o u g h t h e smallest c o n c e n t r a t i o n s t e s t e d (5 and 10 m M ) a p p e a r e d to slightly s t i m u l a t e digestibility, d i f f e r e n c e s w e r e n o t significant
{P > 0.05). When D M D values w e r e c o m p a r e d w i t h t h e i r c o r r e s p o n d i n g O M D values n o d i f f e r e n c e s w e r e d e t e c t e d (P > 0.05). A t c o n c e n t r a t i o n s higher t h a n 20 m M t h e e x t e n t o f i n h i b i t i o n increased w i t h increasing c o n c e n t r a t i o n s o f N a L a c . A s e x p e c t e d , D M D and O M D in t h e a b s e n c e o f r u m e n m i c r o o r g a n i s m s (leaching o f forage soluble c o m p o n e n t s in artificial saliva} was l o w e r t h a n in i n o c u l a t e d t u b e s (leaching + m i c r o b i a l digestion o f f o r a g e components). Values o f % i n h i b i t i o n o f M D M D a n d M O M D s h o w e d t h a t t h e m i c r o b i a l c o n t r i b u t i o n t o t h e digestion o f forage was a f f e c t e d b y b o t h N a L a c a n d
165
NaC1 (Table I). Inhibition of microbial digesting activity increased between 20 and 200 mM NaLac. Concentrations below 20 mM appeared to slightly increase both MDMD and MOMD, but differences were not significant (P > 0.05). Almost 65% of microbial digestion of forage was inhibited by 200 mM NaLac vs. only 35% by the same concentration of NaC1. Medium pH was not substantially altered by the additions of either NaC1 or NaLac. Forage ADF digestibility was also impaired by both NaLac and NaC1 (Table II). Complete inhibition of MADFO was obtained with 200 mM NaLac, but the inhibition provoked by 200 mM NaC1 was considerably lower. Significant differences in medium pH were detected only at the end of the incubation. NaLac 80 mM was the only addition that modified pH with respect to the values of the inoculated control. Percentage inhibitions of both MDMD and MOMD were positively correlated with NaLac concentrations as follows: % Inhibition of MDMD = --7.6 + 0.37 (raM NaLac), (r 2 = 0.86, P ~< 0.05) % Inhibition of MOMD = --7.3 + 0.37 (raM NaLac), (r: = 0.87, P ~< 0.05) DISCUSSION According to the preceding equations, the minimum concentration of NaLac concentration of about 2.7 mM will elicit a 1% depression of both plete inhibition of microbial digestibility would be obtained with NaLac concentrations greater than 290 raM. Above 20 mM NaLac, every increase in NaLac concentration of about 2.7 mM will elicit a 1% depression of both MDMD and MOMD. Fiber digesting activity of rumen microbes seems to be more sensitive to high NaLac concentrations since complete inhibition of ADF digestion was obtained with only 200 mM of the salt. The inclusion of gains or other high-energy feeds in the diet usually provokes a decrease in tureen pH as more VFA and lactic acid are produced by microbial fermentation (Counotte et al., 1983; Mackie et al., 1984). This pH drop has been suggested to be the main cause for the observed decrease in fiber digestion after grain supplementation because ceUuloytic activity of rumen microbes is maximal at pH 6.8--7.1 and markedly decreases below these values (Mertens, 1979). In the present study, lactic acid was added to the medium as its sodium salt and during the assays pH remained within the reported optimum range for ceUulolysis; a depression of digestion due to a pH change, therefore, can be ruled out. Lactate concentrations that originate in the rumen when gains or other concentrates are included in the diet normally do not exceed 20 mM (Malestein et al., 1981; Mackie et al., 1984). According to our results, these concentrations would not be high enough to depress forage digestibility provided that pH is kept above 6.8 (Mertens, 1979). However, Grau (1981) showed that the inhibitory effect of lactic acid on growth of some nontureen bacteria increases when pH decreases from 6.1 to 5.5, and we can speculate that the inhibition of rumen microbes by lactate may also be
166
potentiated when pH falls as a consequence of grain supplementation. Furthermore, a pH drop may result in higher ruminal levels of lactic acid due to reduced microbial degradation of this compound below pH 6 (Malenstein et al., 1981) compared to degradation at higher pH values. When grains are supplied in excessive amounts, either ground or without a previdus adaptation of the animals to these feeds, ruminal concentrations of lactic acid rapidly increase and can reach levels well over 20 mM (Counotte and Prins, 1981; Muir et al., 1981). According to results of this study, these high concentrations of lactate will be inhibitory for the microbial digesting activity even at pH values within the normal range in the tureen. Increased osmolality reduces cellulose digestibility by rumen microbes (Bergen, 1972) and in this work increasing concentrations of NaLac induced increasing inhibitions of A. elongatum digestion in vitro. Nevertheless, the inhibition of MDMD, MOMD and MADFD by 200 mM NaC1 was only half that caused by an equivalent concentration of NaLac which clearly indicates that lactate per se depress forage digestion by rumen microbes. The mechanisms of the inhibition of forage digestion by lactate could be similar to those proposed by E1-Shazly et al. (1961) and by Mertens (1979) to explain the depression of cellulose digestion by starch. In addition, the possibility that lactate itself may be toxic for fiber digesting rumen microbes cannot be ruled out since this compound has been shown to be toxic to some microorganisms (Grau, 1981). We conclude that lactate is a potential inhibitor of forage digestion by rumen microbes and, therefore, it may contribute to the reported decrease of fiber digestion in grain-supplemented ruminants. ACKNOWLEDGEMENTS
Thanks are due to Dr. H. Fernandez for his help with the statistical analysis, and to M. Patriarca, C. Bastarrechea and M.E. Giuliano for their technical assistance. This work was supported in part by a grant from the Consejo Nacional de Investigaciones Cientlficas y T~cnicas de la Repfiblica Argentina (CONtCET). J.P. Fay is a Career Investigator of the CONICET. REFERENCES Bergen, W.G., 1972. Role of osmolality in feed intake control in sheep. J. Anita. Sci., 34: 1054--1060. Burroughs, W., Gerlaugh, P., Edgington, B.H. and Bethke, R.M., 1949. The influence of corn starch upon roughage digestion in cattle. J. Anita. Sci., 8: 271--278. Counotte, G.H.M. and Prins, R.A., 1981. Regulation of lactate metabolism in the rumen. Vet. Res. Commun., 5: 101--115. Counotte, G.H.M., Lankhorst, A. and Prins, R.A., 1983. Role of DL-Lactic acid as an intermediate in rumen metabolism of dairy cows. J. Anita. Sci., 56: 1222--1235. EI-Shazly, K., Dehority, B.A. and Johnson, R.R., 1961. Effect of starch on the digestion of cellulose in vitro and in vivo by tureen microorganisms. J. Anita. Sci., 20 : 268--273.
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