Supplementation of grass hay with molasses in crossbred (Bos taurus × Bos indicus) non-lactating cows: effect of level of molasses on feed intake, digestion, rumen fermentation and rumen digesta pool size

Animal Feed Science and Technology, 41 ( 1993 ) 23-38

23

Elsevier Science Publishers B.V., Amsterdam

Supplementation of grass hay with molasses in crossbred (Bos taurus X Bos indicus ) nonlactating cows: effect of level of molasses on feed intake, digestion, rumen fermentation and rumen digesta pool size

H. Khalili International Livestock Centre for Africa (ILCA), P.O. Box 5689, Addis Ababa, Ethiopia (Received 26 March 1992; accepted 12 October 1992)

ABSTRACT Khalili, H., 1993. Supplementation of grass hay with molasses in crossbred (Bos taurus X Bos indicus) non-lactating cows: effect of level of molasses on feed intake, digestion, rumen fermentation and rumen digesta pool size. Anim. FeedSci. Technol., 41: 23-38. This 5 X 5 Latin square experiment evaluated the effects of the level of sugar-cane molasses on feed intake, diet digestibility, dry matter (DM) degradation of grass hay, rumen pool sizes and rumen fermentation. Five ruminally fistulated crossbred (Bos taurus X Bos indicus) cows were given a basal diet of grass hay ad libitum and 2.0 kg DM of cottonseed cake day-l (Diet M0). The other four diets were supplemented with 1.5 (Diet ML), 3.0 (Diet MM) or 4.5 kg DM of molasses (Diet MH), or the MH diet supplemented with 0.6 kg of sodium bicarbonate (Diet MHB ). The voluntary intake of hay decreased from 8.2 to 5.6 kg DM (linear effect, P < 0.001 ) with the level of molasses. The apparent digestibility of DM increased from 0.66 to 0.73 (linear effect, P < 0.01 ) but that of neutral detergent fibre (NDF) decreased from 0.68 to 0.60 (linear effect, P<0.01 ) with the level of molasses. Sodium bicarbonate did not affect the intake of hay or the digestibilities of different dietary constituents (P> 0.05 ). The disappearance of hay DM from nylon bags decreased (linear effect, P < 0.001 ) with the level of molasses, so that for Diet MM depression was less than for Diet MH (quadratic effect, P < 0.05 ). The rumen pool sizes of total ingesta (90 vs. 80 kg, linear effect, P < 0.05 ) and DM were smaller ( 10.4 vs 8.3 kg DM, P < 0.01 ) with molasses diets. The rumen pool size of NDF was not affected (P> 0.05 ) by the treatments. Molasses reduced the mean value of rumen pH from 6.6 to 6.2 (linear, P < 0.001, and quadratic effects, P < 0.05) and concentration of ammonia from 9.5 to 5.8 mmol l - ~ (linear effect, P < 0.001 ). The molar proportion of acetate decreased from 726 to 573 mmol mol-~ (linear effect, P < 0.001 ), and those of propionate and butyrate increased (from 160 to 205 mmol m o l - ~, P < 0.0 l, and from 106 to 205 mmol tool- ', P < 0.001 ) with the level of molasses.

Correspondence to." H. Khalili, International Livestock Centre for Africa (ILCA), P.O. Box 5689, Addis Ababa, Ethiopia.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0377-8401/93/$06.00

24

H. KHALILI

INTRODUCTION

In the sub-tropics and tropics, the voluntary intake and quality of basal feeds (pasture, hay or crop residues) are usually low. Poor-quality forages have a high fibre content, and rumen fill is an important factor in controlling voluntary intake (Campling et al., 1962; Campling, 1966; Aitchison et al., 1986). Low-quality forage diets are limited in the supply of both energy and amino acids. In addition, a shortage of glucose precursors may limit gluconeogenesis and the utilization of acetate (Cronje et al., 1991 ). Therefore, both energy and protein supplements are needed to support animal production with diets based on low-quality forages. Sugar-cane molasses is a potential energy supplement available in many countries in sub-Saharan Africa. Soluble carbohydrates given at moderate levels increase the amount of energy in the diet, improve the utilization of nitrogen in the rumen and may increase ruminal outflow rate (Stern and Hoover, 1979; Huhtanen, 1987; Rooke et al., 1987; Khalili and Huhtanen, 1991 a). Soluble carbohydrates have also been found to increase molar proportion of propionate in rumen volatile fatty acids (VFA) (Sutton, 1968; Kellogg and Owen, 1969), which, in addition to enhancing the supply of amino acids from microbial protein synthesis, increases gluconeogenesis, thereby ensuring a more efficient utilization of acetate when low-quality forages are fed (Cronje et al., 1991 ). On the other hand, soluble carbohydrates have been found to have negative effects by decreasing fibre digestibility (England and Gill, 1985; Huhtanen, 1987; Brown et al., 1987; Rooke et al., 1987) and forage intake (Lofgreen and Otagaki, 1960a, b; Ahmed and Kay, 1975 ). However, information on the optimum level of molasses with low-quality roughage based diets used in Africa is limited. An ideal situation would be for soluble carbohydrates to improve nitrogen utilization and provide more glucose precursors without depressing fibre digestion or intake of roughage. The objective of this study was to compare the effects of various levels of molasses supplements on the utilization of grass hay based diets. The relative importance ofpH and carbohydrate effects on neutral detergent fibre (NDF) digestion and the relation between forage intake, NDF digestion and rumen pool size of NDF were studied. The effects of various levels of molasses on rumen ammonia concentration and rumen fermentation patterns were monitored. Sodium bicarbonate supplement with the highest level of molasses was used to observe the importance of pH effect on rumen fermentation pattern and NDF digestion and to see if bicarbonate would alleviate the possible negative effects of molasses on NDF digestion. MATERIALS A N D M E T H O D S

Animals, management, experimental design and treatments Five non-lactating rumen fistulated Friesian (Bos taurus) × Zebu (Boran, Bos indicus) crossbred cows (liveweight mean 510 kg; SD+ 31 kg), housed

EFFECT OF MOLASSES SUPPLEMENT LEVEL ON FEED UTILIZATION

25

and fed individually, were used. The experimental design was a partially balanced 5 × 5 Latin square. The animals were fed twice daily (at 07:00 and 19:00 h) a control diet (Diet M0) of native grass hay ad libitum and 2.0 kg dry matter ( D M ) of cottonseed cake. In the other four diets, the basal diet was supplemented with 1.5 (Diet ML), 3.0 (Diet MM) or 4.5 kg DM of sugarcane molasses (Diet MH), or 4.5 kg DM of molasses and 0.6 kg of sodium bicarbonate (Diet MHB). Hay was predominantly Pennisetum uliginosum, Andropogon species and Danthonia sulubata. Native legumes, mainly Trifolium reuplianum, Cyperus species and Forbes species formed the undergrowth. The hay was harvested at early maturity. Cows were fed hay separately from the supplements so that there was always hay present in front of the cows and the amount of orts of hay was at least 25-30% of what was offered. Molasses and cottonseed cake were given separately but simultaneously so that molasses was fed from the bucket and cottonseed cake was fed from the feeding trough. Sodium bicarbonate (0.3 kg) was administered at the same time as molasses and cottonseed cake were given, via a rumen cannula to ensure that the effect of bicarbonate aimed at the rumen fermentation was not risked by the cow refusing to consume the bicarbonate. Cows had free access to water and mineral licks. Daily feed intakes were recorded individually, and feed refusals were removed before the morning feeding and weighed. Each experimental period lasted about 28 days, of which the first 3 weeks served as an adaptation period, and data collection took place during the last week.

Sampling and analyses Feed samples were taken weekly and analysed for DM at 100°C for 24 h. Three sub-samples from each collection period were pooled to provide a sample for other analyses, dried at 60°C, ground and stored at room temperature for subsequent analyses. Apparent digestibilities of the diets were estimated using acid-insoluble ash (AIA) as a natural marker (Van Keulen and Young, 1977 ). On 5 days in each collection period faecal grab samples were taken at 07:00, 11:00, 15:00 and 19:00 h, pooled for each cow and period, and frozen. The pooled samples were subsequently thawed and dried at 60 ° C and stored at room temperature for subsequent analyses. The effects of the five diets on the rate of disappearance of DM of grass hay were determined by using the nylon bag technique. The hay was ground to pass a 2.0 m m screen. From Day 20 of each period onwards, duplicate bags (Polymon, Zurich, Switzerland; dimensions 6 cm × 12 cm; pore size 41/tm ) containing about 2.5 g DM of hay were incubated in the rumen of each cow. The incubation periods were 6, 12, 24, 48, 72 and 96 h. After withdrawal from the rumen, the bags were machine washed (Tefal Alternatic, Finland) with

26

H. KHALILI

cold tap water. The washing procedure took 30 min, and consisted of five rinsing cycles. A '0 h wash value' for the calculation of the lag time of DM degradation was determined during each collection period in three bags. After washing, the bags were dried in a forced draft oven at 60°C (for about 48 h) to constant weight and then weighed. Rumen fluid samples were taken on day 24 before feeding and 1, 2, 3, 4, 6, 8 and 10 h after feeding. The pH was measured immediately (Kent EIL 7045/ 46, ABB Kent-Taylor, Gloucestershire ). A portion ( 100 ml) of each rumen sample was stored in a bottle containing acid (0.4 ml of 98% H2SO4) at 20 ° C for later analyses of ammonia and volatile fatty acids (VFA). The total weights of rumen contents were estimated by manually emptying the rumen of each cow before the morning feeding and 6 h after, on the last day of each experimental period. Rumen contents were collected into two (60 1) plastic buckets which were put in a warm (about 45°C) water container. The materials were then weighed, mixed thoroughly by hand and sampled (a total of 2.5-3.0 kg). The remainder was returned to the rumen as soon as possible. The entire procedure seldom exceeded 10-15 min per cow and the rumen was 'empty' for only 3-4 min. Samples from the experimental feeds were analysed for organic matter (OM) by ashing at 500°C, and for nitrogen (Kjeldahl-N) and NDF (Goering and Van Soest, 1970). The rumen fluid was deproteinized with a solution containing metaphosphoric acid (2.4%) and isocapric acid (0.32%) and immediately assayed for VFA using gas-liquid chromatography (GLC; Pye Unicam 304, Pye Unicam, Cambridge, U K ) . Ammonia was steam distilled using micro-Kjeldal equipment (Tecator 1028/1026, Tecator, Hrgan~is) and titrated against 0.1 N HC1. Faecal samples were analysed for nitrogen and NDF, and samples of rumen contents for NDF. To measure the potential digestibility of rumen ingesta, samples of rumen contents were incubated in nylon bags for 336 h in the rumen of three cows fed 2 kg DM of cottonseed cake and grass hay ad libitum. Nylon bag residues after 336 h were considered to be rumen-indigestible DM (IDM), and the degradable fractions rumendigestible DM ( D D M ) . -

Calculation of results and statistical analyses The disappearance of DM from nylon bags was fitted to the equations of McDonald ( 1981 ). The degradation curve is described as: within a lag time T, Y=A (i.e. the initial washing loss )

( 1)

beyond time T, Y2= a + b ( 1 - e - ct)

(2 )

A represents the '0 h wash value', Y2 is the proportionate amount of DM disappearing after time T(h) and where a, b and c are degradation constants fitted by an iterative procedure. In eqn. (2), a is the rapidly soluble fraction,

EFFECT OF MOLASSESSUPPLEMENT LEVELON FEED UTILIZATION

27

b the amount which in time will degrade and c the fractional rate constant at which the fraction b will be degraded per hour. The kinetics of rumen NDF intake, passage and digestion were calculated using the model presented by Robinson et al. ( 1987 ), assuming steady-state conditions in the rumen: rate of intake ( ki, h - ~) = ( 1/24 ) × ( intake, kg day- 1) / ( pool size, kg ) rate of passage (kp, h - ~) = ( 1/24) × (faecal flow, kg day- 1) / (pool size,

kg) rate of digestion (ko, h - 1 ) = k i - kp This model was used, bearing in mind that the assumption concerning the steady-state conditions may not be totally true. However, the relative differences between diets should be valid and the mean values of two evacuation times which will show the average situation are presented. The limitations of using faecal flow values should not change the relative differences between dietary effects on rumen kinetics. In addition, faecal flow is often used to calculate these values because of the difficulties associated with duodenal cannulation. For example, Robinson et al. ( 1987 ) divided the faecal flow by 0.85. The analysis of variance technique for Latin square experiments was applied to the data. The effects of the level of molasses were further separated into single degrees of freedom using orthogonal contrasts (Snedecor and Cochran, 1980). The contrasts used were (1) linear (L) and (2) quadratic (Q). The effect of bicarbonate was tested by a non-orthogonal contrast of Diet MH vs. Diet MHB. Rumen fluid data (pH, ammonia, VFA) and rumen evacuation data were analysed using a repeated measures analysis of variance. The analysis used the split-plot approach with the Greenhouse-Geiser approximate (conservative) significance tests (Littell et al., 1992 ). RESULTS

The chemical composition of the experimental feeds is given in Table 1. The grass hay used was a typical sub-tropical mature hay that had low crude protein and high N D F concentrations. The intakes and digestibilities of feeds and dietary constituents are sumTABLE 1 Chemical composition of the experimental feeds (g kg-~ DM ) Grass hay

Cottonseed cake

Molasses

Dry matter (g kg- ~)

880

890

750

In dry matter Ash CP NDF

86 61 704

79 369 471

120 40

28

H. KHALILI

TABLE2

Intake of feeds and dietary constituents and digestibilities of dry matter ( D M ) , organic matter ( O M ) , crude protein (CP) and neutral detergent fibre ( N D F ) in crossbred cows given grass hay and cottonseed cake

Diet M0

SEM ML

MM

MH

MHB

Statistical significance of Level of

molasses LI

Effect of NaHCO3

Q2

In take (kg DM) Hay Cottonseed cake Molasses NaHCO3 Total DM OM CP NDF

8.17 2.00

10.15 9.29 1.23 6.68

7.35 2.00 1.50 10.83 9.86 1.24 6.10

6.55 2.00 3.00 11.53 10.45 1.25 5.54

5.62 2.00 4.50 12.10 10.92 1.25 4.86

5.72 2.00 4.50 0.60 12.80 11.01 1.26 4.96

0.364 . . . . 0.366 0.334 0.022 0.257

*** . . ** ** NS ***

0.706 0.713 0.544 0.564

0.0168 0.0164 0.0336 0.0167

** * NS **

NS

NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

. .

Digestibility DM OM CP NDF

0.657 0.687 0.623 0.684

0.707 0.734 0.647 0.689

0.721 0.741 0.644 0.648

0.729 0.748 0.619 0.600

~L, Linear contrast. 2Q, Quadratic contrast. Significance: NS, non-significant; *P< 0.05; **P< 0.01 ; ***P< 0.001.

marized in Table 2. Increasing the level of molasses decreased (linear effect, P < 0.001 ) the intake of hay but increased ( P < 0.01 ) the intake of total DM. The apparent digestibility of DM increased ( P < 0.01 ) but that of N D F decreased (linear effect, P<0.01 ) with the level of molasses. Including NaH C O 3 in the diet (Diet MHB) had no significant ( P > 0.05 ) influence on the intake of hay or the digestibilities of the various dietary constituents compared with the corresponding diet without NaHCO3. The disappearance of hay DM from nylon bags is shown in Table 3. Increasing the amount of molasses decreased the disappearance of hay DM from nylon bags at all incubation periods (linear effect, P < 0.001 or P < 0.01 ). The first level of molasses (ML) did not affect the hay DM disappearance but the depressing effect of molasses was stronger when the level was increased from Diet MM to Diet MH (quadratic effect, P < 0 . 0 5 or P<0.01 from the 24 h period onwards). The potential degradability of hay DM (a + b) was not affected by molasses supplementation. There was a trend towards a linear decrease in the rate constant (c) and an increase in the lag time with increasing level of molasses. NaHCO3 did not affect significantly ( P > 0.05 ), the values

EFFECT OF MOLASSES SUPPLEMENT LEVEL ON FEED UTILIZATION

29

TABLE3

Effect of level of molasses supplements on the disappearance of grass hay D M ( m g g - ~) from nylon bags incubated in the rumen of crossbred cows given grass hay and cottonseed cake and the degradation parameters (a, b, c and lag time) Diet

M0

SEM

ML

MM

MH

MHB

Statistical significance o f Level o f

Effect of

molasses NaHCO3 LI

Q2

*** ** *** *** ...... *** NS

NS NS * *

NS NS NS NS

** NS

** NS

NS NS

NS NS

NS NS

Incubation period (h) 6 12 24 48 72 96

a+b c

260 350 487 637 701 719 748 0.0374 2.26

249 348 490 636 697 729 750 0.0368 2.54

232 304 426 578 651 690 747 0.0317 3.33

218 292 358 470 521 584 671 0.0290 3.57

210 273 363 511 591 646 715 0.0210 3.89

5.3 16.7 14.3 19.7 20.4 14.5 45.1 0.0061 0.50

Lag time

(h) L, Linear contrast. 2Q, Quadratic contrast. Significance: NS, non-significant; * P < 0.05; **P< 0.01: ***P< 0.001.

of the rate constant (c) or lag time, but the disappearance of DM was significantly higher from the 72 h period onwards with the NaHCO3 diet. The disappearance of cottonseed cake DM from nylon bags was also measured (results not shown ) and the results were in line with the disappearance of hay DM. The potential degradability (a+b, average 845 mg g - l ) or the rate constant (c, average 0.044) of cottonseed cake DM were not affected by molasses supplementation. The mean values for two evacuation times are summarized in Table 4. The data from different evacuations were not reported separately because there were no significant ( P > 0.05 ) interactions of diet with time. The standard error of means of these different times were similar. Not surprisingly, the rumen pool size of N D F was greater at 6 h after feeding than before feeding. Increasing the level of molasses decreased the rumen pool sizes of total ingesta (linear effect, P < 0.01 ) and DM ( P < 0.05 ). The potential digestibility of rumen ingesta increased with the level of molasses ( P < 0.001 ). Sodium bicarbonate increased the pool sizes of total ingesta and D D M (P<0.01; P < 0.05). The rumen pool size of N D F was not affected ( P > 0.05) by the

87.8 104 9.17 674 6.25 2.93 6.14 0.0437 0.0141 0.0297

90.1 120 10.39

641 6.68 3.72 6.54 0.0449 0.0150 0.0299

ML

700 6.13 2.61 5.98 0.0407 0.0147 0.0260

85.2 105 8.74

MM

IL, Linear contrast. 2Q, Quadratic contrast. Significance: NS, non-significant; *P< 0.05; **P< 0.01 ; ***P< 0.001.

ka

Rumen pool size Total ingesta (kg) Dry matter (g kg- ~) DM (kg) DM degradability (mg g - i ) DDM (kg) IDM (kg) NDF (kg) NDF Ki ( h - l )

M0

Diet

727 6.02 2.27 6.07 0.0339 0.0137 0.0202

79.7 105 8.29

MH

726 7.13 2.71 6.61 0.0309 0.0138 0.0171

96.4 104 9.84

MHB

9.5 0.296 0.208 0.224 0.00252 0.00110 0.00182

3.21 5.0 0.452

SEM

*** NS *** NS ** NS **

* NS **

Ll

Level o f molasses

NS NS NS NS NS NS NS

NS NS NS

Q2

NS * NS NS NS NS NS

** NS *

Effect of NaHCO3

Statistical significance of

Effect of level of molasses supplements on rumen pool size of ingesta, dry matter, the potential digestibility of rumen ingesta and NDF, and on digestion kinetics of NDF in crossbred cows given grass hay and cottonseed cake

TABLE 4

EFFECT OF MOLASSES SUPPLEMENT LEVEL ON FEED UTILIZATION

31

TABLE 5

Effect of level of molasses supplements on rumen pH, concentrations of ammonia and total VFA, and the molar proportions of acetic, propionic and butyric acid in crossbred cows given grass hay and cottonseed caket Diet

M0

SEM

ML

MM

MH

MHB

Statistical significance o f Levelof

Effect o f

molasses N a H C O 3

pH Ammonia (mmoll-~) TotalVFA (mmoll -~)

6.62 9.45 103.2

6.57 7.71 102.4

6.57 6.29 89.4

6.24 5.75 100.6

6.59 4.48 101.4

573 205 205

580 192 212

0.056 0.579 3.87

L2

Q3

*** *** NS

* NS NS

*** NS NS

*** ** ***

NS NS NS

NS NS NS

Molar proportion of VFA (mmol mol- 1) Aceticacid Propionic acid Butyric acid

726 160 106

677 184 128

648 174 164

9.0 8.8 10.3

~The values for pH, ammonia and VFA are averages of eight sampling times. 2L, Linear contrast. 3Q, Quadratic contrast. Significance: NS, non-significant; * P < 0.05; **P< 0.01 ; ***P <: 0.001.

treatments. Increasing the level of molasses reduced linearly the values of digestion rate (kd) of N D F ( P < 0 . 0 5 ) . Sodium bicarbonate did not affect ( P > 0.05 ) the digestion kinetics of NDF. The results for rumen fermentation are presented in Table 5. Increasing the amount of molasses decreased the mean values of rumen pH (linear effect, P < 0.001 ), with Diet MH clearly lower (quadratic effect, P < 0.05 ). Rumen ammonia concentration decreased ( P < 0.001 ) with the level of molasses. The molar proportion of acetate decreased ( P < 0.001 ), and propionate and butyrate increased ( P < 0.01, P < 0.001 ), with the level of molasses. Including sodium bicarbonate in the diet increased the mean values of pH ( P < 0.001 ) but did not change the fermentation pattern. The minimum pH value was obtained with all molasses diets 1 h after feeding. Only with the highest level of molasses (Diet MH) were the pH values below 6.0 during the first 4 h after feeding. Post-prandial changes in rumen fermentation parameters increased with the level of molasses. Interaction of level of molasses with time was significant for pH (linear effect of level of molasses with time, P < 0.001 ), concentration of total VFA ( P < 0 . 0 5 ) and for the molar proportions of acetate ( P < 0.01 ) and propionate (linear effect, P < 0.001, and quadratic effect, P < 0.05, of level of molasses by time).

32

H. KHALILI

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Fig. 1. The effect of level of molasses supplement on rumen pH, concentration of ammonia, total VFA and molar proportions of acetate, propionate and butyrate in crossbred cows given grass hay and cottonseed cake. i Diet M0; i , Diet ML; Q, Diet MM; ,, Diet MH; A, Diet MHB.

EFFECT OF MOLASSES SUPPLEMENT LEVEL ON FEED UTILIZATION

33

DISCUSSION

Fibre digestion Decreased NDF digestion and DM degradation of hay with increasing level of molasses confirm the results of other studies with molasses or sugar supplements (Ahmed and Kay, 1975; Mould et al., 1983; England and Gill, 1985; Huhtanen, 1987; Rooke et al., 1987; Garrett et al., 1989; Khalili and Huhtanen, 1991b). It was possible to include up to 1.5 kg in the present trial and 2 kg DM of molasses per day in our other trial (Khalili et al., 1993) without depressing digestion of N D F in hay. There were minor changes in intrinsic characteristics of fibre, as the proportion of cottonseed fibre of the total fibre increased with the level of molasses. DM degradation of hay was used to describe the dietary effects on cell-wall degradation, as, according the Khalili and Huhtanen (199 l b), degradation of NDF and ADF of silage reflected a similar pattern to DM degradation. The adverse effects of molasses on hay DM degradation were greater than the differences in NDF digestibility. Hindgut fibre digestion and longer rumen retention time of fibre may provide some explanation. However, retention time in the hindgut was only 4 h in cattle (Kukkonen and Huhtanen, 1991 ). The potential digestibility of rumen ingesta was higher with molasses diets, indicating decreased digestion in the rumen. Rumen evacuation-derived rate of digestion (kd) of N D F was consistent with the lower digestibility of NDF. Similar results were reported by Huhtanen and Khalili (1991) and Huhtanen and Jaakkola (1993). There were no significant differences in rumen pool size of NDF in spite of differences in NDF digestibility and intake. Some reasons for the decrease in NDF digestion need to be pointed out. Cottonseed cake was included in all diets to provide rumen degradable protein because hay alone could not ensure a sufficient amount of degradable nitrogen for rumen microbes. Rumen ammonia concentration was above 5.7 mmol 1-1 for all diets. This level of ammonia N is suggested to be the optimum for digestion of nutrients (Hoover, 1986). Results from the study by Huhtanen and Khalili (1992) indicated that either reduced attachment of fibrinolytic microbes to cell walls or reduction in their enzyme synthesis was involved in the depression of cell-wall digestion when soluble carbohydrates were given. One reason could be the pH effect, as cellulolytic bacteria are very sensitive to low rumen pH, especially for pH below 6.0 (Stewart, 1977; Russell and Dombrowski, 1980). Bicarbonate supplement almost completely alleviated the adverse effect of sucrose on fibre digestion with grass silage (Khalili and Huhtanen, 1991 b). In the present trial, with bicarbonate, fibre digestion in vivo was not improved, indicating that the mean pH with Diet MH was less harmful (6.24 vs. 6.03 ) than with the sucrose diet (Khalili and Huhtanen, 1991 b ). According to Mould and Orskov ( 1983 ), in sheep given

34

H. KHALILI

concentrate, when rumen pH was increased by the infusion of bicarbonate there was no increase in cellulolysis. Possibly increased osmotic pressure with bicarbonate may have also affected fibre digestion (Rogers et al., 1979 ). Digestion of fibre is important because it can influence forage intake (Campling, 1966; Van Soest, 1973 ). The present results suggest that with Diet MM and MHB, depression in fibre digestion was associated mainly with the carbohydrate effect caused by a preference by rumen microbes for soluble carbohydrates (see Russell and Baldwin, 1978; Mould et al., 1983; Russell, 1984). Similarly, continuous intraruminal infusion of sugars, at a constant rate or varying levels (0, 450 or 900 g day- ~), depressed fibre digestion although pH was not affected (Huhtanen, 1987; Rooke et al., 1987). Because of the combined influences of pH and carbohydrate effects with Diet MH, comparison of mechanisms controlling fibre digestion with molasses was difficult. Small increases in DM degradation in cattle given bicarbonate supplement suggest that the decrease in DM degradation with the highest level of molasses was a combined effect of low pH and soluble carbohydrates.

Forage intake In agreement with the results of Lofgreen and Otagaki ( 1960a, b ), Ahmed and Kay ( 1975 ) and England and Gill ( 1985 ) with molasses or sucrose, the intake of forage decreased with increasing levels of molasses. There was no optimum level of molasses because the substitution rates for the intake of hay DM per kg additional DM of molasses were 0.55, 0.53 and 0.62 for increasing levels of molasses. On the other hand, in our other trial 2.0 kg DM of molasses did not decrease forage intake (Khalili et al., 1993). The different results may be related to differences in the quality of the forage. Reduction in forage intake per unit of additional concentrate varied with the type of forage, being higher with better-quality forages (Campling and Murdoch, 1966; Bines, 1985). Given that there was no effect on fibre digestion or passage with Diet ML, hay intake was probably affected by metabolic regulation. Intake of digestible OM in the basal diets was 26% greater in the present trial than in the other trial (Khalili et al., 1993 ). The present results, and similar results of Huhtanen and Jaakkola ( 1993 ), suggest that the effects of rumen fill cannot be ruled out as a factor controlling forage intake in diets supplemented with high amounts of soluble or starch-based carbohydrates. Multiple feedbacks (physical fill and metabolic satiety) may have regulated hay intake with Diets MM and MH. Inclusion of sodium bicarbonate in the diet did not improve hay intake. This was not surprising, because fibre digestion was not improved. The effects of bicarbonate on feed intake have been inconsistent in many reported

EFFECT OF MOLASSES SUPPLEMENT LEVEL ON FEED UTILIZATION

35

experiments (see Rogers et al., 1979; Rogers and Davis, 1982; West et al., 1987; Newbold et al., 1989).

Rumen fermentation In agreement with other studies, soluble carbohydrates decreased rumen ammonia concentration (Syrj~ilL 1972; Chamberlain et al., 1985; Huhtanen, 1987; Rooke et al., 1987; Khalili and Huhtanen, 1991a). The reduction in ammonia concentration with molasses may suggest increased microbial protein synthesis (Huhtanen, 1987; Rooke et al., 1987; Khalili and Huhtanen, 1991 a). There were no differences in ammonia concentration between Diets MM and MH, although the amount of digestible DM was higher with Diet MH. Lower pH may have depressed microbial growth rates and yields (Russell et al., 1979; Strobel and Russell, 1986) or decreased absorption of ammonia from the rumen (Siddons et al., 1985) with Diet MH. In addition to improved microbial protein synthesis, lower ammonia concentration in Diet MHB than in Diet MH may be related to higher rumen pH, increased liquid dilution rate or rumen volume. Chamberlain et al. ( 1985 ) observed that diets supplemented with sucrose and bicarbonate decreased rumen ammonia concentration, but Khalili and Huhtanen ( 199 la) reported no effect of bicarbonate on ammonia concentration or microbial synthesis. The total VFA concentration was not affected, although the intakes of digestible DM were increased with increasing level of molasses. Some reasons may be increased lactic acid production or absorption of VFA. In agreement with other studies, soluble carbohydrates increased molar propionic and/or butyric acid at the expense of acetate (Sutton, 1968; Kellogg and Owen, 1969; Chamberlain et al., 1985; Khalili and Huhtanen, 1991a). Increase in molar proportion of propionate may be beneficial in forage diets producing high proportions of acetate, as it has been proposed that an insufficient supply of glucose relative to acetate may reduce the efficiency of utilization of acetate (Preston and Leng, 1987 ). In several studies, bicarbonate has been found to increase the proportion of acetic acid and decrease that ofpropionic acid (see review by Staples and Lough, 1989). With grass silage diets supplemented with sucrose, bicarbonate has been found to increase the proportion of acetic and propionic acid and decrease that of butyric acid (Chamberlain et al., 1985; Khalili and Huhtanen, 199 la). In the present trial, bicarbonate did not change the fermentation pattern, possibly as a result of differences in microbial populations between silage and hay diets or because changes in pH, with or without bicarbonate, were moderate. In conclusion, the results suggest positive effects of molasses supplementation on nitrogen utilization, increased gluconeogenesis and on the supply of glucose precursors. These could improve the utilization of forage-based diets. However, it was not possible to show any optimum level of molasses for hay

36

H. KHALILI

intake, a l t h o u g h 1.5 kg D M o f m o l a s s e s c o u l d be i n c l u d e d in the diet w i t h o u t d e p r e s s i n g cell-wall digestion. H a y i n t a k e was d e p r e s s e d p r o b a b l y as a result o f c o m b i n e d m e t a b o l i c a n d p h y s i c a l fill r e g u l a t i o n . C o m p a r i s o n b e t w e e n different m e c h a n i s m s a f f e c t i n g fibre d i g e s t i o n w a s difficult b e c a u s e p H a n d carb o h y d r a t e effects w e r e b o t h i n v o l v e d in fibre digestion. It is suggested t h a t w o r k to d e t e r m i n e the f a c t o r s t h a t c o n t r o l r u m e n fill in forage diets s h o u l d c o n t i n u e , a n d this w o r k s h o u l d b e l i n k e d w i t h forage intake. ACKNOWLEDGEMENTS T h e a u t h o r w o u l d like to t h a n k the s t a f f o f I L C A at the D e b r e Z e i t R e s e a r c h S t a t i o n a n d the N u t r i t i o n a n d B i o m e t r i c s U n i t s at H e a d q u a r t e r s in A d d i s A b a b a for t h e i r t e c h n i c a l assistance. T h e a u t h o r w o u l d also like to t h a n k F I N N I D A ( F i n n i s h I n t e r n a t i o n a l D e v e l o p m e n t A g e n c y ) for f i n a n c i a l s u p p o r t .

REFERENCES Ahmed, F.A. and Kay, M., 1975. A note on the value of molasses and tapioca as energy supplements to forage for growing steers. Anim. Prod., 21: 191-194. Aitchison, E.M., Gill, M., Dhanoa, M.S. and Osbourn, D.F., 1986. The effect of digestibility and forage species on the removal of digesta from the rumen and the voluntary intake of hay by sheep. Br. J. Nutr., 56: 463-476. Bines, J.A., 1985. Feeding systems and food intake by housed dairy cows. Proc. Nutr. Soc., 44: 355-462. Brown, W.F., Phillips, J.D. and Jones, D.B., 1987. Ammoniation or cane molasses supplementation of low quality forages. J. Anim. Sci., 1205-1214. Campling, R.C., 1966. The effect of concentrates on the rate of disappearance of digesta from the alimentary tract of cows given hay. J. Dairy Res., 33:13-23. Campling, R.C. and Murdoch, J.C., 1966. The effect of concentrates on the voluntary intake of roughages by cows. J. Dairy Res., 33:1-11. Campling, R.C., Freer, M. and Balch, C.C., 1962. Factors affecting the voluntary intake of food by cows. Br. J. Nutr., 16:115-124. Chamberlain, D.G., Thomas, P.C., Wilson, W., Newbold, C.J. and MacDonald, J.C., 1985. The effects of carbohydrate supplements on ruminal concentrations of ammonia in animals given diets of grass silage. J. Agric. Sci., 104:331-340. Cronje, P.B., Nolan, J.V. and Leng, R.A., 1991. Acetate clearance rate as a potential index of the availability of glucogenic precursors in ruminants fed on roughage-based diets. Br. J. Nutr., 66: 301-312. England, P. and Gill, M., 1985. The effect of fish meal and sucrose supplementation on the voluntary intake of grass silage and live-weight gain in young cattle. Anim. Prod., 40: 259265. Garrett, J.E., Guessous, F. and Eddebbarh, A., 1989. Utilization of sugar beet molasses and monensin for finishing dairy bullocks. Anim. Feed Sci. Technol., 25:11-21. Goering, H.K. and Van Soest, P.J., 1970. Forage Fibre Analyses. Agriculture Handbook No. 379. US Department of Agriculture, Washington, DC, pp. 1-20. Hoover, W.H., 1986. Chemical factors involved in ruminal fiber digestion. J. Dairy Sci., 69: 2755-2766.

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Huhtanen, P., 1987. The effects of intraruminal infusions of sucrose and xylose on the nitrogen and fiber digestion in cattle receiving diets of grass silage and barley. J. Agric. Sci. Finl., 59: 405-424. Huhtanen, P, and Jaakkola, S., 1993. The effects of the forage preservation method and the proportion of concentrate on digestion of cell wall carbohydrates and rumen digesta pool size in cattle. Grass Forage Sci., in press. Huhtanen, P. and Khalili, H., 1991. Sucrose supplements in cattle given grass silage-based diet. 3. Rumen pool size and digestion kinetics. Anim. Feed Sci., Technol., 33: 275-287. Huhtanen, P. and Khalili, H., 1992. The effect of sucrose supplements on particle-associated carboxymethylcellulase and xylanase activities in cattle given grass silage based diet. Br. J. Nutr., 67: 245-255. Kellogg, D.W. and Owen, F.G., 1969. Alterations of in vitro tureen fermentation patterns with various levels of sucrose and cellulose. J. Dairy Sci., 52:1458-1460. Khalili, H. and Huhtanen, P., 1991a. Sucrose supplements in cattle given grass silage-based diet. 1. Digestion of organic matter and nitrogen. Anim. Feed Sci. Technol., 33:247-261. Khalili, H. and Huhtanen, P., 1991b. Sucrose supplements in cattle given grass silage-based diet. 2. Digestion of cell wall carbohydrates. Anim. Feed Sci. Technol., 33: 263-273. Khalili, H., Varvikko, T. and Osuji, P.O., 1993. Supplementation of grass hay with molasses in crossbred (Bos taurus X Bos indicus) non-lactating cows: effect of timing of molasses supplements on feed intake, digestion, DM degradation and rumen fermentation. Anim. Feed Sci. Technol., 41: 39-50. Kukkonen, U. and Huhtanen, P., 1991. Rate of passage calculations based on marker or rumen evacuation technique. Anim, Prod., 52: 592-593. (Abstract). Littell, R.C., Freund, R.J. and Spector, P.C., 1992. SAS System for Linear Models, 3rd edn. SAS Series in Statistical Applications. SAS Institute, Cary, NC, 329 pp. Lofgreen, G.P. and Otagaki, K.K., 1960a. The net energy of blackstrap molasses for lactating dairy cows. J. Dairy Sci., 43: 220-230. Lofgreen, G.P. and Otagaki, K.K., 1960b. The net energy of blackstrap molasses for fattening steers as determined by a comparative slaughter technique. J. Anim. Sci., 19: 392-403. McDonald, I., 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci., 96:251-252. Mould, F.L. and Orskov, E.R., 1983. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anita. Feed Sci. Technol., 10: 1-14. Mould, F.L., Orskov, E.R. and Mann, S.O., 1983. Associative effect of mixed feeds. I. Effects of type and level of supplementation and the influence of the rumen fluid pH on cellulolysis in vivo and dry matter digestion of various roughages. Anita. Feed Sci. Technol., l 0:15-30. Newbold, C.J., Thomas, P.C. and Chamberlain, D.G., 1989. A note on the effects of the method of inclusion of sodium bicarbonate and diet composition on the intake of diets based on silage by dairy cows. J. Anim. Prod., 48:611-615. Preston, T.R. and Leng, R.A., 1987. Matching Ruminant Production Systems with Available Resources in the Tropics and Sub-Tropics. CTA, Wageningen, Netherlands; Penambul Books, Armidale, N.S.W., 245 pp. Robinson, P.H., Tamminga, S. and van Vuuren, A.M., 1987. Influence of declining level of feed intake and varying the proportion of starch in the concentrate on rumen ingesta quantity, composition and kinetics ofingesta turnover in dairy cows. Livest. Prod. Sci., 17: 37-62. Rogers, J.A. and Davis, C.L., 1982. Rumen volatile fatty acid production and nutrient utilization in steers fed a diet supplemented with sodium bicarbonate and monensin. J. Dairy Sci., 65: 944-952. Rogers, J.A., Marks, B.C., Davis, C.L. and Clark, J.H., 1979. Alteration ofrumen fermentation in steers by increasing rumen fluid dilution rate with mineral salts. J. Dairy Sci., 62:15991605.

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Rooke, J.A., Lee, N.H. and Armstrong, D.G., 1987. The effects of intraruminal infusions of urea, casein, glucose syrup and mixture of casein and glucose syrup on nitrogen digestion in the rumen of cattle receiving grass silage diets. Br. J. Nutr., 57: 89-98. Russell, J.B., 1984. Factors influencing competition and composition of rumen bacterial flora. In: F.M.C. Gilchrist and R.I. Mackie (Editors), Herbivore Nutrition in the Tropics and Subtropics. Science Press, Pretoria, pp. 313-345. Russell, J.B. and Baldwin, R.L., 1978. Substrate preferences in rumen bacteria: evidence of catabolite regulatory mechanisms. Appl. Environ. Microbiol., 36:319-329. Russell, J.B. and Dombrowski, D.B., 1980. Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture. Appl. Environ. Microbiol., 39: 604-610. Russell, J.B., Sharp, W.M. and Baldwin, R.L., 1979. The effects of pH on maximum bacterial growth rate and its possible role as a determinant of bacterial competition in the rumen. J. Anim. Sci., 48: 251-255. Siddons, R.C., Nolan, J.V., Beever, D.E. and Macrae, J.C., 1985. Nitrogen digestion and metabolism in sheep consuming diets containing contrasting forms and levels of N. Br. J. Nutr., 54: 175-187. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods, 7th edn. Iowa State University Press, Ames, 507 pp. Staples, C.R. and Lough, D.S., 1989. Efficacy of supplemental dietary neutralizing agents for lactating dairy cows. A review. Anim. Feed Sci. Technol., 23: 277-303. Stern, M.D. and Hoover, W.H., 1979. Methods for determining and factors affecting rumen microbial protein synthesis: a review. J. Anim. Sci., 49:1590-1603. Stewart, C.S., 1977. Factors affecting the cellulolytic activity ofrumen contents. Appl. Environ. Microbiol., 33: 497-502. Strobel, H.J. and Russell, J.B., 1986. Effect of pH and energy spilling on bacterial protein synthesis by carbohydrate-limited cultures of mixed rumen bacteria. J. Dairy Sci., 69: 29412947. Sutton, J.D., 1968. The fermentation of soluble carbohydrates in rumen contents of cows fed diets containing a large proportion of hay. Br. J. Nutr., 22:689-712. Syrj~il~i,L., 1972. Effect of different sucrose, starch, and cellulose supplements on the utilization of grass silages by ruminants. Ann. Agric. Fenn., 11:199-276. Van Keulen, J. and Young, B.A., 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci., 44: 282-287. Van Soest, P.J., 1973. The uniformity and nutritive availability of cellulose. Fed. Proc. Fed. Am. Soc. Exp. Biol., 32: 1804-1808. West, J.W., Coppock, C.E., Nave, D.H., Labore, J.M. and Greene, L.W., 1987. Effects of potassium carbonate and sodium bicarbonate on rumen function in lactating Holstein cows. J. Dairy Sci., 70:81-90.