EFFECT OF DIET ON IN V I T R O A N D IN V I V O RUMEN LACTATE DISAPPEARANCE RATE IN SHEEP 1,2 W. E. Kunkle 3,4, A. W. Fetter s and R. L. Preston 3,6
SUMMARY
Four fistulated sheep were fed either chopped grass hay, hay plus two levels of lactic acid or a corn based concentrate diet in a 4 x 4 extra-period latin square design with 21 day experimental periods. Disappearance rates of infused lactic acid in an in vitro fermentation were measured on day 7, 11, 14 and 21 of each period. On day 15 of each period, lactate was infused into the rumen of sheep to measure in vivo disappearance rates. Both in vitro and in vivo disappearance rates were lower by at least 50% when the hay diet was fed compared to the hay plus lactic acid and concentrate diets. The in vitro disappearance rates of lactate in ruminal fluid from sheep fed hay, hay plus lactic acid (two levels) and concentrate were 19, 44, 57 and 127 rag/100 ml/hr, respectively, on the 21st day. In contrast, the in vivo lactate disappearance rates were 154, 486, 500 and 313 mg/lO0 ml/hr, respectively. The use of whole instead of strained ruminal contents as inocula resulted in in vitro treatment rankings similar to those obtained in vivo. (Key Words: Sheep, Hay, Concentrate, Lactic
Acid, Lactic Acid Disappearance, Lactic Acid Isomers.) INTRODUCTION
Lactic acid has been demonstrated to disappear from the rumen (Phillipson and McAnally, 1942; Waldo and Schuhz, 1956; Heuter et al., 1956; Emery et al., 1966). Lactic acid given to ruminants by infusion or in natural diets such as silages was found to disappear from the rumen in 1 to 8 hr depending on the quantity given and the experiment. Lactic acid accumulates in the rumen of unadapted animals that consume large quantities of grain (Dunlop, 1972). However, large accumulation of lactic acid can be avoided if ruminants are converted gradually to a high concentrate diet. Caldwell and Bryant (1966) and Latham et al. (1971) found that a large portion of the bacterial isolates from the rumen were capable of metabolizing lactate in ruminants fed concentrates as compared to those fed forage diets. Jayasuriya and Hungate (1959) reported that ruminal contents of steers fed grain has a higher lactate turnover rate than hay-fed steers. These results indicate that part of the adaptive changes to a high concentrate diet involves a shift in the relative amounts of some ruminal microorganisms which results in Supported in party by NIH grant No. 5 SO1 RR an increased rate of lactate utilization. 054 63-11 and Central Division, Consolidated Coal Dunlop (1972) noted that a large proportion Co., Cadiz, Ohio. Approved for publication as Journal Article No. 100-74 of the Ohio Agricultural Research of the accumulated lactic acid may be the D(--) and Development Center, Wooster. isomer. Schaadt and Johnson (1968) reported 2The authors thank the Monsanto Co., St. Louis, that the D(--) isomer usually comprised more Missouri for donating the lactic acid used in this study. than 50% of the lactic acid in corn silage. The The assistance of Dr. C. R. Weaver in the statistical D(--) isomer has been shown to be metabolized analyses of the data is gratefully acknowledged. by ruminal contents in vitro (Jayasuriya and 3DeparWnentof Animal Science. 4Present address: Department of Animal Science, Hungate, 1959) but little information is availJull Hall, University of Maryland, College Park, 20742. able to indicate if one isomer is preferentially $Veterinary Pathobiology and Veterinary Clinical metabolized. Sciences. The objective of this research was to meaPresent address: Department of Animal Sciences, sure lactate disappearance rates in vitro and in Washington State University, Pullman, 99163. 1256 JOURNAL OF ANIMAL SCIENCE, Vol. 42, No. 5, 1976
Downloaded from https://academic.oup.com/jas/article-abstract/42/5/1256/4698077 by Iowa State University user on 17 January 2019
Obio Agricultural Research and Development Center, Wooster, 44691 and The Obio State University, Columbus, 43210
DIETARY EFFECTS ON RUMEN LACTATE DISAPPEARANCE
1257
period.
acid, or concentrate diets.
In Vitro Studies. Lactic acid disappearance was determined on days 7, 11, 14 and 21 of each period. Ruminal contents were removed via the fistula from each sheep at 3 hr postfeeding and strained by squeezing through a double layer of cheesecloth. Strained rumen fluid (20 ml) was added to a carbon dioxidegassed tube containing media and lactic acid. The media was similar to that described by Johnson (1966) except that PABA (100 mcg/ml) and cysteine (4 mg/ml) were added at .5 m i l l 0 0 ml, sodium sulfate was substituted for potassium sulfate, and 1/2 the amount of biotin was used (J9 H. Cline, personal communication). A racemic mixture of food grade lactic acid which contained no measurable polymer was added to each tube to give a final concentration of 800 mg/100 ml in a total volume of 50 milliliters. The lactic acid was from the same source as that fed with the hay. The media and lactic acid were mixed, gassed with CO2, adjusted to pH 6.9 and incubated in a 39 C water bath prior to addition of strained tureen fluid. The tubes were bubbled with carbon dioxide continuously and maintained at 39 C throughout the incubation. Samples (1 to 2 ml)
EXPERIMENTAL PROCEDURE
Four male yearling sheep weighing approximately 50 kg and fitted with permanent rumihal cannulas were housed in a temperature controlled room in individual elevated metal stalls with expanded metal floors. The diets listed in table 1 were formulated to meet requirements for maintenance (N.R.C., 1968). Diets B and C were mixed daily and all diets were fed once daily. Lactic acid was added without attempting to adjust the nutrient content of diets B and C; thus energy, protein and other nutrients were not equal between diets. The sheep were given fresh water and trace mineralized salt blocks ad libitum. Prior to starting the experiment, all sheep were wormed with L-Ripercol (American Cyanamid Co.) and injected intramuscularly with 2.5 ml of BoSe (Vitamin E and Selenium, Burns Pharmaceutical), 500,000 units of Vitamin A and 75,000 units of Vitamin D. The experimental design was a 4 x 4 extra period latin square with 21-day periods. The diets were switched in equal increments over the first 10 days of each
TABLE 1. COMPOSITION OF DIETS Ingredients
Orchardgrass, hay S-C, cut 1 chopped (1), REF No. 1-03-433 (chopped Grass Hay)c Lactic acid (50% syrup) d (4) Corn grain, REF No. 4-02-879 (shelled corn, No. 2) Soybean seeds, solv-extd grnd, mx 7% fiber (5) REF No. 5-04-604 (44% soybean meal) Limestone, grnd, mn 33% calcium (6), REF No. 6-02-632 (ground limestone) Potassium sulfate (6), REF No. 6-08-098 Potassium chloride, KCI, commercial, (6), REF No. 6-03-755
Ha
H(L)a
H(2L) a
Cb
%
%
%
%
100.00 9 99
85.00 15.00
70.00 30.00
... . ..
.
.
.
.
.
.
.
.
.
88.18 10.20 19 9
.
~
9 9
al,300 g offered daily. b930 g offered daily9 Soybean meal, minerals and enough corn to make 150 g daily was ground, mixed and pelleted ; the remainder of the corn was fed as whole shelled corn. CAna|ysis: dry matter, 90.0%; crude protein, 10.8%; calcium, .48% ; phosphorus, .44%. dFeed grade lactic acid, supplied b v Monsanto Company, St. Louis, Missouri9Contained approximately equal amounts of the D(-) and L(+~ isom ith no measurable quantities of polymerized lactic acid.
Downloaded from https://academic.oup.com/jas/article-abstract/42/5/1256/4698077 by Iowa State University user on 17 January 2019
vivo when sheep were fed hay, hay plus lactic
1258
KUNKLE, FETTER AND PRESTON
~Advanced Instruments Inc., 55 Kenneth Street, Newton Highlands, Massachusetts 02161.
had a significant quadratic component, however, in these cases the linear effect was several magnitudes greater than the quadratic effect. Therefore, only the linear regression coefficient was used in our analyses. The carryover effects were determined by coding the data for previous diet. The differences in disappearance rates and percentage isomeric composition were compared by least square analyses (Harvey, 1960). The osmolality of the in vivo samples was measured after thawing and centrifuging at 29,000 g for 20 min in a refrigerated centrifuge. The supernatant was used for osmolality determination in an Advanced Wide Range Osometer 7. RESULTS AND DISCUSSION
In Vitro Studies. The lactate disappearance rates in vitro are presented in table 2. One sheep on H(2L) refused to eat during the latter part of the period and the sheep also rubbed their cannulas out sporadically; therefore these divergent data were omitted from the analysis thus giving somewhat different standard errors of the mean. Inocula from sheep on diet H, compared to diets (H(L), H(2L) and C, had significantly (P
SUMMARY
Four fistulated sheep were fed either chopped grass hay, hay plus two levels of lactic acid or a corn based concentrate diet in a 4 x 4 extra-period latin square design with 21 day experimental periods. Disappearance rates of infused lactic acid in an in vitro fermentation were measured on day 7, 11, 14 and 21 of each period. On day 15 of each period, lactate was infused into the rumen of sheep to measure in vivo disappearance rates. Both in vitro and in vivo disappearance rates were lower by at least 50% when the hay diet was fed compared to the hay plus lactic acid and concentrate diets. The in vitro disappearance rates of lactate in ruminal fluid from sheep fed hay, hay plus lactic acid (two levels) and concentrate were 19, 44, 57 and 127 rag/100 ml/hr, respectively, on the 21st day. In contrast, the in vivo lactate disappearance rates were 154, 486, 500 and 313 mg/lO0 ml/hr, respectively. The use of whole instead of strained ruminal contents as inocula resulted in in vitro treatment rankings similar to those obtained in vivo. (Key Words: Sheep, Hay, Concentrate, Lactic
Acid, Lactic Acid Disappearance, Lactic Acid Isomers.) INTRODUCTION
Lactic acid has been demonstrated to disappear from the rumen (Phillipson and McAnally, 1942; Waldo and Schuhz, 1956; Heuter et al., 1956; Emery et al., 1966). Lactic acid given to ruminants by infusion or in natural diets such as silages was found to disappear from the rumen in 1 to 8 hr depending on the quantity given and the experiment. Lactic acid accumulates in the rumen of unadapted animals that consume large quantities of grain (Dunlop, 1972). However, large accumulation of lactic acid can be avoided if ruminants are converted gradually to a high concentrate diet. Caldwell and Bryant (1966) and Latham et al. (1971) found that a large portion of the bacterial isolates from the rumen were capable of metabolizing lactate in ruminants fed concentrates as compared to those fed forage diets. Jayasuriya and Hungate (1959) reported that ruminal contents of steers fed grain has a higher lactate turnover rate than hay-fed steers. These results indicate that part of the adaptive changes to a high concentrate diet involves a shift in the relative amounts of some ruminal microorganisms which results in Supported in party by NIH grant No. 5 SO1 RR an increased rate of lactate utilization. 054 63-11 and Central Division, Consolidated Coal Dunlop (1972) noted that a large proportion Co., Cadiz, Ohio. Approved for publication as Journal Article No. 100-74 of the Ohio Agricultural Research of the accumulated lactic acid may be the D(--) and Development Center, Wooster. isomer. Schaadt and Johnson (1968) reported 2The authors thank the Monsanto Co., St. Louis, that the D(--) isomer usually comprised more Missouri for donating the lactic acid used in this study. than 50% of the lactic acid in corn silage. The The assistance of Dr. C. R. Weaver in the statistical D(--) isomer has been shown to be metabolized analyses of the data is gratefully acknowledged. by ruminal contents in vitro (Jayasuriya and 3DeparWnentof Animal Science. 4Present address: Department of Animal Science, Hungate, 1959) but little information is availJull Hall, University of Maryland, College Park, 20742. able to indicate if one isomer is preferentially $Veterinary Pathobiology and Veterinary Clinical metabolized. Sciences. The objective of this research was to meaPresent address: Department of Animal Sciences, sure lactate disappearance rates in vitro and in Washington State University, Pullman, 99163. 1256 JOURNAL OF ANIMAL SCIENCE, Vol. 42, No. 5, 1976
Downloaded from https://academic.oup.com/jas/article-abstract/42/5/1256/4698077 by Iowa State University user on 17 January 2019
Obio Agricultural Research and Development Center, Wooster, 44691 and The Obio State University, Columbus, 43210
DIETARY EFFECTS ON RUMEN LACTATE DISAPPEARANCE
1257
period.
acid, or concentrate diets.
In Vitro Studies. Lactic acid disappearance was determined on days 7, 11, 14 and 21 of each period. Ruminal contents were removed via the fistula from each sheep at 3 hr postfeeding and strained by squeezing through a double layer of cheesecloth. Strained rumen fluid (20 ml) was added to a carbon dioxidegassed tube containing media and lactic acid. The media was similar to that described by Johnson (1966) except that PABA (100 mcg/ml) and cysteine (4 mg/ml) were added at .5 m i l l 0 0 ml, sodium sulfate was substituted for potassium sulfate, and 1/2 the amount of biotin was used (J9 H. Cline, personal communication). A racemic mixture of food grade lactic acid which contained no measurable polymer was added to each tube to give a final concentration of 800 mg/100 ml in a total volume of 50 milliliters. The lactic acid was from the same source as that fed with the hay. The media and lactic acid were mixed, gassed with CO2, adjusted to pH 6.9 and incubated in a 39 C water bath prior to addition of strained tureen fluid. The tubes were bubbled with carbon dioxide continuously and maintained at 39 C throughout the incubation. Samples (1 to 2 ml)
EXPERIMENTAL PROCEDURE
Four male yearling sheep weighing approximately 50 kg and fitted with permanent rumihal cannulas were housed in a temperature controlled room in individual elevated metal stalls with expanded metal floors. The diets listed in table 1 were formulated to meet requirements for maintenance (N.R.C., 1968). Diets B and C were mixed daily and all diets were fed once daily. Lactic acid was added without attempting to adjust the nutrient content of diets B and C; thus energy, protein and other nutrients were not equal between diets. The sheep were given fresh water and trace mineralized salt blocks ad libitum. Prior to starting the experiment, all sheep were wormed with L-Ripercol (American Cyanamid Co.) and injected intramuscularly with 2.5 ml of BoSe (Vitamin E and Selenium, Burns Pharmaceutical), 500,000 units of Vitamin A and 75,000 units of Vitamin D. The experimental design was a 4 x 4 extra period latin square with 21-day periods. The diets were switched in equal increments over the first 10 days of each
TABLE 1. COMPOSITION OF DIETS Ingredients
Orchardgrass, hay S-C, cut 1 chopped (1), REF No. 1-03-433 (chopped Grass Hay)c Lactic acid (50% syrup) d (4) Corn grain, REF No. 4-02-879 (shelled corn, No. 2) Soybean seeds, solv-extd grnd, mx 7% fiber (5) REF No. 5-04-604 (44% soybean meal) Limestone, grnd, mn 33% calcium (6), REF No. 6-02-632 (ground limestone) Potassium sulfate (6), REF No. 6-08-098 Potassium chloride, KCI, commercial, (6), REF No. 6-03-755
Ha
H(L)a
H(2L) a
Cb
%
%
%
%
100.00 9 99
85.00 15.00
70.00 30.00
... . ..
.
.
.
.
.
.
.
.
.
88.18 10.20 19 9
.
~
9 9
al,300 g offered daily. b930 g offered daily9 Soybean meal, minerals and enough corn to make 150 g daily was ground, mixed and pelleted ; the remainder of the corn was fed as whole shelled corn. CAna|ysis: dry matter, 90.0%; crude protein, 10.8%; calcium, .48% ; phosphorus, .44%. dFeed grade lactic acid, supplied b v Monsanto Company, St. Louis, Missouri9Contained approximately equal amounts of the D(-) and L(+~ isom ith no measurable quantities of polymerized lactic acid.
Downloaded from https://academic.oup.com/jas/article-abstract/42/5/1256/4698077 by Iowa State University user on 17 January 2019
vivo when sheep were fed hay, hay plus lactic
1258
KUNKLE, FETTER AND PRESTON
~Advanced Instruments Inc., 55 Kenneth Street, Newton Highlands, Massachusetts 02161.
had a significant quadratic component, however, in these cases the linear effect was several magnitudes greater than the quadratic effect. Therefore, only the linear regression coefficient was used in our analyses. The carryover effects were determined by coding the data for previous diet. The differences in disappearance rates and percentage isomeric composition were compared by least square analyses (Harvey, 1960). The osmolality of the in vivo samples was measured after thawing and centrifuging at 29,000 g for 20 min in a refrigerated centrifuge. The supernatant was used for osmolality determination in an Advanced Wide Range Osometer 7. RESULTS AND DISCUSSION
In Vitro Studies. The lactate disappearance rates in vitro are presented in table 2. One sheep on H(2L) refused to eat during the latter part of the period and the sheep also rubbed their cannulas out sporadically; therefore these divergent data were omitted from the analysis thus giving somewhat different standard errors of the mean. Inocula from sheep on diet H, compared to diets (H(L), H(2L) and C, had significantly (P
