DETERMINATION OF RUMEN FILL, RETENTION TIME AND RUMINAL TURNOVER RATES OF INGESTA AT DIFFERENT STAGES OF LACTATION IN DAIRY COWS 1,2

Transcription

1 DETERMINATION OF RUMEN FILL, RETENTION TIME AND RUMINAL TURNOVER RATES OF INGESTA AT DIFFERENT STAGES OF LACTATION IN DAIRY COWS 1,2 Gary F. HartneU 3 and Larry D. Satter University of Wisconsin, Madison SUMMARY Rumen fill and turnover rates of liquid, grain, and hay in the gastrointestinal tract were measured using rare-earth elements as multiple markers in four rumen fistulated cows during the dry period and throughout lactation. Apparent digestibilities of dry matter, crude protein, neutral-detergent fiber and aciddetergent fiber were also measured. Hay to grain ratios of 82.5:17.5, 45:55, 57:43, and 67:33 (dry matter basis) were fed during the dry period and during weeks 0 to 12, 13 to 24, and 25 to 44 of lactation, respectively. Apparent digestibilities of DM, CP, ADF and NDF were 64.1, 67.2, 68.3, 66.4; 77.2, 70.9, 73.0, 73.7; 41.0, 34.5, 35.6, 38.2;and 51.6, 36.7, 44.0 and 47.1 for the four phases of the experiment. For the dry period, 0 to 12, 13 to 24, and 25 to 44 weeks of lactation, rumen dry matter and liquid content (kg) were 9.6, 62.1; 16.0, 79.4; 16.0, 89.3; and 15.7 and Mean ruminal turnover rates across periods for liquid, grain and hay were 8.1, 4.4, and 3.9% per hour. Experimental period had no effect on turnover rate. However, stage of lactation, level of intake and hay:grain ratio all changed with each experimental period. The greatest difference in turnover rate (twofold) was among individual cows. There were small differences noted among stage of lactation periods in total mean retention time of liquid, grain and bay fractions 1 Research supported by the College of Agricultural and Life Sciences, the University of Wisconsin Graduate Research Committee, and by Federal Hatch Project a The technical assistance of Paul Fritschel, Lyle Holschbach and Ric Grummer and the assistance in neutron activation analysis by Richard J. Cashwell and Stephen M. Matusewic are gratefully acknowledged. 3Present address: Allied Mills Inc., Research and Development Center, P. O. Box 459, Libertyville, IL within the digestive tract. Grab sampling of feces as compared to total feces collection resulted in similar estimates of liquid, grain and hay fractions within the digestive tract. Grab sampling of feces as compared to total feces collection resulted in similar estimates of liquid, grain and hay turnover rates in the reticulorumen. Comparisons were made between stained feed particles and rare-earth elements as markers of ingesta flow. (Key Words: Turnover Rate, Rare-earth Elements, Retention Time, Rate of Passage, Digestibility.) INTRODUCTION Increasing the turnover rate of substrate or ingesta has increased yields of rumen microbial protein in vitro (lsaacson et al., 1975; Stouthamer and Bettenhaussen, 1973), in sheep (Harrison et al., 1975; Walker et al., 1975) and in steers (Cole et al., 1976). The amount of feed protein escaping fermentation might be expected to increase with increased turnover rates of the rumen liquid phase also (Harrison et al., 1975). In addition, an increased passage rate of undigested forage residue out of the rumen may be associated with increased feed intake with high forage rations (Baumgardt, 1970). This, however, is often accompanied by a depression in digestibility (Blaxter, 1969). The suitability of using rare-earth elements as markers of particulate matter flow in the gastro-intestinal tract has been discussed (Hartnell and Satter, 1979). The purpose of this study was to determine: (1) the range of ingesta turnover rates and weights of ruminal ingesta (fill) in dairy cows during the dry period and throughout lactation, (2) the effect of stage of lactation on fiber, protein and dry matter digestibilities, (3) the difference between grab sampling and total feces collection in deriving ingesta turnover rates, and (4) the difference 381 JOURNAL OF ANIMAL SCIENCE, Vol. 48, No. 2, 1979

2 382 HARTNELL AND SATTER between stained particle technique and rareearth elements as nutrient markers. MATERIALS AND METHODS Four rumen fistulated Holstein cows which calved within one month of each other were fed second crop alfalfa hay (IRN ) and grain (table 1) ad libitum in the ratios of 82.5:17.5, 45:55, 57:43 and 67:33 during the dry period (-8 to 0 weeks), early lactation (0 to 12 weeks), midqactation (13 to 24 weeks), and late lactation (25 to 44 weeks), respectively. The cows were first fed the 82.5:17.5 hay:grain ratio 8 weeks prior to calving. Enough alfalfa hay was harvested from one field to last the entire experiment. Hay and grain were analyzed for crude protein using the Kjeldahl procedure and for acid- and neutral-detergent fiber using the procedure of Goering and Van Soest, One-third of the daily grain allotment was fed at 0400 hr before the morning milking, again at mid-morning (1030 hr) and again at 1700 hr after the afternoon milking. Feed and water intakes were recorded daily. Water intakes were measured using water meters placed in the water line between the drinking cup and the main water line. Body weights were taken once a week at 0745 hours. Weight of total rumen contents was measured four times over a 4- to 6-week period in the middle of each phase. Two hours after the mid-morning feeding, the reticulorumens were manually emptied. The rumen contents were weighed, and numerous samples were taken as the contents were placed back into the rumen. Digesta samples were composited and dried in a forced air oven at 60 C for 72 hr to determine dry matter content. Digestibility. and rate of passage measurements were performed during a 2.5-week sampling period in the middle of each phase as illustrated in figure 1. Samarium (Sm) and lanthanum (La) solutions were prepared by dissolving 1.16 g of samarium oxide (99.9% pure) or 1.17 g of lanthanum oxide (99.9% pure) 4 in 4 ml of concentrated hydrochloric acid and diluting with water to 30 milliliters. The cerium solution was prepared by dissolving 3.92 g of ceric ammonium nitrate 4Research Chemicals, P. O. Box 14588, Phoenix, AZ s G. Frederick Smith Chemical Company, Columbus, OH. TABLE 1. COMPOSITION OF THE GRAIN MIXTURE Ingredient I RN a Percent Ground shelled corn Ground oats Soybean oil meal (44%) b Beet pulp Dried cane molasses Dicalcium phosphate Trace mineralized saltc Vitamin ADE premix d aatlas of nutritional data on United States and Canadian feeds National Academy of Sciences, Washington, DC. blinseed meal ( ) substituted for soybean meal during the lactation phases of the experiment. CThe mineral mix contained: NaCI (95-99%); Mn (>.20%); Ferrous (Fe), (>.16%); Ferric (Fe), (>.14%); Cu (>.033%); Zn (>.010%); I (>.007%); and Co (>.005%). dvitamin premix contained 2,000,000 IU of vitamins A and D and 200 IU of vitamin E per kilogram. (99.9% pure) s in 10 ml of water and diluting with water to 30 milliliters. The resulting solutions contained approximately 1 g of element per 30 milliliters. Solutions were stored in polyethylene containers until needed. The marked hay was prepared by mixing.5 kg of crystal violet stained alfalfa hay (Balch, 1950) with.5 kg of unstained alfalfa hay. The mixture was sprayed with 30 ml (approximately 1 g of element) of each of the rare-earth solutions with each cow receiving a different combination of elements. The reason for using different combinations of elements was to verify that all of the elements behaved similarly. Stained hay was included to allow comparison of the two methods of marking. After spraying, the hay was air dried and placed into plastic garbage bags until needed for feeding. Marked grain was prepared as described for the hay except.25 kg of basic fuchsin stained grain (Balch, 1950) was mixed with.75 kg of unstained grain before spraying with La, Ce and/or Sm solutions. Thus, the marked meal fed each cow consisted of 1 kg of treated hay and 1 kg of treated grain. One liter of Cr-EDTA (2.6 to 3.0 g Cr) prepared according to the procedure of Binnerts et al. (1968) was placed into the rumen via the fistula at the time of feeding the marked meal. Marked hay and

3 RUMEN FILL, RETENTION AND TURNOVER ON LACTATION 383 Day i i0 Ii A 71 B B c ;hi D C "~1 E r - ~1 Figure 1. Description of 2.5 week sampling period during which time all ingesta and feces samples were collected. A) Feed and water intakes were recorded daily. B) Marked hay and grain were fed with 1 liter of Cr-EDTA at 1030 hours. C) Grab samples of rumen contents were taken at O, 6, 12, 18, 24, 30, 36, 48, 60, 72, 96, 120 and144 hr after feeding the marked meal. Grab samples of feces were taken at O, 12, 18, 24, 30, 36, 48, 60, 72, 84, 96, 108, 120, 144 and 168 hr after feeding the marked meal. D) Measured rumen fill. Three additional measurements of rumen fill were obtained just prior to or following this 2.5-week period. E) Attached feces collecting apparatus. F) Attached feces bag. The bags were changed every 6 hr and the 12 hr collections composited through 7 days. G) Removed feces collecting apparatus. G grain that was not consumed by the cow after 45 min were manually broken into 10 cm pieces and placed directly into the rumen via the fistula. Usually this amount did not exceed 10 to 20% of the total marked feed. Grab samples of rumen contents, consisting of a composite of samples taken from six different locations in the rumen, and grab samples of feces were weighed, dried at 60 C for 48 hr, weighed, and ground through a 40 mesh screen using a Wiley mill. Portions of the ground samples (2.5 to 4.0 g) were weighed into 4-dram polyethylene vials for neutron activation analysis of Cr, Sm, Ce and La. In obtaining the feces, the animal was induced to defecate and the last portion of feces excreted was taken as the sample. Total feces collections were performed using the feces collecting apparatus described by Gorski et al. (1957). Using this apparatus, the cows could go outside for exercise and be milked in the parlor during the collection. No urine was collected. Twelve-hour feces composites and grab samples of feces for the 7-day collection period were prepared for neutron activation analysis of Cr, Sm, Ce and La as described by Hartnell and Satter (1978). The ground dried feces from the total feces collection were composited into 24-hr samples. These samples were analyzed for crude protein (Kjeldahl method) and acid- and neutral-_ detergent fiber (Goering and Van Soest, 1970). The stained particle technique employed was similar to that used by Balch (1950) and Castle (1956) except that.1 g of dried ground feces was spread out over a 12.5 cm grid and then counted with the aid of a microscope. Wet feces were not washed over a piece of cheesecloth and counted, as Balch recommended, since cheesecloth withholds only those particles of diameter greater than the pores of the cloth, whereas no particles were lost using a dried ground sample. Liquid, hay and grain ruminal turnover rates, total mean retention times and transit times were calculated by fitting a two compartmental model (Brandt and Thacker, 1958) to the fecal excretion data (grab samples and total feces collection) using the procedure of Grovum and Williams (1973b). A completely randomized design was used in calculating the analysis of variance. The means were compared using Duncan's new multiple range test (Duncan, 1955). RESULTS AND DISCUSSION Dry matter, crude protein, neutral-detergent fiber and acid-detergent fiber contents in hay and grain which were fed to the cows are reported in table 2. Table 3 contains body weight, milk production, water and feed intake

4 384 HARTNELL AND SATTER TABLE 2. DRY MATTER (DM), CRUDE PROTEIN (CP), NEUTRAL-DETERGENT FIBER (NDF), AND ACID-DETERGENT FIBER (ADF) ANALYSIS OF RATION INGREDIENTS Feedstuff DM CP NDFa ADFb (%) Hay Grain aneutral-detergent fiber (Goering and Van Soest, 1970). bacid-detergent fiber (Goering and Van Soest, 1970). and ration digestibility data for each phase of the experiment. In evaluating the results, it must be kept in mind that the effects of feed intake, hay to grain ratio and stage of lactation are confounded. Water and dry matter intakes were lower (P<.05) (25% and 43%) during the dry period but were different (P<.05) during the periods of milk production. It was observed that during very hot (T>32 C) days cows would reduce their dry matter consumption without changing water intake. Apparent dry matter digestibility found in the dry period was less (P<.05) than the three periods in which the animals were lactating. This was probably due to the lower proportion of grain fed during the dry period. Apparent digestibility of crude protein increased (P<.05) from 70.9% after parturition to 77.2% during the dry period. Apparent digestibility of neutral-detergent fiber increased (P<.05) as the proportion of the hay in the diet increased. This was also evident for the acid-detergent fiber. Campling (1966) reported that addition of grain to the ration increased dry matter digestibility, but decreased crude fiber digestibility. Measurements of rumen fill are presented in table 4. There was 40% less dry matter in the reticulo-rumen during the dry period compared to the milking periods. The liquid portion of the rumen contents varied from 62.1 kg during the dry period to 89.3 kg during weeks 13 to 24 of lactation. There was a 10 kg increase in total ruminal ingesta weight from the middle of the 0 to 12 week period to the middle of the 13 to 24 week period. On average, total rumen fill was closely related to dry matter intake. It is important to bear in mind the changes in ingesta weight or gut fill when examining changes in overall body weight during early lactation. A cow may actually be losing body tissue during this time, but the increase in gut fill (24 kg in this study) would obscure this. Therefore, body weights must be used carefully when trying to assess depletion of body stores during early lactation. When the rumens were emptied, it was noted that among the four cows, 2273 seemed to have the smallest rumen, and 2136 the most spacious rumen. The dry matter content in each was TABLE 3. BODY WEIGHT, MILK PRODUCTION, WATER AND DRY MATTER INTAKE, AND APPARENT DIGESTIBILITY OF RATION COMPONENTS AT DIFFERENT STAGES OF LACTATION Stage of lactation (weeks) (Dry) Component SE Forage:Grain 82.5: :55 57:43 67:33... Body weight (kg) Milk production (kg/day) Water intake (kg/day) 60.5 a 82.9 b 83.8 b 78.6 b 2.7 Dry matter intake (kg/day) 10.8a 16.9 b 19.8 b 17.6 b.9 Apparent digestibility, % Dry matter 64. la 67.2 b 68.3 b 66.4 b.6 Crude protein 77.2c 70.9 a 73.0ab 73.7 b.7 Acid-detergent fiber Neutral-detergent fiber 51.6b 36.7a 44.0b 47.1 b 3.1 a'b~cmeans in the same row which do not have a common letter in the superscript are different (P<.05).

5 - ruminal - total RUMEN FILL, RETENTION AND TURNOVER ON LACTATION 385 TABLE 4. RUMEN FILL AT DIFFERENT STAGES OF LACTATION Stages of lactation (weeks) (Dry) Component SE (kga) Dry matter 9.6 b 16.0c 16.0 c 15.7 c.9 Liquid 62.1 b 79.4 c 89.3 d 86.0 ~d 3.2 Total 71.7 b 95.4c I05.3 c c 3.1 aeach value is a mean of four cows. b'c'dmeans in the same row which do not have a common letter in the superscript are different (P<.05). approximately the same, even though cow 2273 had a very congested, filled reticulo-rumen and cow 2136 had ample space above the ingesta for additional material. There were large differences among cows, particularly in liquid fill of the rumen and in turnover of liquid ingesta (table 5). Cow 2136 had up to 30 kg more liquid than cow 2273 during the lactation TABLE 5. INDIVIDUAL COW DATA FOR RATE OF PASSAGE MEASUREMENTS DURING PERIODS OF LOWEST AND HIGHEST DRY MATTER INTAKES (Dry) -8-0 Stage of lactation (weeks) Cow identification (Lactating) Parameter Body weight, kg Milk production, kg/day Dry matter intake kg/day 11.2 " Water intake, kg/day Rumen fill, kg Dry matter Liquid K1, %/hra Liquid Grain Hay K 2, %/hr b Liquid Grain Hay TMRT, hr c Liquid Grain Hay TT, hr d Liquid Grain Hay ak 1 bk 2 CTM RT dtt turnover rate. - thought to be turnover rate of contents in the hindgut or cecum and proximal colon or artifact due to mixing. mean retention time. - time for first appearance of marker in feces.

6 386 HARTNELL AND SATTER periods. Water intake was about the same for these two cows during lactation. Differences between cows in rumen liquid volume may be due to differences in saliva production, liquid turnover rate and/or ruminal capacity. If faster fluid turnover rates in the rumen are correlated with greater efficiency of microbial growth (Isaacson et al., 1975; Walker et al., 1975), then cows 2273 and 2146 might be expected to have a larger supply of microbial protein than cows 2149 and Dry matter intake as well as dry matter content in the rumen were reduced during the dry period, and the question might be raised whether the increase in size of the fetus reduced rumen fill and limited feed intake. In this study, rumens were emptied at 8, 5 and 2 or 1 week prior to parturition. There were no differences in the amount of dry matter or liquid in the reticulo-rumens at any time during the dry period. Lamberth (1969) reported nonsignificant differences in rumen fill between nonpregnant and pregnant twin heifers. Samarium (Sm), cerium (Ce)and lanthanum (La) were used as particulate markers because they adsorb tenaciously to feed particles, are inexpensive, have nonoverlapping decay patterns upon activation, and have sufficiently long half lives to enable counting 7 to 10 days after neutron activation (Hartnell, 1977; Hartnell and Satter, 1979). Therefore, excretion of the markers should be representative of the diet component marked. The exception might be that fraction of marker which was adsorbed onto particles which were subsequently digested away. In general, close to 100% of the elements were recovered in the feces, with the extreme range of recovery equaling 92% and 115%. Urine and milk were monitored for the elements throughout the experiment, and no rareearth element was detected. In calculating and measuring ingesta turnover rates and total mean retention times in the gastrointestinal tract, the method of Grovum and Williams (1973b) was used to fit the two compartmental model described by the following equation (Brandt and Thacker, 1958) to the fecal excretion data: y = Ae-ka (T-TT) _Ae-k2 (T-TT) y = o T>TT T-<< TT where y is the concentration of marker in the feces; kl represents the turnover rate of marker from the reticulo-rumen; T is the time elapsed since feeding of the marked meal; TT denotes the time of first appearance of marker in the feces; A is biologically undefined; and k2 is thought to be related to the turnover rate of marker post ruminally or in the cecum and proximal colon (Grovum and Williams, 1973b), or to be an artifact which is dependent upon mixing within the rumen (Leverett et al., 1977). The importance of this approach is that ruminal turnover rates of ingesta can be calcu- lated based on fecal excretion of marker. Computer programs (Hartnell, 1977) were used in calculating the rate constants and transit times based on the procedure used by Grovum and Williams (1973b). Chi-square was used as an index of dispersion in determining the equation parameters that best fit the semiqog plot of actual excretion data. The turnover rates and total mean retention times for liquid, grain and hay at various stages of lactation were obtained by the following methods of calculation: 1) determining the best fit of the two compartmental model to the data obtained by grab sampling feces; 2) determining the best fit of the two compartmental model to the actual fecal excretion curve using the total feces collection data; 3) determining the best fit of the two compartmental model to the cumulative percentage recovery curve using the total feces collection data; 4) determining the mean retention time using the stained particle technique; and 5) grab sampling of rumen contents to determine the rate of marker disappearance (k~) from the rumen. Method 1 is perhaps the preferred method of calculation, and it is illustrated with the following example. Excretion of the hay component in feces using La as the marker for cow 2146, phase 4, is presented in table 6. First, the concentration of La detected in the feces was corrected by substracting background levels. Secondly, the concentration values were then transformed into their respective natural logarithms (table 6). If the rate of removal of marker from the reticulo-rumen is exponential with time, then the plot of the natural logarithm of the marker concentration versus time should be linear after a certain amount of time, normally 24 to 36 hr in this experiment. y=at e "kt T lny=ina~ -k~ T The absolute value of the regression coefficient (slope) obtained from the natural logar-

7 RUMEN FILL, RETENTION AND TURNOVER ON LACTATION 387 TABLE 6. CONCENTRATION OF LA IN GRAB SAMPLES OF FECES COLLECTED IN PHASE 4 FOR COW 2146 AFTER FEEDING A MEAL CONTAINING LA MARKED HAY Sampling La La Sample time feces feces No. (hr) (ppm) (ln ppm) a asamples 2-14 have had the zero time concentration subtracted from them. The zero time concentration was considered background. ithms of the marker concentration in the feces in the linear portion of the curve is k~. The y intercept of the regression line yields the value for lna,. The data point with which to start the linear regression analysis was selected so that the values in the rising or peak portion of the curve did not appear to bias the regression coefficient. Generally, the highest or peak value was used as the starting point (sample 5 in table 6). Next, various sets of sample values were used in the linear regression analysis to obtain values for kl and A1 (table 7). Initially, all of the values from the peak onward (samples 5 to 14) were used, then values near the peak and at the tail end of the curve were deleted and the regression parameters recalculated. The resuhing kl and A1 values are presented in table 7. The coefficient of determination for each regression line was very high (.986). A computer program (HartneU, 1977) was used in determining the best k2, TT and A for each kl value. The kl, AI, an initial k2 value (generally kx +.03) was used) and the final excretion time were read into the computer along with the natural logarithms of the marker concentrations in the feces (table 6, column 4). As an example,.03469(kl ), 254.4(A1), (k2) and 144 hr (tables 6 and 7) would be read into the computer. The program holds kl TABLE 7. DETERMINATION OF K 1 VALUES USING VARIOUS SETS OF SAMPLES IN A LINEAR REGRESSION ANALYSIS a (GRAB SAMPLING METHOD) Set of sample values used b r 2 lnal A 1 (ppm) k alny = lna, -k, T; where y is the concentration of marker in the excreted feces, T is the sampling time, A, is the antilogarithm of the intercept of the regression line on the Y axis and k 1 is the absolute value of the regression coefficient (slope): r 2 is the coefficient of determination. bsample values used are from table 6. constant. TT is initially set to a time which is estimated to be before the time of first appearance of marker. In table 6, La was detected at 12 hr, therefore an initial TT of 8.25 hr might be selected. Thus the actual Tr should be between 8.25 hr and hr. While holding kl and TT constant, k2 is varied. The sampling times (12, 18, 24, 30, 36, 48, 60, 72, 84, 96, 108, 120, 144 and 150 hr) are then computed and stored. The A value is computed as being equal to Ale-k1TT (Grovum and Williams, 1973b). Then at each sampling time through the final sampling time read in, the natural logarithm of fecal marker concentration will be calculated using the following equation: In y = In y = 0 ln(ae "kl (T-TT)--Ae-k2 (T-TT))T>TT T~ TT The predicted In y for each sampling time is compared with the In (marker concentration) actually detected in the feces by using chisquare. The k I, k2, TT and chi-square are then stored. Still holding kl and TT constant, k2 is incremented by.01 units and a new set of In y values are calculated. If the chi-square value is less than the chi-square value calculated with the previous k2, then the current kl, k2, TT and chi-square values are stored and k2 is again incremented by.01 until k2 has been incremented 52 times. If the chi-square value is greater than the stored value, the parameters are not stored and the program proceeds to increment k2 and calculate new values. Afte~

8 388 HARTNELL AND SATTER k2 has been increased 52 times as indicated in the do loop, the stored kl, k2, TT, chi-square and predicted in y at each sampling time are printed out. The computer then increments TT by.25 hr while holding kl constant. The program starts over again with the same kl and the new TT value held constant and the k2 varied. This procedure is repeated until a predetermined TT value is reached. This predetermined time is the sampling time in which marker is first detected in the feces. Thus, the computer printout contains a list of parameters with k 1 being constant and the best k2 determined with each increment of TT (table 8). If TT was varied from 8.25 to 1200 hr at.25-hr increments, there would be 16 parameters printed out (table 8). Of the parameters printed out, the set with the minimum chi-square value would be the one that resulted in the best fit of the model to the data for that particular kl. In the example, this would be.03469(kl ),.01469(k2), hr (TT) and.133 (chi square) as presented in table 8. When TT was equal to 1200 hr, the chi-square was lower than when TT was equal to hr (table 8). This oc- TABLE 8. VALUES OF K 2 AND TT RESULTING FROM THE BEST FIT OF THE TWO COMPART- MENTAL MODEL TO THE DATA WHEN TT WAS INCREMENTED AND K 1 HELD CONSTANT (GRAB SAMPLING METHOD) Set kt k 2 TT Chi-square a b aminimum chi-square. bwhen TT equals 12, there is no comparison made between the actual value and predicted value because the predicted value is set to equal zero. Thus the chi-square is incorrect if the actual value is not zero. curred because when TT equalled hr, the predicted value was set to zero and thus no comparison was made between the predicted and actual values. If the actual value would have been zero, then the chi-square would have been correct. So far, k2 and TT have been varied with kl being held constant. In comparing the parameters obtained when the three kl values from table 7 were used, was the best k I value to be used. Table 10 compares the predicted values using the three sets of parameters in Table 9 versus the in (actual excretion of marker) detected. Therefore,.03559(kl),.09560(k2), hr (TT) and ppm (A) were the parameters that resulted in the overall best fit. These parameters were then used in calculating turnover time (1/kl ; 1/k2), total mean retention time (TT + 1/kl + 1/k2) and half-rimes (.693/kl ;.693/k2). The procedure used for methods 2 and 3 was essentially the same as that used for method 1 (Hartnell, 1977), except in method 3 the cumulative percentage of marker recovered was predicted according to the equation of Grovum and Phillips (1973): PC = 100[(k2 - kl + kle -k2 (T-TT) --k2e -kl (T--TT))/(k2 -- kl )] when T>TT PC=0 whent<tt where PC is the cumulative percentage of excreted marker and the other symbols are the same as described earlier. Figure 2 demonstrates how well this model actually fits the total feces collection data for the cumulative excretion of Cr (liquid), Sm (hay) and La (grain). The symbols represent actual data and the smooth curves represent the best fit of the model to the data. The cumulative TABLE 9. COMPUTER CALCULATED BEST FIT VALUES FOR K2, TT AND A AT THREE DIFFERENT K 1 VALUES (GRAB SAMPLING METHOD) Set k 1 ka TT A Chi-square (hr) (ppm)

9 RUMEN FILL, RETENTION AND TURNOVER ON LACTATION 389 TABLE 10. COMPARISON OF THE COMPUTER PREDICTED VALUES USING THREE DIFFERENT K 1 VALUES WITH THE ACTUAL VALUES OF LA DETECTED IN THE FECES (GRAB SAMPLING METHOD) Calculated La excreted A ctual,. La in feces usingk 1 k 2 A. ' ' Sample Sampling excreted and TT values m table 9. no. time in feces In (ppm) 1 o [607 2[643 2[579 2[ , percent recovery curves (figure 2) might be considered to represent the average of a family of curves for each of the hay, grain and liquid fractions. Since the hay particles that were sprayed with Sm were of different particle sizes, one would expect a different rate of passage for the small particles as compared to Lu IO0 ~4Q 2 % t 9 LIQUID I I I ~80~-1 IooI I I I I,/~ t TIME AFTER FEEDING MARKED MEAL (HR) Figure 2. Cumulative percentage recovery curves of Cr (liquid), Sm (Hay) and La (Grain) where the symbols (0, +, zx) represent the actual data and the smooth curves represent the best fit of the model to the actual data. the large. Thus the excretion curve of Sm is reflective of the mean rate of passage for the hay component. Calculations used in method 4 for obtaining the R values are described by Caste (1956). Method 5 simply assumed that feed components passed out of the reticulorumen at an exponential rate (Y = Ae-kt), and that a linear regression of a plot of lny versus time would give k, the rate of disappearance from the rumen. Examples of calculations for each of the five methods are available (Hartnell, 1977). The turnover rates and total mean retention times (TMRT) for the liquid, grain and hay during the dry period and various stages of lactation are presented in table 11. The values are means obtained by averaging results from cows obtained by method 1 (grab sampling of feces). This would perhaps be the method of choice in most situations. If we assume that kl obtained from the two compartmental model represents the turnover rate of the marked fraction within the reticulorumen (Grovum and Williams, 1973b; Grovum and Phillips, 1973), then turnover rates of the liquid, hay and grain fractions did not significantly differ with stage of lactation. Stage of lactation, of course, is confounded here with level of feed intake and forage to concentrate ratio. High levels of dry matter intake might be expected to increase turnover rate. The possible reduction in cellulose hydrolysis with high grain feeding would tend to decrease the turnover rate. Thus, these factors may balance each other out. Another factor that may be involved is calorie demand. Kennedy et al. (1976) reported that sheep challenged with an increased calorie demand as a result of cold exposure had an increased rate of digesta passage. It is conceivable that in early lactation, when'the cow is mobilizing body tissue to supply calories, that a similar situation might exist in the dairy cow. Either stage of lactation did not have a marked effect on turnover rate, or the high grain level and its adverse effect on cellulose hydrolysis tended to compensate for the influence that higher levels of dry matter intake and calorie demand would be expected to have on increasing turnover rate. Whatever the explanation, ruminal turnover rates of ration components were relatively unchanged over the period of time the cows were lactating. The k2 values up to this point in time have uncertain biological meaning. Originally, T~Ak 2 was thought to be the half-time of marker in

10 390 HARTNELL AND SATTER TABLE 11. COMPONENT TURNOVER RATES, TOTAL MEAN RETENTION TIMES AND TRANSIT TIMES FOR RATION CONSTITUENTS AT DIFFERENT STAGES OF LACTATION a Stage of lactation (weeks) (Dry) Component SE Hay:Grain 82.5:17.5 f 45:55g 57:43 h 67:33 i... k~, %/hr b Liquid ,54 Grain Hay ,39 k2, %/hr c Liquid Grain 11.0 f 31.1 h 28.9gh 15.8fg 4.70 Hay 7.7f 13.1g 14.4g 8.8 f 1.47 TMRT, hr d Liquid Grain Hay "IT, hr e Liquid Grain Hay acalculated by method 1. bk I Ck 2 dtmrt ett - ruminal turnover rate. - thought to be turnover rate of contents in the hindgut or cecum and proximal colon or artifact due to mixing. - total mean retention time. - time of first appearance of marker in the feces. f'g'h'imeans in the same row which do not have a common letter in the superscript are different (P<.05). the hind gut, but when Grovum and Williams (1973b) injected markers into the rumen and collected feces they found that T%k: on the average was about 66% of the half-time (Ty2a) computed by injecting the marker into the abomasum. One would expect the two methods to give similar values. If there was a diurnal pattern of digesta mixing in the caecum and proximal colon, Ty2k~ might be affected differently from TY2a values because TtAk 2 was determined over a shorter period of time. When Ty~k 2 was measured in a model digestive tract, there was no difference between it and the expected value (Grovum and Phillips, 1973) indicating that something different was happening in vivo. The effect of time of injection has not been evaluated. However, Grovum and Williams (1973b) found good fits between the concentrations of ~44Pr and S lcr-edta in feces and the calculated curves which indicates that T%k 2 does describe the way marker and digesta passed through the hind gut. Since the half times of 144 Pr and s 1 Cr-EDTA were relatively small in the abomasum, 37 and 17 min, compared with those in the caecum and proximal colon, 413 and 406 min (Grovum and Williams, 1973a), there is reason to associate T~k2 with the kinetics of digesta flow through the caecum and proximal colon. The changes in Ty2k2 may therefore reflect differences in the times that digesta spends in the caecum and colon, but the values for average retention times (1/k~) may not be the true times that digesta actually spends in these organs (Grovum and Williams, 1973b). Finally, the differences of magnitude between Ty2k2 and T89 in vivo may be due to imperfect mixing of digesta in the caecum and proximal colon of the sheep (Grovum and Williams, 1973a). Leverett, Marls and Ellis (1977) suggested that k2 is an artifact due to mixing in the reticulo-rumen, or in other words, that mixing in the rumen represents the second compartment (k2) with rate of passage out of the rumen as the first compartment (ki). However, there is perhaps more evidence at this time to

11 RUMEN FILL, RETENTION AND TURNOVER ON LACTATION 391 suggest that k2 reflects retention of ingesta in a post ruminal compartment than of a mixing phenomenon. In this experiment, k2 values tended to be greatest for hay and grain when 55% of the ration was grain, and decreased as the proportion of grain in the ration was reduced. The liquid turnover rate remained fairly constant except for an unexplainable large value during the third phase of the experiment. The k2 value was always found to be greater than kl which agrees with Grovum and Williams (1973b). The k2 value has limited meaning in this experiment because it is based on only two to three samples or data points. To obtain more reliable estimates of k2, at least six samples should be obtained during the first 24 hr after feeding the marked meal. The total mean retention time (TMRT) was calculated as TMRT = TT + l/k1 + l/k2. The R-value was found to be at least 97.8% of the TMRT value. The TMRT of liquid and hay fractions did not differ significantly from period to period (table 11). Grain tended to spend more time in the digestive tract when the ration contained relatively small amounts of grain. The time of first appearance (TT) of liquid, grain or hay in the feces were not different (P<.05) with regard to stage of lactation, however they tended to be shorter during the dry period. Table 12 contains comparisons of turnover rates obtained by using feces grab sampling (method 1) or the two methods (methods 2 and 3) using the total feces collection data. The values are means of the data collected over all periods of the experiments. The kl values calculated from these data are also compared with the k value obtained from the rumen grab sampling method (method 5). There were no differences (P<.05) between the fecal grab sampling method and the total feces collection methods in determining kl or k2. The significantly lower k2 value for the liquid phase using method 2 is unexplainable. Rumen grab sampiing tended to yield slightly higher values for kl, especially for the liquid component. The reliability of the rumen grab sample values is limited because representative samples were difficult to obtain and a limited number of samples (3 or 4) were taken during the descent of the exponential curve. The TMRT values were used in comparing the stained particle technique with the use of TABLE 12. COMPARISON OF METHODS FOR DETERMINING RATE OF PASSAGE PARAMETERS Method a Parameters ka, %/hr Liquid 8.7 c 7.8 c 8.0c 9.7 d Grain Hay c cd cd d k2, %/hr Liquid 48.1 d 28.1 c 46.3 d Grain Hay TMRT, hr Liquid 27.8 e 25.1 d 22.1 c Grain 42.3d 40.5 d 36.0c 41.5 d Hay 52.6 d 48.6 d 45.3 c 53.6 d R-value b amethod 1 : determining the best fit of the model to the data obtained by grab sampling feces. Method 2: determining the best fit of the model to the actual marker excretion curve (total feces collection). Method 3: determining the best fit of the model to the cumulative percent recovery curve (total feces collection). Method 4: stained particle technique. Method 5: grab sampling of tureen contents. bcalculated as described by Castle (1956). c'd'emeans in the same row which do not have a common letter in the superscript are different (P<.05). rare-earth elements in either feces grab samples or the two total feces collection methods. There was no difference among the grab sampling method, stained particle method and the total feces collection method using the best fit of the In (marker concentration) to the actual value except for the liquid component. The total feces collection method using the best fit of cumulative percentage recovery values to the actual cumulative percentage recovery values (method 3 in table 12) tended to yield lower total mean retention time. The reason for the difference is yet unexplained. On the basis of the relative differences among the methods of analysis, grab sampling of feces from the rectum would be the desired method to use. The important point with this method is that the animal should be induced to defecate and the last excrement taken as the sample to avoid any bias due to storage of feces in the rectum. Using this method with rareearth elements as multiple component markers within a ration, one can use more animals without the laborious task of making a total feces collection. The method also enables one

12 392 HARTNELL AND SATTER to calculate the ruminal turnover rates for each component marked and the overall mean retention time just from the marker concentrations in the feces. It is suggested that at least 6 to 8 feces samples be collected with the first 24 hr if a reliable estimate of k2 is to be obtained. The k2 value must be viewed with caution, however, until its biological meaning is better described. The rare-earth elements are easily applied to the ration without altering the chemical or physical properties of the ration, which of course happens when feeds are boiled in water during the staining process. Furthermore, the rare-earth elements are easily analyzed by using neutron activation analysis. These methods should be of help in answering questions pertaining to rate of ingesta passage, and how such things as level of intake, forage to grain ratio, associative effects of feeds, and physical form of diet influence ingesta retention time. Also, if increased fluid turnover rate in the rumen is indeed correlated with microbial protein production, then these methods will provide the means for isolating the animals, feeds or conditions which promote greater turnover rates. LITERATURE CITED Balch, C. C Factors affecting the utilization of food by dairy cows. I. Rate of passage of food through the digestive tract. Brit. J. Nutr. 4:361. Baumgardt, B. R Control of feed intake in the regulation of energy balance. P In A. T. Phillipson (Ed.) Physiology of Digestion and Metabolism in the Ruminant. Binnerts, W. T., A. th. Van't Klooster and A. M. Frens Soluble chromium indicator measured by atomic absorption in digestion experiments. Vet. Record, 82:470. Blaxter, K. L The efficiency of energy transformations in ruminants. P. 21. In K. L. Blaxter, J. Kielanowski and Greta Thorbek (Ed.). Energy Metabolism of Farm Animals. Oriel Press Ltd., Newcastle upon Tyne, England. Brandt, C. S. and E. J. Thacker A concept of rate of food passage through the gastro-intestinal tract. J. Anim. Sci. 17:218. Campling, R. C The effect of concentrates on the rate of disappearance of digesta from the alimentary tract of cows given hay. J. Dairy Res. 33:313. Castle, E. J The rate of passage of foodstuffs through the alimentary tract of the goat. I. Studies on adult animals fed on hay and concenn.ates. Brit. J. Nun'. 10:15. Cole, N. A., P. R. Johnson, F. N. Owens and J. R. Males Influence of roughage level and corn processing method on microbial protein synthesis by beef steers. J. Anim. Sci. 43:497. Duncan, D. B Multiple range and multiple F tests. Biometrics 1 : 11. Goering, H. K. and P. J. Van Soest Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agriculture Handbook No Agriculture Research Service, USDA. Gorski, J., T. H. Blosser, F. R. Murdock, H. S. Hodgson, B. K. Soni and R. E. Erb A urine and feces collecting apparatus for heifers and cows. J. Anita. Sci. 16:100. Gray, D. H. and J. R. Vogt Neutron activation analysis of stable heavy metals as multiple markers in nutritional monitoring. J. Agr. Food Chem. 22:144. Grovum, W. L. and G. D. Phillips Rate of passage of digesta in sheep. V. Theoretical considerations based on a physical model and computer simulation. Brit. J. Nutr. 30:377. Grovum, W. L. and V. J. Williams. 1973a. Rate of passage of digesta in sheep. IIL Differential rates of water and dry matter from reticulorumen, abomasum, caecum, and proximal colon. Brit. J. Nutr. 30:231. Grovum, W. L. and V. J. Williams. 1973b. Rate of passage of digesta in sheep. IV. Passage of marker through the alimentary tract and the biological relevance of the rate-constants derived from the changes in concentrations of marker in feces. Brit. J. Nutr. 30:313. Harrison, D. G., D. E. Beever, D. J. Thomson and D. F. Osbourn Manipulation of rumen fermentation in sheep by increasing the rate of flow of water from the tureen. J. Agr. Sci. 85:93. Hartnell, G. F Measurement and significance of ingesta turnover rates in dairy cattle using rareearth elements. Ph.D. Thesis, Univ. of Wisconsin, Madison. Hartnell, G. F. and L. D. Satter Extent of particulate marker (samarium, lanthanum and cerium) movement from one digesta particle to another. J. Anim. Sci. 48:375. Isaacson, H. R., F. C. Hinds, M. P. Bryant and F. N. Owens Efficiency of energy utilization by mixed rumen bacteria in continuous culture. J. Dairy Sci. 58:1645. Kennedy, P. M., R. J. Christopherson and L. P. Milligun The effect of cold exposure of sheep on digestion, rumen turnover time, and efficiency of microbial synthesis. Brit. J. Nutr. 36:231. Lamberth, J. L The effect of pregnancy in heifers on voluntary intake, total rumen contents, digestibility and rate of passage. Australian J. Expti. Agr. Anita. Husb. 9:493. Leverert, E. A., J. H. Marls and W. C. Ellis Dosing techniques in measuring gastrointestinal flow. Abstr. J. Anita. Sci. (69th Annu. Meet.). p Stouthamer, A. H. and C. Bettenhaussen utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochem. Biophys. Acta. 301:53. Walker, D. J., A. R. Egan, C. J. Nader, M. J. Ulyatt and G. B. Storer Rumen microbial protein synthesis and proportions of microbial and nonmicrobial nitrogen flowing to the intestines of sheep. Australian J. Agr. Res. 26:699.