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The effects of increasing the proportion of molasses in the diet of milking dairy cows on milk production and composition
Animal Feed Science and Technology 78 (1999) 189±198
The effects of increasing the proportion of molasses in the diet of milking dairy cows on milk production and composition J.J. Murphy* Dairy Production, Department, Teagasc, Moorepark Research Centre, Fermoy Co. Cork, Ireland Received 19 May 1998; accepted 23 December 1998
Abstract The objective of this experiment was to measure the effects on milk production and composition of including different levels of molasses, which is a good source of rumen-fermentable energy, in the diet. The experiment was a balanced Latin square design with four treatments and four periods, each of 4 weeks duration. There were five squares using a total of 20 Holstein/Friesian cows. At the start of the experiment cows were on average 32 days in milk (range 18±47 days), had an average body weight of 591 kg (range 516±716 kg) and an average milk yield of 27.5 kg/day (range 21.9± 32.5 kg/day). The treatments consisted of 5 kg/day of a concentrate supplement plus one of the following molasses/grass±silage mixtures (g/kg) fed to appetite: (1) 0 molasses/1000 grass±silage, (2) 50 molasses/950 grass±silage (139 g/kg molasses on a DM basis), (3) 100 molasses/900 grass± silage (254 g/kg molasses on a DM basis), and (4) 150 molasses/850 grass±silage (351 g/kg molasses on a DM basis). Milk yield was measured daily, milk composition on three successive morning and evening milkings weekly and blood samples were taken once before an evening milking in the final week of each period. Protein composition was measured on a composite of one successive evening and morning milk sample in the final week of each period from twelve cows (3 squares). Intake was measured daily. Milk and constituent yields, milk composition and intake data from the final 2 weeks of each period were used to compare the treatments. There was a significant linear increase in milk yield (p < 0.001), protein yield (p < 0.001), protein concentration (p < 0.01), casein concentration (p < 0.05) and total dry matter intake (p < 0.001) with increasing level of molasses inclusion. Plasma b-hydroxy butyrate levels were significantly higher (p < 0.05) in diets containing molasses. It is concluded that including rumen-fermentable energy in the form of molasses in a grass±silage-based diet for dairy cows significantly increased milk production and improved composition. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Molasses; Dairy cows; Milk protein * Tel.: +353-25-42222; fax: +353-25-42340; e-mail: [email protected] 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 0 7 - 3
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1. Introduction Protein is now the most valuable component of milk because of market demand for protein-containing dairy products and because of quota restrictions on milk fat. However, cows fed diets based on high proportions of grass±silage produce milk with a low protein content and it has been shown that the protein concentration of milk is significantly lower on grass±silage compared to the parent herbage from which the silage was conserved (Keady et al., 1995). In Ireland, the UK and many parts of Northern Europe grass±silage is the main source of winter forage and therefore in these regions, a relatively high proportion of milk is produced from grass±silage-based diets. Grass±silage, being a fermented feed is limited in its energy supply to the rumen microbes and the level of microbial protein synthesis in unwilted grass±silage is significantly lower than on fresh grass (Younge et al., 1997). Microbial protein is a good source of amino acids for milk protein synthesis but if this is limited on silage diets then it is a likely reason for low milk proteins. Keady and Murphy (1998) have shown that supplementing silage with sucrose (10 g/kg) significantly increased milk protein concentration. Therefore, it appears that stimulating microbial protein synthesis and capturing a greater proportion of the degradable N in silage could lead to increased milk protein. Such a strategy would have the added benefit of reducing losses of N to the environment on such diets. Cane molasses would seem to be a good source of rumen fermentable energy for the rumen microbes. It contains approximately 640 g of sugar/kg of DM. The data of Krohn et al. (1985) indicated a positive effect on milk yield of including high levels of molasses (320 g/kg DM) in a silage-based diet. The levels of inclusion were such (up to 480 g/kg DM) that it would be difficult, and possibly dangerous, to feed them except in a complete diet situation or at least mixed evenly through the silage. More recently, Yan et al. (1997) have shown responses in milk and protein with midlactation cows by including up to 468 g/kg DM of molasses in the diet. Milk protein concentration and yield increased from 31.6 g/kg and 0.49 kg/day at 156 g/kg DM molasses inclusion up to 33.6 g/kg DM and 0.59 kg/day at 468 g/kg DM molasses inclusion. Therefore, from a theoretical point of view and from the small amount of experimental data available, molasses feeding would appear to have potential as a means of raising both milk protein concentration and yield on grass±silage-based diets. The objective of the experiment reported here was to determine the magnitude of the response in milk yield and milk protein concentration, composition and yield to a range of molasses inclusion in the diet. 2. Materials and methods 2.1. Experimental design The experiment was a balanced Latin square design with four treatments and four periods each of 4 weeks duration. Twenty Holstein/Friesian cows were blocked into
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Table 1 The ingredient and chemical composition (g/kg DM) of the concentrate mixtures fed on each treatment Treatment 0M
50M
100M
150M
Unmolassed beet pulp Soyabean meal Rapeseed meal Maize gluten feed Fat Mineralsa
500 100 100 260 15 25
430 180 100 250 15 25
350 295 100 220 10 25
225 410 100 230 10 25
Dry matter (g/kg) Crude Protein Crude fibre Neutral detergent fibre Oil Ash Sugarb
929 203 133 328 31 97 66
929 236 124 304 34 94 68
929 284 114 279 27 91 73
925 337 97 234 32 92 75
a The minerals consisted of 12.5 kg of di-calcium phosphate in 0M, 50M and 100M and 15 kg in 150 m; 5 kg of mono-ammonium phosphate in 0M, 50M and 100M and 2.5 kg in 150M; 5 kg of salt and 2.5 kg of calcined magnesite. The following trace minerals and vitamins per tonne were added to the 4 concentrates: manganous oxide, 80 g; copper sulphate, 150 g; zinc oxide, 118 g; sodium selenite, 2 g; potassium iodate, 12 g; cobalt sulphate, 6 g; vitamin A, 7.5 miu; vitamin D3, 1.5 miu; vitamin E, 7500 iu. b Estimated from tabular values according to R.&H. Hall Technical Bulletin, Issue No. 5, 1996.
groups of four on the basis of days in milk and then subjected to the four treatments in a balanced Latin square design. Thus, there were five squares used. At the start of the experiment the cows were on average 32 days in milk (range 18±47 days), had an average body weight of 591 kg (range 516±716 kg) and an average milk yield of 27.5 kg/day (range 21.9±32.5 kg/day). The treatments consisted of 5 kg/day, as fed, of a concentrate supplement plus one of the following molasses/grass±silage mixtures: 1. 2. 3. 4.
0 g/kg molasses/1000 g/kg grass±silage (0M) 50 g/kg molasses 950 g/kg grass±silage ± 139 g/kg molasses on a DM basis (50M) 100 g/kg molasses/900 g/kg grass±silage ± 254 g/kg molasses on a DM basis (100M) 150 g/kg molasses/850 g/kg grass±silage ± 351 g/kg molasses on a DM basis (150M)
Concentrates were formulated with different levels of crude protein so that the crude protein concentrations in the total diets would be similar. The ingredient and chemical compositions of the concentrates fed with each molasses/ grass±silage mixture is shown in Table 1. The rapeseed and soyabean meals used were solvent-extracted and the maize gluten feed contained solubles. The fat included was lard. The chemical composition of the silage fed is shown in Table 2. The molasses fed had a dry matter concentration of 700 g/kg ( 5.5) a crude protein concentration of 70 g/kg DM ( 23) an ash concentration of 172 g/kg DM ( 15) and a sugar concentration of 607 g/kg DM ( 37). The silage was a second cut material from a mainly perennial ryegrass sward mown with a mower conditioner (Krohn) on the afternoon of 6 July 1994
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Table 2 The chemical composition of the silage (g/kg DM unless stated otherwise) Dry matter (g/kg) Crude Protein Modified acid detergent fibre Neutral detergent fibre Ash Dry matter digestibility Digestible organic matter Ammonia nitrogen pH Lactic acid Acetic Acid Propionic acid Butyric acid Ethanol Water soluble carbohydrates
228 17.7 166 7.4 324 10.7 550 11.6 100 4.9 736 10.9 644 11.1 3.69 0.89 3.95 0.22 97.9 12.5 23.5 7.4 1.54 2.37 2.43 2.72 3.87 0.77 20 1.95
and picked up with a precision chop harvester (Pottinger Max V) in the afternoon of 8 July 1994. The herbage was treated with formic acid (Acid-Safer, 3 l/t). The silage and molasses were mixed in the correct proportions in a feeder wagon (Keenan) and offered to cows individually through Calan electronic doors, once daily at between 1000 and 1100 h, in sufficient quantities to allow a refusal of 50 to 100 g/kg of intake. Refusals were weighed daily between 0900 and 1000 h. Concentrates were fed on top of the silage or silage/molasses mixtures in two feeds per day one after each milking. Milking times commenced at 0730 and 1530 h. 2.2. Measurements and analysis Milk yields were recorded daily and milk fat, protein and lactose concentrations were determined on one successive PM and AM milking in the first 2 weeks of each period and on three successive PM and AM milkings in the final 2 weeks of each period. The milk constituents were analysed by automated infra-red analysis using a Milkoscan 203 (Foss Electric, Denmark). The milk protein nitrogen (N) was fractionated into casein N, whey protein N and non-protein N (NPN) on three of the five squares (12 cows) selected randomly. This was done on a composite of one successive PM and AM milk sample in the final week of each period. Total protein, casein, whey and NPN were measured using the Kjelfoss automatic 16210 instrument (AS/N Foss Electric Denmark). Filtrates were prepared for NPN based on a modification of the IDF standard 20B: 1993 and for NCN based on a modification of the IDF standard 29: 1964. The silage and silage/molasses mixtures, offered and refused, were sampled twice weekly and these samples were composited by treatment into one sample per week. They were stored frozen until analysis. Concentrates and molasses were sampled once weekly. The dry matter content of the concentrates was determined by drying in an oven overnight at 1038C. Silage was dried at 408C for 48 h. The dried concentrates and silages were milled using a 1 mm screen (Tecator Clycotec 1093 mill) and analysed for crude
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protein content using the Kjelfoss automatic 16210 (AS/N Foss Electric Denmark), ash content by burning in a muffle furnace at 5508C for 12 h, MADF by the method of Clancy and Wilson (1966), NDF by the method of Van Soest and Wine (1967) and CF by the method outlined in the European Community Regulations (1984) using a Tecator Fibertec instrument. Oil was determined in the concentrate using the Foss±Let instrument (AS/N Foss Electric, Denmark). The dry matter contents of the silage and molasses were determined by toluene distillation (Dewar and McDonald, 1961) and the silage toluene DM was adjusted for silage ethanol content. The total sugar content of molasses was measured by the method outlined in the European Community Regulations (1984) and the water-soluble carbohydrate content of the silage by the method of Wilson (1978). Silage pH was determined on expressed juice using a pH electrode. Ammonia N was estimated colorimetrically by a modification of the phenol-hypochlorite technique (O'Keeffe and Sherington, 1983). Lactic acid concentration was analysed on a Ciba±Corning Express clinical analyser using the method of Boehringer Mannheim (Catalogue No. 139004) and VFAs and ethanol by gas liquid chromatography (Ranfft, 1973). Blood samples were taken into heparinised vacutainers from the coccygeal vessels of each cow once before an evening milking in the final week of each period. These samples were analysed for glucose, b-hydroxy butyrate, total protein, albumin and urea using a Cobas Mira biochemical analyser (Roche Diagnostics, Basle, Switzerland). Globulin was calculated as the difference between total protein and albumin. 2.3. Statistical analysis The mean results of the last 2 weeks of the periods for milk yields, constituent yields, milk composition and dry matter intakes were used to compare the treatments. Milk yields, milk constituent yield, milk composition, protein nitrogen fractions and blood metabolites were analysed using the GLM procedure of SAS (1991) (version 6.04) taking out the treatment, cow, period, cow within square and treatment by square effects. The data were tested for linear and quadratic effects and an orthogonal comparison was made between the control diet and the three diets containing molasses (no molasses vs. molasses). 3. Results The milk yield, milk constituent yield and milk composition on the four treatments are show in Table 3. Milk yield was significantly higher in the three diets with molasses (50M, 100M and 150M) compared to the control (0M) (p < 0.001) and there was a significant linear effect with the increasing level of molasses inclusion (p < 0.001). Fat yield was not significantly different between treatments. Protein and lactose yields were significantly higher in the molasses diets compared to the control (p < 0.001) and protein was significantly higher in treatment 150M compared to treatments 50M and 100M
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Table 3 The effect of increasing levels of molasses in the diet on milk, fat, protein and lactose yields, milk composition and intake Treatment Milk yield (kg/day) Fat yield (g/day) Protein yield (g/day) Lactose yield (g/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Molasses DMI Silage/molasses DMI (kg/day) Total DM1 (kg/day)
0M
50M
100M
150M
SSED
Effecta
22.1 834 668 1008 38.1 30.6 45.7 0 10.2 14.8
23.2 860 712 1066 37.4 30.9 45.9 1.7 12.0 16.6
23.3 859 715 1072 37.3 31.0 46.1 3.2 12.7 17.4
23.7 846 739 1088 36.1 31.4 45.8 4.8 13.6 18.2
0.27 19 8 13 0.71 0.25 0.16 ± 0.34 0.34
IL (p < 0.001) IL (p < 0.001) IL (p < 0.001) I(p < 0.05)L(p < 0.01) I(p < 0.05)L(p < 0.01) IQ(p < 0.05) IL(p < 0.001) IL(p < 0.001)
a I: dietary inclusion of molasses (0M vs. mean of 50M, 100M and 150M. L: linear effect of molasses inclusion. Q: quadratic effect of molasses inclusion.
(p < 0.01). There was a significant linear effect of increasing molasses inclusion on protein and lactose yields (p < 0.001). Milk fat concentration was significantly lower in molasses diets compared to the control (p < 0.05). Protein and lactose concentrations were significantly higher in the molasses diets than in the control (p < 0.05) and there was a significant linear effect of increasing molasses inclusion on decreasing fat concentration (p < 0.01) and increasing protein concentration (p < 0.01). There was a significant quadratic effect of molasses inclusion on lactose concentration. The intake of the silage/molasses mixture and of total DM increased linearly with increasing molasses inclusion (p < 0.001). Diets containing molasses had significantly higher intakes than the control (p < 0.001). The estimated sugar intakes (kg/cow/day) were 0.51 1.54 2.50 and 3.43 on the 0M, 50M, 100M and 150M diets, respectively, with the molasses contributing 0, 0.66, 0.79 and 0.85 of the sugar. The effect of increasing molasses level in the diet on milk N fractions is shown in Table 4. Casein N in milk showed a significant linear increase with increasing levels of molasses (p < 0.05) and was higher in the molasses diets compared to the control (p 0.056). Molasses inclusion had no effect on whey protein N in milk or on the proportions of whey protein N or casein N in total milk N. Non-protein N expressed as either g/kg milk or g/kg total milk N was significantly lower in molasses diets compared to the control (p < 0.01 and p < 0.001, respectively). Increasing levels of molasses inclusion gave a significant linear reduction in non-protein N concentration in milk and in non-protein nitrogen as a proportion of total milk N (p < 0.001). Blood metabolites measured on the four diets are shown in Table 5. The concentration of b-hydroxy butyrate was significantly higher in the molasses diets compared to the control (p < 0.05) and there was a significant linear effect (p < 0.05) of the increasing molasses level. There was no significant difference between treatments in the concentrations of glucose, protein, albumin, globulin or urea.
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Table 4 The effect of increasing levels of molasses in the diet on milka nitrogen (N) fractions Treatment Total N (g/kg) Casein N (g/kg) Whey protein N(g/kg) Non-protein N (g/kg) Casein N (g/kg total N) Whey protein N (g/kg total N) Non-protein N (g/kg total N)
OM
50M
100M
150M
SED
Effectb
4.51 3.45 0.79 0.27 763 177 60.6
4.55 3.49 0.79 0.26 767 175 58.5
4.58 3.51 0.81 0.25 766 179 55.2
4.63 3.58 0.81 0.25 771 174 54.5
0.04 0.05 0.02 0.006 4.6 4.4 1.5
I(p < 0.05) L(p < 0.01) I(p 0.056) L(p < 0.05) I(p < 0.01) L(p < 0.001) IL (p<0.001)
a
Milk nitrogen fractions were determined on 3 squares (12 cows). I: dietary inclusion of molasses (0M vs. mean of 50M, 100M and 150M). L: linear effect of molasses inclusion. b
Table 5 The effect of increasing levels of molasses in the diet on blood metabolites Treatment Glucose (mmol/l) b-Hydroxy butyrate (mmol/l) Protein (g/l) Albumin (g/l) Globulin (g/l) Urea (mmol/l)
OM
50M
100M
150M
SED
3.66 0.56 73.3 34.0 39.3 5.82
3.50 0.61 75.1 35.0 40.0 5.52
3.67 0.65 74.2 35.1 39.0 5.64
3.57 0.65 76.2 35.1 41.2 5.62
0.10 0.04 1.69 0.86 1.38 0.19
Effecta IL (p < 0.05)
a I: dietary inclusion of molasses (OM vs. mean 50M, 100M and 150M). L: linear effect of molasses inclusion.
4. Discussion The carbohydrates in molasses consist of approximately 600 g/kg sucrose, 120 g/kg each of free glucose and fructose, 70 g/kg of starches and other polysaccharides, 34 g/kg of trisaccharides with the remainder being made up of aconitic acid and volatile fatty acids (Blake, 1993). Most of these carbohydrates would be fermented rapidly in the rumen supplying energy to the microbes and this should lead to more efficient utilisation of the rapidly degradable nitrogen in silages and greater microbial protein synthesis. As microbial protein is a good source of amino acids for milk protein production it would be anticipated that molasses-supplemented diets would result in higher milk protein production. The results of the present study would indicate that this is so, with milk protein concentration and yield increasing linearly with increasing levels of molasses inclusion. In this study, molasses DM accounted for 0 100, 186 and 262 g/kg of total DM intake, corresponding to daily intakes of 0, 1.67, 3.24 and 4.77 kg of molasses DM per cow/day. An earlier study by Krohn et al. (1985) showed that milk protein concentration increased with increasing molasses in the diet up to 480 g/kg DM (9.27 kg/cow/day) and protein
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yield increased with increasing molasses inclusion in the diet up to the 320 g/kg DM (6.03 kg/cow/day). Also, milk yield increased with increasing molasses DM inclusion up to 320 g/kg DM. In the trial of Yan et al. (1997), milk yield, protein yield and protein concentration increased when molasses DM was raised from 125 to 250 g/kg DM corresponding to molasses DM intakes of 1.6 and 4.05 kg/cow/day. All of these data suggest that milk yield and protein yield increase by including molasses in the diet up to a maximum of 250±320 g/kg DM. The data also agree that such inclusions of molasses also increase total DM intake. In the current experiment there was a significant linear effect of increasing molasses inclusion on DM intake and in the studies of Krohn et al. (1985) and Yan et al. (1997) total DM intake also increased with increasing molasses. Therefore, in all these experiments improved production and composition cannot be ascribed to any intrinsic effect of molasses per se but is more likely to be due to an indirect effect of molasses inclusion on dry matter intake. This is borne out by the results of some studies where molasses was used to replace cereal in the concentrate portion of the diet and where total DM intake was not affected (Butler, 1974; Mayne, 1989). In this study and those of Krohn et al. (1985) and Yan et al. (1997) the molasses was either added in a complete diet or included with the silage in a mixture, which was fed ad libitum, and because molasses increased the energy density of the diets intakes increased. Increased protein concentration was paralleled by an increase in casein concentration and a decrease in non-protein nitrogen concentration in milk. Casein as a proportion of total protein was not affected but non-protein nitrogen as a proportion of total protein decreased. In the study of Yan et al. (1997), casein concentration was not significantly affected by the level of molasses inclusion and non-protein nitrogen increased. In that study, however, a diet without molasses was not included and therefore their lowest molasses diet was similar to the 50M diet in this study in terms of the quantity of molasses fed. The differences in casein nitrogen between the 50M, 100M and 150M diets in this study were not statistically significant and therefore the result is not different from that of Yan et al. (1997). However, the non-protein nitrogen data are different. It is difficult to explain the increase in milk non-protein nitrogen in the study of Yan et al. (1997). As stated earlier greater capture of silage-degradable nitrogen would be expected with greater molasses inclusion in the diet resulting in less rumen ammonia and therefore less urea in blood and milk. Theoretically, lower milk non-protein nitrogen would be expected, as observed in this study. Blood urea was not significantly different between treatments in this experiment but was numerically lower in diets containing molasses (p 0.149). This trend would support a more efficient capture of degradable nitrogen in the rumen due to increased fermentable metabolizable energy (FME) intake with the molasses diets. The estimated FME intakes were 126.9 (8.6 MJ/kg DM), 149.9 (9.0 MJ/ kg DM), 164.5 (9.5 MJ/kg DM) and 178.8 MJ/day (9.8 MJ/kg DM) on the 0M, 50M, 100M and 150M diets, respectively. The plasma b-hydroxy butyrate concentration increased significantly with increasing molasses inclusion. Numerically higher levels of b-hydroxy butyrate were also reported by Yan et al. (1997) in diets with higher levels of molasses. Rather than this being an indication of greater body fat oxidation it probably reflects a greater proportion of butyric acid in the rumen volatile fatty acids on the molasses diets.
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Although milk fat yield was not significantly affected in this study the concentration of milk fat was significantly reduced in diets containing molasses and the effect was linearly significant. This could be a reflection of the decreasing fibre concentration in the diet with increasing molasses inclusion. The NDF concentrations were 481, 426, 373 and 325 g/kg DM in diets 0M 50M, 100M and 150M, respectively. Beauchemin et al. (1994) observed a linear decrease in milk fat concentration as NDF concentration in the diet decreased from 400 to 320 g/kg. Overall, the results from this experiment show that including molasses in the silage portion of the diet up to 150 g/kg of silage fresh weight (351 g/kg on a DM basis) significantly increases DM intake, milk yield, milk protein and casein concentrations and protein yield. Acknowledgements The author wishes to thank, Mr. Noel Byrne, Mr. Michael Nolan and Ms. Claire Gorry for technical help. Part funding from E.U. Structural Funds, Irish Dairy Farmers and Premier Molasses is gratefully acknowledged. References Beauchemin, K.A., Farr, B.I., Rode, L.M., Schaalje, G.B., 1994. Optimal neutral detergent fibre concentration of barley-based diets for lactating dairy cows. J. Dairy Sci. 77, 1013±1029. Blake, J., 1993. Cane molasses ± nutritional liquid gold, Feed Compounder, October, pp. 30±31. Butler, T.M., 1974. Comparison of sugarcane molasses and barley as energy feeds for dairy cattle. Ir. J. Agric. Res. 13, 197±202. Clancy, M.J., Wilson, R.K., 1966. Development and application of a new chemical method for predicting the digestibility and intake of herbage samples, Proc. 10th Int. Grassl. Congr., Helsinki, pp. 445±452. Dewar, A.R., McDonald, P., 1961. Determination of dry matter in silage by distillation with toluene. J. Sci. Fd. Agric. 12, 700±795. European Community (Marketing of Feedingstuff) Regulations, 1984. Statutory Instruments S.I. No. 200. International Dairy Federation, 1964. In: Determination of the casein content of milk. IDF (FIL-IDF Standard no. 29), Brussels. International Dairy Federation, 1993. Milk: determination of nitrogen content, IDF (FIL-IDF Standard no. 20B), Brussels. Keady, T.W.J., Murphy, J.J., Harrington, D., 1995. The effects of ensiling on dry matter intake and milk production by lactating dairy cattle given forage as the sole feed. Gr. For. Sci. 51, 131±141. Keady, T.W.J., Murphy, J.J., 1998. The effects of ensiling and supplementation with sucrose and fishmeal on forage intake and milk production of lactating dairy cows. Anim. Sci. 66, 9±20. Krohn, C.C., Anderson, P.E., Hvelplund, T., 1985. Stigende maengdr roemolasse i fuldfoder til malkekoer Statens Husdyrbrugsforsog Meddelelser, No. 568. Mayne, C.S., 1989. Effect of molasses and sodium bicarbonate on the performance of January±March calving cows offered low levels of a high protein supplement in addition to grass silage, Occ. Public. Br. Grassland. Soc., No. 23, pp. 81±85. O'Keeffe, M., Sherington, J., 1983. Comparison of three methods for determination of urea in compound feed and silage. Analyst 108, 1374±1379. Ranfft, K., 1973. Determination by gas chromatography of short chain fatty acids in ruminal fluids. Archiv. Tierernahrung 23, 343±352.
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SAS Users' Guide: Statistics, 1991. SAS Inst., Inc., Cary, NC. Van Soest, P.J., Wine, R.H., 1967. Use of detergents in the analysis of fibrous feeds. IV. Determination of plant cell wall constituents. J. Assoc. Off. Analyt. Chem. 50, 50±55. Wilson, R.K., 1978. Estimation of water soluble and individual carbohydrate in grass samples, Proc. Euroanalysis Confr., Dublin, no. 3, p. 46. Yan, T., Roberts, D.J., Higginbotten, J., 1997. The effects of feeding high concentration of molasses and supplementary with nitrogen and unprotected tallow on intake and performance of dairy cows. Anim. Sci. 64, 17±24. Younge, B.A., Murphy, J.J., Rath, M., 1997. Intestional nutrient flows on grass silage and fresh grass-based diets (abstract). Ir. J. Agric. Food Res. 36, 129.
The effects of increasing the proportion of molasses in the diet of milking dairy cows on milk production and composition J.J. Murphy* Dairy Production, Department, Teagasc, Moorepark Research Centre, Fermoy Co. Cork, Ireland Received 19 May 1998; accepted 23 December 1998
Abstract The objective of this experiment was to measure the effects on milk production and composition of including different levels of molasses, which is a good source of rumen-fermentable energy, in the diet. The experiment was a balanced Latin square design with four treatments and four periods, each of 4 weeks duration. There were five squares using a total of 20 Holstein/Friesian cows. At the start of the experiment cows were on average 32 days in milk (range 18±47 days), had an average body weight of 591 kg (range 516±716 kg) and an average milk yield of 27.5 kg/day (range 21.9± 32.5 kg/day). The treatments consisted of 5 kg/day of a concentrate supplement plus one of the following molasses/grass±silage mixtures (g/kg) fed to appetite: (1) 0 molasses/1000 grass±silage, (2) 50 molasses/950 grass±silage (139 g/kg molasses on a DM basis), (3) 100 molasses/900 grass± silage (254 g/kg molasses on a DM basis), and (4) 150 molasses/850 grass±silage (351 g/kg molasses on a DM basis). Milk yield was measured daily, milk composition on three successive morning and evening milkings weekly and blood samples were taken once before an evening milking in the final week of each period. Protein composition was measured on a composite of one successive evening and morning milk sample in the final week of each period from twelve cows (3 squares). Intake was measured daily. Milk and constituent yields, milk composition and intake data from the final 2 weeks of each period were used to compare the treatments. There was a significant linear increase in milk yield (p < 0.001), protein yield (p < 0.001), protein concentration (p < 0.01), casein concentration (p < 0.05) and total dry matter intake (p < 0.001) with increasing level of molasses inclusion. Plasma b-hydroxy butyrate levels were significantly higher (p < 0.05) in diets containing molasses. It is concluded that including rumen-fermentable energy in the form of molasses in a grass±silage-based diet for dairy cows significantly increased milk production and improved composition. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Molasses; Dairy cows; Milk protein * Tel.: +353-25-42222; fax: +353-25-42340; e-mail: [email protected] 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 0 7 - 3
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1. Introduction Protein is now the most valuable component of milk because of market demand for protein-containing dairy products and because of quota restrictions on milk fat. However, cows fed diets based on high proportions of grass±silage produce milk with a low protein content and it has been shown that the protein concentration of milk is significantly lower on grass±silage compared to the parent herbage from which the silage was conserved (Keady et al., 1995). In Ireland, the UK and many parts of Northern Europe grass±silage is the main source of winter forage and therefore in these regions, a relatively high proportion of milk is produced from grass±silage-based diets. Grass±silage, being a fermented feed is limited in its energy supply to the rumen microbes and the level of microbial protein synthesis in unwilted grass±silage is significantly lower than on fresh grass (Younge et al., 1997). Microbial protein is a good source of amino acids for milk protein synthesis but if this is limited on silage diets then it is a likely reason for low milk proteins. Keady and Murphy (1998) have shown that supplementing silage with sucrose (10 g/kg) significantly increased milk protein concentration. Therefore, it appears that stimulating microbial protein synthesis and capturing a greater proportion of the degradable N in silage could lead to increased milk protein. Such a strategy would have the added benefit of reducing losses of N to the environment on such diets. Cane molasses would seem to be a good source of rumen fermentable energy for the rumen microbes. It contains approximately 640 g of sugar/kg of DM. The data of Krohn et al. (1985) indicated a positive effect on milk yield of including high levels of molasses (320 g/kg DM) in a silage-based diet. The levels of inclusion were such (up to 480 g/kg DM) that it would be difficult, and possibly dangerous, to feed them except in a complete diet situation or at least mixed evenly through the silage. More recently, Yan et al. (1997) have shown responses in milk and protein with midlactation cows by including up to 468 g/kg DM of molasses in the diet. Milk protein concentration and yield increased from 31.6 g/kg and 0.49 kg/day at 156 g/kg DM molasses inclusion up to 33.6 g/kg DM and 0.59 kg/day at 468 g/kg DM molasses inclusion. Therefore, from a theoretical point of view and from the small amount of experimental data available, molasses feeding would appear to have potential as a means of raising both milk protein concentration and yield on grass±silage-based diets. The objective of the experiment reported here was to determine the magnitude of the response in milk yield and milk protein concentration, composition and yield to a range of molasses inclusion in the diet. 2. Materials and methods 2.1. Experimental design The experiment was a balanced Latin square design with four treatments and four periods each of 4 weeks duration. Twenty Holstein/Friesian cows were blocked into
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Table 1 The ingredient and chemical composition (g/kg DM) of the concentrate mixtures fed on each treatment Treatment 0M
50M
100M
150M
Unmolassed beet pulp Soyabean meal Rapeseed meal Maize gluten feed Fat Mineralsa
500 100 100 260 15 25
430 180 100 250 15 25
350 295 100 220 10 25
225 410 100 230 10 25
Dry matter (g/kg) Crude Protein Crude fibre Neutral detergent fibre Oil Ash Sugarb
929 203 133 328 31 97 66
929 236 124 304 34 94 68
929 284 114 279 27 91 73
925 337 97 234 32 92 75
a The minerals consisted of 12.5 kg of di-calcium phosphate in 0M, 50M and 100M and 15 kg in 150 m; 5 kg of mono-ammonium phosphate in 0M, 50M and 100M and 2.5 kg in 150M; 5 kg of salt and 2.5 kg of calcined magnesite. The following trace minerals and vitamins per tonne were added to the 4 concentrates: manganous oxide, 80 g; copper sulphate, 150 g; zinc oxide, 118 g; sodium selenite, 2 g; potassium iodate, 12 g; cobalt sulphate, 6 g; vitamin A, 7.5 miu; vitamin D3, 1.5 miu; vitamin E, 7500 iu. b Estimated from tabular values according to R.&H. Hall Technical Bulletin, Issue No. 5, 1996.
groups of four on the basis of days in milk and then subjected to the four treatments in a balanced Latin square design. Thus, there were five squares used. At the start of the experiment the cows were on average 32 days in milk (range 18±47 days), had an average body weight of 591 kg (range 516±716 kg) and an average milk yield of 27.5 kg/day (range 21.9±32.5 kg/day). The treatments consisted of 5 kg/day, as fed, of a concentrate supplement plus one of the following molasses/grass±silage mixtures: 1. 2. 3. 4.
0 g/kg molasses/1000 g/kg grass±silage (0M) 50 g/kg molasses 950 g/kg grass±silage ± 139 g/kg molasses on a DM basis (50M) 100 g/kg molasses/900 g/kg grass±silage ± 254 g/kg molasses on a DM basis (100M) 150 g/kg molasses/850 g/kg grass±silage ± 351 g/kg molasses on a DM basis (150M)
Concentrates were formulated with different levels of crude protein so that the crude protein concentrations in the total diets would be similar. The ingredient and chemical compositions of the concentrates fed with each molasses/ grass±silage mixture is shown in Table 1. The rapeseed and soyabean meals used were solvent-extracted and the maize gluten feed contained solubles. The fat included was lard. The chemical composition of the silage fed is shown in Table 2. The molasses fed had a dry matter concentration of 700 g/kg ( 5.5) a crude protein concentration of 70 g/kg DM ( 23) an ash concentration of 172 g/kg DM ( 15) and a sugar concentration of 607 g/kg DM ( 37). The silage was a second cut material from a mainly perennial ryegrass sward mown with a mower conditioner (Krohn) on the afternoon of 6 July 1994
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Table 2 The chemical composition of the silage (g/kg DM unless stated otherwise) Dry matter (g/kg) Crude Protein Modified acid detergent fibre Neutral detergent fibre Ash Dry matter digestibility Digestible organic matter Ammonia nitrogen pH Lactic acid Acetic Acid Propionic acid Butyric acid Ethanol Water soluble carbohydrates
228 17.7 166 7.4 324 10.7 550 11.6 100 4.9 736 10.9 644 11.1 3.69 0.89 3.95 0.22 97.9 12.5 23.5 7.4 1.54 2.37 2.43 2.72 3.87 0.77 20 1.95
and picked up with a precision chop harvester (Pottinger Max V) in the afternoon of 8 July 1994. The herbage was treated with formic acid (Acid-Safer, 3 l/t). The silage and molasses were mixed in the correct proportions in a feeder wagon (Keenan) and offered to cows individually through Calan electronic doors, once daily at between 1000 and 1100 h, in sufficient quantities to allow a refusal of 50 to 100 g/kg of intake. Refusals were weighed daily between 0900 and 1000 h. Concentrates were fed on top of the silage or silage/molasses mixtures in two feeds per day one after each milking. Milking times commenced at 0730 and 1530 h. 2.2. Measurements and analysis Milk yields were recorded daily and milk fat, protein and lactose concentrations were determined on one successive PM and AM milking in the first 2 weeks of each period and on three successive PM and AM milkings in the final 2 weeks of each period. The milk constituents were analysed by automated infra-red analysis using a Milkoscan 203 (Foss Electric, Denmark). The milk protein nitrogen (N) was fractionated into casein N, whey protein N and non-protein N (NPN) on three of the five squares (12 cows) selected randomly. This was done on a composite of one successive PM and AM milk sample in the final week of each period. Total protein, casein, whey and NPN were measured using the Kjelfoss automatic 16210 instrument (AS/N Foss Electric Denmark). Filtrates were prepared for NPN based on a modification of the IDF standard 20B: 1993 and for NCN based on a modification of the IDF standard 29: 1964. The silage and silage/molasses mixtures, offered and refused, were sampled twice weekly and these samples were composited by treatment into one sample per week. They were stored frozen until analysis. Concentrates and molasses were sampled once weekly. The dry matter content of the concentrates was determined by drying in an oven overnight at 1038C. Silage was dried at 408C for 48 h. The dried concentrates and silages were milled using a 1 mm screen (Tecator Clycotec 1093 mill) and analysed for crude
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protein content using the Kjelfoss automatic 16210 (AS/N Foss Electric Denmark), ash content by burning in a muffle furnace at 5508C for 12 h, MADF by the method of Clancy and Wilson (1966), NDF by the method of Van Soest and Wine (1967) and CF by the method outlined in the European Community Regulations (1984) using a Tecator Fibertec instrument. Oil was determined in the concentrate using the Foss±Let instrument (AS/N Foss Electric, Denmark). The dry matter contents of the silage and molasses were determined by toluene distillation (Dewar and McDonald, 1961) and the silage toluene DM was adjusted for silage ethanol content. The total sugar content of molasses was measured by the method outlined in the European Community Regulations (1984) and the water-soluble carbohydrate content of the silage by the method of Wilson (1978). Silage pH was determined on expressed juice using a pH electrode. Ammonia N was estimated colorimetrically by a modification of the phenol-hypochlorite technique (O'Keeffe and Sherington, 1983). Lactic acid concentration was analysed on a Ciba±Corning Express clinical analyser using the method of Boehringer Mannheim (Catalogue No. 139004) and VFAs and ethanol by gas liquid chromatography (Ranfft, 1973). Blood samples were taken into heparinised vacutainers from the coccygeal vessels of each cow once before an evening milking in the final week of each period. These samples were analysed for glucose, b-hydroxy butyrate, total protein, albumin and urea using a Cobas Mira biochemical analyser (Roche Diagnostics, Basle, Switzerland). Globulin was calculated as the difference between total protein and albumin. 2.3. Statistical analysis The mean results of the last 2 weeks of the periods for milk yields, constituent yields, milk composition and dry matter intakes were used to compare the treatments. Milk yields, milk constituent yield, milk composition, protein nitrogen fractions and blood metabolites were analysed using the GLM procedure of SAS (1991) (version 6.04) taking out the treatment, cow, period, cow within square and treatment by square effects. The data were tested for linear and quadratic effects and an orthogonal comparison was made between the control diet and the three diets containing molasses (no molasses vs. molasses). 3. Results The milk yield, milk constituent yield and milk composition on the four treatments are show in Table 3. Milk yield was significantly higher in the three diets with molasses (50M, 100M and 150M) compared to the control (0M) (p < 0.001) and there was a significant linear effect with the increasing level of molasses inclusion (p < 0.001). Fat yield was not significantly different between treatments. Protein and lactose yields were significantly higher in the molasses diets compared to the control (p < 0.001) and protein was significantly higher in treatment 150M compared to treatments 50M and 100M
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Table 3 The effect of increasing levels of molasses in the diet on milk, fat, protein and lactose yields, milk composition and intake Treatment Milk yield (kg/day) Fat yield (g/day) Protein yield (g/day) Lactose yield (g/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Molasses DMI Silage/molasses DMI (kg/day) Total DM1 (kg/day)
0M
50M
100M
150M
SSED
Effecta
22.1 834 668 1008 38.1 30.6 45.7 0 10.2 14.8
23.2 860 712 1066 37.4 30.9 45.9 1.7 12.0 16.6
23.3 859 715 1072 37.3 31.0 46.1 3.2 12.7 17.4
23.7 846 739 1088 36.1 31.4 45.8 4.8 13.6 18.2
0.27 19 8 13 0.71 0.25 0.16 ± 0.34 0.34
IL (p < 0.001) IL (p < 0.001) IL (p < 0.001) I(p < 0.05)L(p < 0.01) I(p < 0.05)L(p < 0.01) IQ(p < 0.05) IL(p < 0.001) IL(p < 0.001)
a I: dietary inclusion of molasses (0M vs. mean of 50M, 100M and 150M. L: linear effect of molasses inclusion. Q: quadratic effect of molasses inclusion.
(p < 0.01). There was a significant linear effect of increasing molasses inclusion on protein and lactose yields (p < 0.001). Milk fat concentration was significantly lower in molasses diets compared to the control (p < 0.05). Protein and lactose concentrations were significantly higher in the molasses diets than in the control (p < 0.05) and there was a significant linear effect of increasing molasses inclusion on decreasing fat concentration (p < 0.01) and increasing protein concentration (p < 0.01). There was a significant quadratic effect of molasses inclusion on lactose concentration. The intake of the silage/molasses mixture and of total DM increased linearly with increasing molasses inclusion (p < 0.001). Diets containing molasses had significantly higher intakes than the control (p < 0.001). The estimated sugar intakes (kg/cow/day) were 0.51 1.54 2.50 and 3.43 on the 0M, 50M, 100M and 150M diets, respectively, with the molasses contributing 0, 0.66, 0.79 and 0.85 of the sugar. The effect of increasing molasses level in the diet on milk N fractions is shown in Table 4. Casein N in milk showed a significant linear increase with increasing levels of molasses (p < 0.05) and was higher in the molasses diets compared to the control (p 0.056). Molasses inclusion had no effect on whey protein N in milk or on the proportions of whey protein N or casein N in total milk N. Non-protein N expressed as either g/kg milk or g/kg total milk N was significantly lower in molasses diets compared to the control (p < 0.01 and p < 0.001, respectively). Increasing levels of molasses inclusion gave a significant linear reduction in non-protein N concentration in milk and in non-protein nitrogen as a proportion of total milk N (p < 0.001). Blood metabolites measured on the four diets are shown in Table 5. The concentration of b-hydroxy butyrate was significantly higher in the molasses diets compared to the control (p < 0.05) and there was a significant linear effect (p < 0.05) of the increasing molasses level. There was no significant difference between treatments in the concentrations of glucose, protein, albumin, globulin or urea.
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Table 4 The effect of increasing levels of molasses in the diet on milka nitrogen (N) fractions Treatment Total N (g/kg) Casein N (g/kg) Whey protein N(g/kg) Non-protein N (g/kg) Casein N (g/kg total N) Whey protein N (g/kg total N) Non-protein N (g/kg total N)
OM
50M
100M
150M
SED
Effectb
4.51 3.45 0.79 0.27 763 177 60.6
4.55 3.49 0.79 0.26 767 175 58.5
4.58 3.51 0.81 0.25 766 179 55.2
4.63 3.58 0.81 0.25 771 174 54.5
0.04 0.05 0.02 0.006 4.6 4.4 1.5
I(p < 0.05) L(p < 0.01) I(p 0.056) L(p < 0.05) I(p < 0.01) L(p < 0.001) IL (p<0.001)
a
Milk nitrogen fractions were determined on 3 squares (12 cows). I: dietary inclusion of molasses (0M vs. mean of 50M, 100M and 150M). L: linear effect of molasses inclusion. b
Table 5 The effect of increasing levels of molasses in the diet on blood metabolites Treatment Glucose (mmol/l) b-Hydroxy butyrate (mmol/l) Protein (g/l) Albumin (g/l) Globulin (g/l) Urea (mmol/l)
OM
50M
100M
150M
SED
3.66 0.56 73.3 34.0 39.3 5.82
3.50 0.61 75.1 35.0 40.0 5.52
3.67 0.65 74.2 35.1 39.0 5.64
3.57 0.65 76.2 35.1 41.2 5.62
0.10 0.04 1.69 0.86 1.38 0.19
Effecta IL (p < 0.05)
a I: dietary inclusion of molasses (OM vs. mean 50M, 100M and 150M). L: linear effect of molasses inclusion.
4. Discussion The carbohydrates in molasses consist of approximately 600 g/kg sucrose, 120 g/kg each of free glucose and fructose, 70 g/kg of starches and other polysaccharides, 34 g/kg of trisaccharides with the remainder being made up of aconitic acid and volatile fatty acids (Blake, 1993). Most of these carbohydrates would be fermented rapidly in the rumen supplying energy to the microbes and this should lead to more efficient utilisation of the rapidly degradable nitrogen in silages and greater microbial protein synthesis. As microbial protein is a good source of amino acids for milk protein production it would be anticipated that molasses-supplemented diets would result in higher milk protein production. The results of the present study would indicate that this is so, with milk protein concentration and yield increasing linearly with increasing levels of molasses inclusion. In this study, molasses DM accounted for 0 100, 186 and 262 g/kg of total DM intake, corresponding to daily intakes of 0, 1.67, 3.24 and 4.77 kg of molasses DM per cow/day. An earlier study by Krohn et al. (1985) showed that milk protein concentration increased with increasing molasses in the diet up to 480 g/kg DM (9.27 kg/cow/day) and protein
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yield increased with increasing molasses inclusion in the diet up to the 320 g/kg DM (6.03 kg/cow/day). Also, milk yield increased with increasing molasses DM inclusion up to 320 g/kg DM. In the trial of Yan et al. (1997), milk yield, protein yield and protein concentration increased when molasses DM was raised from 125 to 250 g/kg DM corresponding to molasses DM intakes of 1.6 and 4.05 kg/cow/day. All of these data suggest that milk yield and protein yield increase by including molasses in the diet up to a maximum of 250±320 g/kg DM. The data also agree that such inclusions of molasses also increase total DM intake. In the current experiment there was a significant linear effect of increasing molasses inclusion on DM intake and in the studies of Krohn et al. (1985) and Yan et al. (1997) total DM intake also increased with increasing molasses. Therefore, in all these experiments improved production and composition cannot be ascribed to any intrinsic effect of molasses per se but is more likely to be due to an indirect effect of molasses inclusion on dry matter intake. This is borne out by the results of some studies where molasses was used to replace cereal in the concentrate portion of the diet and where total DM intake was not affected (Butler, 1974; Mayne, 1989). In this study and those of Krohn et al. (1985) and Yan et al. (1997) the molasses was either added in a complete diet or included with the silage in a mixture, which was fed ad libitum, and because molasses increased the energy density of the diets intakes increased. Increased protein concentration was paralleled by an increase in casein concentration and a decrease in non-protein nitrogen concentration in milk. Casein as a proportion of total protein was not affected but non-protein nitrogen as a proportion of total protein decreased. In the study of Yan et al. (1997), casein concentration was not significantly affected by the level of molasses inclusion and non-protein nitrogen increased. In that study, however, a diet without molasses was not included and therefore their lowest molasses diet was similar to the 50M diet in this study in terms of the quantity of molasses fed. The differences in casein nitrogen between the 50M, 100M and 150M diets in this study were not statistically significant and therefore the result is not different from that of Yan et al. (1997). However, the non-protein nitrogen data are different. It is difficult to explain the increase in milk non-protein nitrogen in the study of Yan et al. (1997). As stated earlier greater capture of silage-degradable nitrogen would be expected with greater molasses inclusion in the diet resulting in less rumen ammonia and therefore less urea in blood and milk. Theoretically, lower milk non-protein nitrogen would be expected, as observed in this study. Blood urea was not significantly different between treatments in this experiment but was numerically lower in diets containing molasses (p 0.149). This trend would support a more efficient capture of degradable nitrogen in the rumen due to increased fermentable metabolizable energy (FME) intake with the molasses diets. The estimated FME intakes were 126.9 (8.6 MJ/kg DM), 149.9 (9.0 MJ/ kg DM), 164.5 (9.5 MJ/kg DM) and 178.8 MJ/day (9.8 MJ/kg DM) on the 0M, 50M, 100M and 150M diets, respectively. The plasma b-hydroxy butyrate concentration increased significantly with increasing molasses inclusion. Numerically higher levels of b-hydroxy butyrate were also reported by Yan et al. (1997) in diets with higher levels of molasses. Rather than this being an indication of greater body fat oxidation it probably reflects a greater proportion of butyric acid in the rumen volatile fatty acids on the molasses diets.
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Although milk fat yield was not significantly affected in this study the concentration of milk fat was significantly reduced in diets containing molasses and the effect was linearly significant. This could be a reflection of the decreasing fibre concentration in the diet with increasing molasses inclusion. The NDF concentrations were 481, 426, 373 and 325 g/kg DM in diets 0M 50M, 100M and 150M, respectively. Beauchemin et al. (1994) observed a linear decrease in milk fat concentration as NDF concentration in the diet decreased from 400 to 320 g/kg. Overall, the results from this experiment show that including molasses in the silage portion of the diet up to 150 g/kg of silage fresh weight (351 g/kg on a DM basis) significantly increases DM intake, milk yield, milk protein and casein concentrations and protein yield. Acknowledgements The author wishes to thank, Mr. Noel Byrne, Mr. Michael Nolan and Ms. Claire Gorry for technical help. Part funding from E.U. Structural Funds, Irish Dairy Farmers and Premier Molasses is gratefully acknowledged. References Beauchemin, K.A., Farr, B.I., Rode, L.M., Schaalje, G.B., 1994. Optimal neutral detergent fibre concentration of barley-based diets for lactating dairy cows. J. Dairy Sci. 77, 1013±1029. Blake, J., 1993. Cane molasses ± nutritional liquid gold, Feed Compounder, October, pp. 30±31. Butler, T.M., 1974. Comparison of sugarcane molasses and barley as energy feeds for dairy cattle. Ir. J. Agric. Res. 13, 197±202. Clancy, M.J., Wilson, R.K., 1966. Development and application of a new chemical method for predicting the digestibility and intake of herbage samples, Proc. 10th Int. Grassl. Congr., Helsinki, pp. 445±452. Dewar, A.R., McDonald, P., 1961. Determination of dry matter in silage by distillation with toluene. J. Sci. Fd. Agric. 12, 700±795. European Community (Marketing of Feedingstuff) Regulations, 1984. Statutory Instruments S.I. No. 200. International Dairy Federation, 1964. In: Determination of the casein content of milk. IDF (FIL-IDF Standard no. 29), Brussels. International Dairy Federation, 1993. Milk: determination of nitrogen content, IDF (FIL-IDF Standard no. 20B), Brussels. Keady, T.W.J., Murphy, J.J., Harrington, D., 1995. The effects of ensiling on dry matter intake and milk production by lactating dairy cattle given forage as the sole feed. Gr. For. Sci. 51, 131±141. Keady, T.W.J., Murphy, J.J., 1998. The effects of ensiling and supplementation with sucrose and fishmeal on forage intake and milk production of lactating dairy cows. Anim. Sci. 66, 9±20. Krohn, C.C., Anderson, P.E., Hvelplund, T., 1985. Stigende maengdr roemolasse i fuldfoder til malkekoer Statens Husdyrbrugsforsog Meddelelser, No. 568. Mayne, C.S., 1989. Effect of molasses and sodium bicarbonate on the performance of January±March calving cows offered low levels of a high protein supplement in addition to grass silage, Occ. Public. Br. Grassland. Soc., No. 23, pp. 81±85. O'Keeffe, M., Sherington, J., 1983. Comparison of three methods for determination of urea in compound feed and silage. Analyst 108, 1374±1379. Ranfft, K., 1973. Determination by gas chromatography of short chain fatty acids in ruminal fluids. Archiv. Tierernahrung 23, 343±352.
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SAS Users' Guide: Statistics, 1991. SAS Inst., Inc., Cary, NC. Van Soest, P.J., Wine, R.H., 1967. Use of detergents in the analysis of fibrous feeds. IV. Determination of plant cell wall constituents. J. Assoc. Off. Analyt. Chem. 50, 50±55. Wilson, R.K., 1978. Estimation of water soluble and individual carbohydrate in grass samples, Proc. Euroanalysis Confr., Dublin, no. 3, p. 46. Yan, T., Roberts, D.J., Higginbotten, J., 1997. The effects of feeding high concentration of molasses and supplementary with nitrogen and unprotected tallow on intake and performance of dairy cows. Anim. Sci. 64, 17±24. Younge, B.A., Murphy, J.J., Rath, M., 1997. Intestional nutrient flows on grass silage and fresh grass-based diets (abstract). Ir. J. Agric. Food Res. 36, 129.