The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets Hannele Khali li Agricultural Research Centre ofFinland, North-Savo Research Station, FIN-71750 Maaninka, Finland, current address: Agricultural Research Centre ofFinland, Animal Production Research, FIN-31600 Jokioinen, Finland, e-mail: hannele.khalili@mtt.fi Tuomo Varvikko, Vesa Toivonen Agricultural Research Centre ofFinland, AnimalProduction Research, FIN-31600 Jokioinen, Finland Kari Hissa Finn Feed Ltd, PO Box 105, FIN-00241 Helsinki, Finland Marjatta Suvitie Agricultural Research Centre ofFinland, North-Savo Research Station, FIN-71750 Maaninka, Finland The addition of glycerol or free fatty acids either alone or in combination to concentrate was studied for the effects on feed intake, milk production, rumen fermentation, blood metabolites and diet di- gestibility in dairy cows given grass silage ad libitum. The study was conducted on 12 mid-lactating cows, four of them ruminally cannulated. Barley-based concentrate(control diet, C) was given 7 kg/d as fed. In the other three diets, 36 g/kg of barley was replaced by glycerol (G) or a mixture of free fatty acids (FA) or by a combination of the two, making a total of 72 g/kg (GFA). The experimental design consisted of balanced 4x4 Latin squares with a 2 x 2 factorial arrangement of diets: the effects of G, FA and G*FA interaction. The FA diets significantly decreased silage intake, increased milk yield, decreased milk protein content, increased the concentrations of C |Bo, CIB | , and C2Ol and decreased those of C816, and C |g3 fatty acids in milk fat. The FA diets also increased the concentration of nonesterified fatty acids in plasma, and decreased the digestibility of organic matter and neutral detergent fibre but increased that of fat. Glycerol decreased the molar proportion of acetate and in- creased the molar proportions of propionate and butyrate in the rumen, but the addition of glycerol did not have any effect on silage intake, milk yield or milk composition. Milk yield was highest when glycerol and free fatty acids were given together, showing a positive interaction. Key words: blood metabolites, digestibility, fatty acids, glycerol, grass silage milk production, rumen fermentation © Agricultural and Food Science inFinland Manuscript received May 1997 349 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=X-g04CMetHDbx7OT.FOGUGZX4sq6m0QqsOMNDCA.LCbxzsK5UD7sleXI5mGv0vRggBTcdJ5p_2dKqmMZp7g9X2NVyQJrVhDpJ_4aCn7phqjB_lYZmFBV7YCppkJAl21tKi3vrpE6u7BL6XJ-nh709bzQt3CbGbYUDPygp1JtEOSX12kGbqtvE1_hfS-q6TO3dFbHQpkaW4HNvse6WwJ6_lPSu-MGsCY7JZNT2lp3DAMoLmiJ8WauwQdIJ4urmoCrXhR1XbfCyR4BLhYvEvX7VaSt9-Fddr03mNi_CDC8_VqrKXxvidRZBSMJY-HGUD4H1ztOHK-EGtdTUEo8628wFbW_eRamaezRiEI6XW_Ct-Yes09FHvH7u85SJL6rYA Introduction In Finland dairy cows are typically fed grass si- lage with barley based supplement. Due to the fermentation type in the rumen however, this diet is not optimal if the aim is to decrease the fat content in milk without simultaneous decreas- ing the milk protein content (Huhtanen 1988, Jaakkola and Huhtanen 1993). Grass silage with barley produces generally a rumen fermentation pattern relatively high in butyrate and low in pro- pionate, which explains the high milk fat con- tent. In addition, glucose supply may be limited in dairy cows, and glucogenic amino acids may then be used for glucose production. As a result, smaller amounts of amino acids will be availa- ble for milk protein synthesis. Therefore, grass silage-based diets should be supplemented with a concentrate mixture producing more propion- ate and less butyrate in the rumen in order to increase the glucose supply to the host animal. Energy-yielding components other than car- bohydrates or their by-products are alternative sources of ingredients that could be included in the concentrate mixture. We look here at the ef- fects of glycerol and unprotected long-chain free fatty acids as dietary supplements. The use of glycerol and fatty acids separately in concentrate mixtures has commercial interest, for instance if these ingredients affect milk yield and com- position. In reported trials glycerol has been fer- mented under different conditions and therefore the proportions of the end products of rumen fermentation have also varied. Information is lacking on the effects of glycerol supplementa- tion on the performance of dairy cows fed grass silage-based diets. Glycerol in the diet could have the potential to increase the glucose sup- ply if the proportion of propionic acid is mainly increased at the expense of other acids. Glucose carbon can be supplied by propionate or any odd- carbon compounds or by glucogenic amino ac- ids (Bergman and Pell 1985). Carbon sources other than propionate include glycerol from mobilized lipids (Van Soest 1994). The addition of fat to the diet is one way of increasing energy intake in order to obtain a higher milk yield pro- vided that feed intake and/or digestibility are not much reduced. The level of supplemental fat is important since high levels cause negative effects but even moderate levels have often been report- ed to reduce milk protein content (Thomas and Chamberlain 1984, Tesfa et al. 1991a). Jenkins (1993) and Doreau and Ferlay (1995) recently showed that unprotected long-chain fatty acids in the diet interfere with the microbial ecosys- tem. Dietary fat also affects the milk fatty acid composition, e.g. by reducing the content of short- and medium-chain fatty acids and increas- ing that of long-chain fatty acids (Palmquist and Jenkins 1980, Coppock and Wilks 1991, Tesfa et al. 1991b). Because, however, dietary fat is modified within the rumen, the profile of long- chain fatty acids in milk may not be like that provided in the diet. Our objective was to study the effects of a low level of free fatty acids because high, and even moderate, levels have negative effects and also because information on the responses of a low level of fatty acids is scarce in grass silage- based diets. We studied the effects of including either glycerol or a mixture of unprotected free fatty acids, mainly C |g | and C |g 2 , or a combina- tion of glycerol and fatty acids on feed intake, milk production and composition, rumen fermen- tation, blood metabolites and diet digestibility in dairy cows fed grass silage and a concentrate mixture. Material and methods Animals, diets, and experimental design The study was conducted on 12 mid-lactating (115 ± 31 days in milk) Friesian cows, of which four were cannulated in the rumen, in their first lactation period. The cows were individually fed and milked twice a day, at 0700 and 1600. A to- tal of 7 kg of barley-based concentrate (control diet, C) was given twice a day (0800 and 1730) 350 Khalili, H. et al. Glyceroland unprotectedfreefatty acids in the diets ofdairy cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND in two equal meals of 3.5 kg each. The concen- trate (C diets) consisted (g/kg) of barley (600), oats (80), sugar beet pulp (150), soya-bean meal (95), molasses (20) and others (55). In other three diets, 36 g/kg of barley was replaced by glycer- ol (G diets) and a mixture of free fatty acids (FA diets) either alone or in combination (GFA) (36 g/kg ofglycerol and 36 g/kg offatty acids), mak- ing a total of 72 g/kg. The fatty acid composi- tion (g/kg) of the mixture of free FA (fractioned vegetable fatty acid blend) was C |6o (126), C ]go (22), C |8;1 (376), C |g.2 (274), C, 8:3 (32) and the rest was a mixture of fatty acids in triglycerides form (170). Wilted grass silage was fed ad libi- tum, and refusals were between 50 to 100 g/kg of the amount offered. Second cut silage was made from a mixture of timothy and meadow fescue, and ensiled with a formic acid based ad- ditive (5 1/1000 kg; 690 g/kg formic acid). The experiment was carried out as three balanced 4x4 Latin squares, each having 21 day periods comprising 14 days for adjustment and 7 days for data collection. Measurements and analytical procedures Milk yield and silage intake were recorded dai- ly. The results presented are based on the data on intake and milk production during the last 6 days of each experimental period. The silage dry matter (DM) content was determined by oven drying at 105°C for 24 h, and the DM content was corrected for the volatile losses according to Huida et al. (1986). Feed samples were col- lected on days 15-19 and pooled within the pe- riod. Feed samples were analysed for organic matter (OM), nitrogen (N), neutral detergent fi- bre (NDF) and HCI fat. The silage samples were also analysed for pH, water-soluble carbohy- drates, lactic acid, ammonia N and soluble N. Diet digestibility was measured using acid in- soluble ash as an internal marker (Van Keulen and Young 1977). During 5 days of each collec- tion period clean (not mixed with urine) faecal grab samples were taken from the four cannu- lated cows at 0700 and 1900, and pooled within each cow and frozen. The pooled samples were subsequently thawed and dried at 60°C and stored at room temperature for chemical analy- ses. Samples from the feeds and faeces were analysed for organic matter by ashing at 600°C for 12 h, nitrogen (Kjeldahl-N), NDF, ADF and lignin (Robertson and van Soest 1981). HCI fat was extracted with diethyl ether (AOAC 1990) after boiling for 1 h in 3 N HCI. Milk samples were taken during four consecutive milkings on days 17 and 18 and analysed for fat, protein and lactose. Milk samples were pooled within diet for analyses. The composition and amount of milk fatty acids were measured after methyla- tion (Anttila and Kankare 1983) using a Hewlett Packard (Böblingen, Germany) HP 5890 gas chromatograph on a 2 mm x 3 m glass column packed with GP 10% SP-2330 on 100/120 Cro- mosorb WAW (Supelco, Bellofonte USA) and equipped with an HP 7673 automatic injector and flame ionization detector. The column tempera- ture was initially 90°C and then programmed to 220°C at B°C/min. The split/slitless injector was used in splitless mode at 250°C and the detector also at 250°C. The carrier gas was helium at a flow rate of 40 ml/min. The fatty acid methyl esters were identified and quantitated utlizing the Nu-Check-Prep GLC-60 milk fat standard (Bast of Copenhagen, Denmark) and HP 3365 Chem- station. Blood samples were collected on day 18 before morning feeding and 3 and 6 h later from the coccygeal vein ofeach cow into 10ml EDTA tubes and placed in ice. Beta-hydroxybutyrate (BHB) was analysed from acid precipitated blood according to the method described by Työppönen and Kauppinen (1980). Plasma was separated by centrifugation and samples were analysed enzymatically using commercial kits for glucose (Boehringer Mannheim GmbH, Ger- many) and for non-esterified fatty acids (NEFA) (Wako Chemicals GmbH, Neuss, Germany; Shimizu et al. 1980). Plasma urea was analysed as describedby Gutmann and Bergmeyer (1974). Rumen fluid samples were collected from the four cannulated cows on day 19 before morning feeding and 1.5, 3, 4.5, 6, 7.5 and 9 h thereafter. pH was measured immediately. Rumen fluid 351 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND samples were first strained through cheese cloth, and then 0.5 ml of saturated mercury(ll)chloride and 2 ml of 1 M sodium hydroxide were added to 5 ml of rumen fluid. The samples were kept frozen until analysed. Before analysis a 0.5 ml sample was acidified with 0.25 ml of formic acid (Riedel-de-Haen 33015), dilutedto 5 ml with dis- tilled water and centrifuged at 2000 g for 10 min. The VFA content was measured by an HP 5890 gas chromatograph (Hewlett Packard, Avondale, USA) provided with an HP 7673 autosampler, a flame ionization detectorand an HP 3365 Chem- station. The capillary column was an HP-FFAP 10 m x 0.53 mm x 1 (im (HP, USA). The sample volume injected was 1 |il. Helium was used as a carrier gas at a flow rate of 9 ml/min. The col- umn temperature was programmed to rise from 60 to 78°C at a rate of 25°C/min, isothermally at a rate of 7.5°C/min to 100°C for 1 min and finally at a rate of 25°C/min to 180°C in the fi- nal time of 3 min. The injector was operated in the split mode (split vent flow 45 ml/min). The injector and detector temperatures were 220 and 260°C, respectively. The results were calculated by an external standard method. Ammonia N was analysed as described by McCullough (1967). The number of protozoa of pooled daily (within each cow) rumen samples were counted, after mixing with methyl green-formalin-saline solu- tion, using a haemo-cytometer. The cows were weighed on two consecutive days at the begin- ning of the experiment and on two days at the end of each period. Statistical methods Data were analysed by ANOVA for a balanced Latin square design. The model was a mixed one; y... —tt+A, + P. + FA. +G. +FA*G + f... + T + J ijlkm “ i j k I kl ijkl m A*T + P*T + FA*T. + G*T. + im jm km Im FA*G*T., + e.... ,klm ijklm’ where (t is the overall mean, A and P are the random effects of animal and period, FA, G and FA*G are the fixed effects of diets, f.„ is the ran-’ ijlk dom error term for whole-plot (mean square of the whole-plot), T is fixed effect time, A*T and P*T are the random effects of animal*time and period*time, G*T, FA*T and G*FA*T are the fixed effects of diets*time, and e.... is the ran-’ ijklm dom error term for sub-plot (mean square of the sub-plot). The random variables, A., P., f u , A*T , P*T and e... , are all assumed to be im jm ijklm independent and normally distributed with zero means and variances a2A , G 2 p , a 2 f , G 2 AT , a 2 pr and o 2 e , respectively. The effects of FA, G and FA*G on intake, milk production and composi- tion, live weight, mean values of blood metabo- lites, and mean values of rumen characteristics and digestibility were tested using the whole-plot error mean square as an error variance in the F test. The effects of FA*time, G*time and FA*G*time on the post-prandial values of blood metabolites and rumen characteristics were test- ed using the sub-plot error mean square as an error variance in the F test. Statistical signifi- cance of treatments was tested 2x2 factorially: the effect of glycerol (G and G*FA v C and FA); the effect of free fatty acids (FA and G*FA v C and G) and their respective interaction effect G*FA (C and G*FA v G and FA). Blood metab- olites and rumen fluid (pH, ammonia, VFA) data were assessed by analysis of variance for repeat- ed measurements. The analysis used the split- plot approach with Greenhouse-Geiser approxi- mate (conservative) significance tests (Littell et al. 1992). Results The chemical composition of experimental feeds is presented in Table 1. The fermentation quali- ty of silage was good. The N contents of all con- centrate mixtures were very similar. The fat con- tents were higher in FA and GFA diets owing additional free fatty acids. FA diets increased (P<0.001) milk yield de- spite reduced silage DM intake (PcO.Ol) (Table 2). However, total DM intake and ME intake were similar in all diets. FA supplementation 352 Khalili, H. et al. Glyceroland unprotectedfreefatty acids in the diets of dairy cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 1.Chemical composition of experimental feeds. Silage 1 CM 2 CM+G CM+FA CM+GFA Dry Matter (g kg 1) 288 885 888 891 895 In dry matter (g kg 1) OM 913 930 927 927 928 N 29.025.8 25.725.6 25.9 Hel fat 58.238.1 36.365.3 68.3 NDF 560 236 219 228 215 ADF 309 88.677.9 80.178.7 Lignin 34.716.2 14.516.9 14.2 Cellulose 275 72.463.4 63.264.5 Hemicellulose 250 148 141 148 136 1 In silage: pH 4.26; in DM (g kg '): water-soluble carbohydrates 48.6; lactic acid 66.3; acetic acid 18.8; propionic acid 0.08; butyric acid 0.47; isovaleric acid 0.028; ethanol 2.6; in total N(g kg'): ammonia N 58.6; soluble N 479. Cellulose digestibility of silage DM 0.752. Cellulose=ADF-lignin, Hemicel- lulose=NDF-ADF. 2 CM=concentrate mixture, CM+G=concentrate mixture inluding glycerol, CM+FA=concentrate mixture inluding unprotected free fatty acids, CM+GFA=concentrate mixture inluding both glycerol and unpro- tected free fatty acids. G=glycerol; FA=free fatty acids; GFA=glycerol and free fatty acids; OM=organic matter; NDF=neutral detergent fibre; ADF=acid detergent fibre Table 2. Effect of various supplements on feed intake, milk yield, milk composition, yield of milk constituents and live weight. Treatment Significance of effect C G FA GFA SEM G FA GxFA Feed intake (kg DM day 1) Silage 10.18 10.06 9.94 9.74 0.108 NS ** NS Supplement 6.09 6.01 6.11 6.15 0.058 Total intake 16.27 16.06 16.05 15.89 0.115 NS NS NS Silage+suppl. g/W075 148 146 147 146 1.1 NS NS NS ME intake (MJ) 187 185 189 188 1.3 NS NS NS ECM 1 yield (kg day 1) 22.52 22.08 23.07 23.19 0.206 NS *** NS Milk yield (kg day') 22.98 22.21 23.47 23.69 0.258 NS *** P=0.07 Milk composition (g kg ') fat 39.1 39.2 40.1 39.8 0,47 NS P=0.09 NS protein 31.8 32.1 31.1 31.2 0.17 NS *** NS lactose 49.7 49.4 49.6 49.9 0.12 NS P=0.09 * Yield of milk constituents (g day 1) fat 891 873 934 937 11.0 NS *** NS protein 726 714 727 740 8.0 NS P=o.lo NS lactose 1148 1124 1170 1195 12.5 NS *** P=0.06 Live weight (kg) mean 527 527 522 523 1.6 NS ** NS change (day 1) +0.424 +0.408 +0.325 +0.143 0.1238 NS NS NS Statistical significance: NS, P>o.lo not significant; * P<0.05; ** P<0.01; *** PcO.ool 1 ECM=energy corrected milk (Sjaunja et al. 1990) C=concentrate mixture (CM); G=CM+glycerol; FA=CM+fatty acids; GFA=CM+glycerol+fatty acids 353 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 3. Effect of various supplements on milk fatty acid composition (g kg 1 fat). Treatment Significance of effect C G FA GFA SEM G FA GxFA C 4 55.3 56.5 56.5 59.3 1.95 NS NS NS C 6 29.0 29.8 28.0 28.5 0.93 NS NS NS C g 16.0 16.0 14,0 14.5 0.50 NS ** NS C lo 36.0 36.3 30.0 30.8 1.04 NS *** NS C |2 41.3 43.0 35.5 35.5 0.89 NS *** NS C |4o 128.5 133.5 118.3 121.3 2.12 NS *** NS C |4’, 15.5 16.3 13.8 14.0 0.29 NS *** NS Total C 4-C 14 321.5 331.3 296.0 303.8 6.59 NS ** NS4 14 C l6o 293.5 278.8 268,8 264.3 10.28 NS P=o.lo NS C |fr, 26.0 27.8 24.8 26.0 0.61 * * NS Total Cl 6 319.5 306.5 293.5 290.3 9.80 NS P=0.07 NS C„. O 111.0 109.0 128.5 129.8 2.54 NS *** NS C,,', 218.0 222.0 252.3 246.8 4.70 NS *** NS C |g2 20.5 22.0 21.3 22.0 1.33 NS NS NS C |g3 7.5 6.8 6.5 6.3 0.20 * »* NS C2ft| 3.3 2.8 4.0 3.5 0.25 P=0.09 * NS Total C|g-C,„ 360.3 362.5 412.5 408.3 6.61 NS *** NS Statistical significance: NS, P>o.lo not significant; * P<0.05; ** P<0.01; *** Po.lo not significant; * Pc0.05; ** PcO.Ol; *** Po.lo not significant; * P<0.05; ** PcO.Ol; *** PcO.ool C=concentrate mixture (CM); G=CM+glycerol; FA=CM+fatty acids; GFA=CM+glycerol+fatty acids lowest 3 h after morning feeding compared with the values before feeding and 6 h after feeding with each diet (results not shows here). FA diets decreased (PcO.Ol) digestibilities of DM, OM and (P<0.05) NDF and tended to de- crease the digestibility ofN (P=0.09) but increase (PcO.OOl) that of ether extract fat (Table 5). Glycerol inclusion had no significant effect on digestibility. An interaction (PcO.Ol) affecting fat digestibility was observed between glycerol and FA . FA diets did not significantly affect the aver- age (seven sampling times) rumen fermentation parameters or numbers of protozoa (Table 6). In contrast, G diets altered the rumen fermentation type, the molarproportion of acetate decreasing (PcO.OOl) and the proportions of propionate, butyrate and valerate increasing (P<0.05 at least). Glycerol decreased (P<0.05) the number ofHol- otrich protozoa. There were no effects of an in- teraction between glycerol and fatty acids on rumen fermentation parameters or protozoal numbers. Postprandial changes in molarproportions of acetate, propionate and butyrate are shown in Figure 1a-c. G diets caused interactions with time for acetate and butyrate, decreasing (G*h PcO.Ol) the postprandial value of acetate and increasing (G*h PcO.OOl) that of butyrate. 355 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 6. Effect of various supplements on rumen fermentation characteristics' (four ruminally cannulated cows, mean values of seven sampling times). Treatment Significance ofeffect C G FA GFA SEM G FA GxFA pH 6.276.29 6.286.37 0.058 NS NS NS NH4 -N (mmol P 1) 10.459.02 9.489.35 0.638 NS NS NS Total VFA(mmol l 1) 120.6122.4 120.6120.4 1.74 NS NS NS Molar proportion of VFA's (mmol mol') Acetate 656 637 655 637 2.9 *** NS NS Propionate 170 176 172 178 2.3 * NS NS Isobutyrate 9.59.4 8.99.8 0.28 NS NS NS Butyrate 133 144 134 142 1.3 * NS NS Isovalerate 12.512.5 11.913.1 0.40 NS NS NS valerate 13.915.4 13.614.8 0.40 ** NS NS Caproate 6.36.5 6.66.5 0.25 NS NS NS Protozoa (xlO4 ml ') Total 19.119.2 18.317.5 1.00 NS NS NS Holotricha 1.41.0 1.81.2 0.20 * NS NS Entodiniomorph 17.718.2 16.516.3 1.17 NS NS NS Statistical significance: NS, P>o.lo not significant; * P<0.05; ** P<0.01; *** PcO.ool 1 Means of seven sampling times C=concentrate mixture (CM); G=CM+glycerol; FA=CM+fatty acids; GFA=CM+glycerol+fattyacids Discussion Effects of free fatty acids Inclusion of unprotected supplementary fats has had both beneficial and adverse effects on dairy cow feeding and production, depending on fac- tors such as the amount and type of fat given. The mixture of unprotected free fatty acids used in the present trial consisted mainly of C 18 (376 g/kg) and C |B2 (274 g/kg) fatty acids. The ef- fects of supplementary fats with very different fatty acid compositions are not therefore referred to in this paper. Feed intake, milk production and composition Replacement of barley by 36 g/kg of a mixture of free fatty acids decreased silage DM intake in the cows given FA diets. Generally, high con- centrations of supplementary fat have decreased feed DM intake but the results of many trials with supplementary fat have been less consistent (Coppock and Wilks 1991). FA supplementation increased milk yield in the trial discussed here. Such an increase has often been observed if the effect on feed intake has been slight or non-neg- ative, resulting in increased energy intake. The estimated ME intake did not increase significant- ly with FA diets and there were no differences in ME balances (intake-requirement). As shown by Coppock and Wilks (1991), FA diets tended to increase milk fat content and decrease milk protein content. A decrease in milk protein con- tent has often, but not invariably, been reported and the reasons for such a decrease are not clear. It is not easy to explain the decrease in milk pro- tein here either, because there were no changes in the concentrations of plasma glucose or ru- men ammonia. Furthermore, the amino acid sup- ply from the small intestine was estimated to be higher than thatrecommended (maintenance and milk production, ignoring live weight change) 356 Khalili, H. et al. Glyceroland unprotectedfreefatty acids in the diets ofdairy cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND (Madsen et al. 1995) in each diet.The main cause of the decrease in milk protein content here was the dilution effect due to increased milk yield. Wu and Huber (1994) attribute the decrease in milk protein concentration associated with sup- plemental fat partly to increased milk yield. Milk fatty acids It is well documented that dietary fats affect milk FA composition (Palmquist and Jenkins 1980). In the present trial, free FA in FA diets modified milk FA composition by increasing long-chain Fig. 1 (a-c). The effects of various supplements on molar proportions of acetate, propionate and butyrate. con- trol diet, glycerol, a mixture of free fatty acids and -*- combination of glycerol and free fatty acids. 357 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND FA derived from the diet. However, because the dietary supply of free fatty acids (main compo- nents in the FA mixture were C lgi| and C |g 2) is modified within the rumen, the long-chain FA profile of milk changed, increasing the propor- tions of C and C ]g.r Unsaturated free fatty acids are rapidly hydrogenated by rumen mi- crobes into more saturated fatty acids at rates depending on factors such as the nature and amount of lipids fed (Jenkins 1993). According to Doreau and Ferlay (1994), the higher the li- noleic acid concentration in the diet the greater is the extent of hydrogenation of linoleic acid. Judging by the lack of an increase in the linoleic acid in milk despite the addition of linoleic acid, this is most probably what occurred here. The hydrogenation of linoleic acid (C |g2n 6) is not complete, but provides stearic acid and differ- ent isomers of C |g-| (Tamminga and Doreau 1991). This hydrogenation was also indicated here by the increase in C |go and C |B., with FA diets. A supply of dietary fatty acids in FA diets could have reduced de novo synthesis ofC |g,0 and C |6o fatty acids by rumen bacteria (Jenkins 1993) probably because of enhanced uptake of exoge- nous lipids by bacteria (Poncet et al. 1995). Fur- thermore, a decrease in the content of C,, in milkI o with FA diets was affected by the supply of C lB fatty acids and their subsequent uptake by the mammary gland, which inhibits de novo synthe- sis of short- chain fatty acids (Palmquist and Jenkins 1980). FA diets increased the intake of free fatty acids by about 170 g/d, which increased the pro- duction of C 8 fatty acids by 62.9 g/d in milk and simultaneously slightly decreased de novo synthesis of short-chain fatty acids in the mam- mary cell. Doreau and Ferlay (1994) concluded that it is currently difficult to predict the amount of absorbable FA, since the factors controlling the synthesis and disappearance ofFA in the ru- men are not well known. Blood metabolites FA diets did not affect the blood BHB concen- tration, a finding supporting the conclusion that BHB concentrations do not usually markedly increase during fat supplementation (Grummer and Carroll 1991). As found in fat studies (Dhi- man et al. 1995, Choi and Palmquist 1996), FA diets did not have any effect on the glucose con- centration in plasma. According to Grummer and Carroll (1991), it seems likely that fat supple- mentation spares glucose from oxidation, but that the metabolic consequences are unknown. They concluded that fat supplementation does not con- sistently increase blood glucose. The plasma NEFA concentrations of lactating cows fed sup- plemental fat almost invariably increase (Grum- mer and Carroll 1991). In the present trial too, FA diets increased the NEFA concentration, which did not indicate increased lipolysis since the cows were in mid-lactation and their energy balance was positive, as shown by the positive live weight change and energy balance. An in- creased plasma NEFA concentration was proba- bly related to an increased dietary supply of ad- ditional free fatty acids rather than to net chang- es in the balance between lipolysis and lipogen- esis. Digestibility FA inclusion has been shown to reduce digesti- bilities of OM and fibre (Palmquist and Jenkins 1980, Tesfa 1992, Jenkins 1993), as was also observed in the present trial with FA diets. How- ever, free fatty acids reduced the digestibilities of OM and NDF in FA diets only slightly, partly due to the high proportion of C |g., in the mixture ofFA used and to the total amount ofFA given. Note that the mixture of free FA included poly- unsaturated FA. According to Chalupa et al. (1984), lipids rich in short-chain, medium-chain or poly-unsaturated fatty acids have a greater negative effect on carbohydrate, especially fibre, digestion than have saturated or mono-unsatu- rated long-chain fatty acids. Here, a slight de- pression in NDF digestibility might be one rea- son for the observed decrease in feed intake. Although several mechanisms may be operative in the rumen to explain the reduction in fibre digestion when fatty acids are given, Palmquist and Jenkins (1980) concluded that most data suggest an inhibitory effect on microbial activi- 358 Khalili, H. et al. Glyceroland unprotectedfree fatty acids in the diets ofdairy cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND ty. When fibre digestibility has been depressed by lipids, the fermentation type, too, has often been modified through an increased proportion of propionate in the rumen. This is probable due to a decrease in the activity of cellulolytic bac- teria. According to a recent review by Doreau and Ferlay (1995), lipid supplements decrease the protozoal counts in the rumen but this effect is very variable. They also noted that the nega- tive effect on protozoa seems to be more marked with free than with esterified fatty acids and that the strongest decrease in protozoal number has been associated with a significant drop in the ammonia concentration in the rumen. Including a low level of free FA in a silage-based diet did not affect the rumen ammonia concentration or the protozoal number in FA diets. In the present trial, FA supplementation did not affect the ru- men fermentation pattern, which is consistent with no change in protozoal numbers. In con- trast, Tesfa (1992) reported a decrease in proto- zoal number, an increase in the proportion of propionate and a decrease in that of butyrate in the rumen in response to a daily supplement of 0.5 kg rapeseed oil. Effects of glycerol In the present trial, inclusion of glycerol did not affect feed intake or milk yield and had only a slight effect on milk fatty acid composition and plasma urea concentration. Few papers have been published on the effects ofglycerol as an energy yielding substrate in dairy cow feeding. Remond et al. (1991) reported no effects on milk yield, milk composition or food intake when glycerol was given in amounts to those in the present tri- al. The ability to increase the proportion of pro- pionate in the rumen at the expense of other vol- atile fatty acids by using glycerol was investi- gated in the present trial with grass silage-based diets. The effects of glycerol on rumen fermen- tation reported elsewhere have varied due to dif- ferences in diets; and the effects have not been studied using grass silage-based diets. Wright (1969) observed increases in acetic and propi- onic acids but Remond et al. (1993) reported that glycerol increased butyrate more than it did pro- pionate at the expense of acetate, as was noticed here, too. Glycerol did not affect either blood glucose or BHB concentrations, but Remond et al. (1991) observed that glycerol increased the plasma 3-hydroxybutyrate concentration. In the present trial the increase in butyrate was not due to the pH effect since the mean value of rumen pH was not affected with G diets. It is not easy to explain why butyrate increased more than did propionate with glycerol supplementation. It is possible, however that propionate formation de- pressed when fermentation proceeds rapidly in the rumen. Our results with grass silage-based diets confirmed the observations of Remond et al. (1993) that an increase in butyrate is not ev- idence of the glucogenic ability of glycerol in dairy cows given maize silage. According to Bro- ster et al. (1970), both glucose and sucrose de- pressed the proportion of acetate and increased the proportions of propionate and butyrate when a basal diet of hay and concentrates was supple- mented with glucose or sucrose in dairy cow feeding. They reported that glucose diets in- creased butyrate more than sucrose. Similarly, when cattle were fed purified diets, glucose di- ets increased the proportion of butyrate more than that of propionate (orskov and Oltjen 1967). Glucose and sucrose supplements tended to in- crease the proportion ofpropionate and butyrate. However, these changes were not statistically significant in goats given a silage diet (Cham- berlain et al. 1985). Conclusions The low level of fatty acids used only slightly decreased silage intake and diet OM digestibili- ty. Fatty acids didnot change the rumen fermen- tation type or the ammonia concentration or pro- tozoal numbers in the rumen. On the other hand, fatty acids increased the milk yield and simulta- neously decreased the milk protein content but 359 Vol. 6 (1997): 349-362. AGRICULTURAL AND FOOD SCIENCE IN FINLAND not the protein yield. Furthermore, milk compo- sition changed and the proportions of C lgo and C |B| fatty acids in milk fat increased. Our findings suggest that the addition ofglyc- erol to the diet does not have any positive ef- fects when cows are fed grass silage-based di- ets. However, when glycerol and fatty acids are given in combination there tended to be a posi- tive effect on milk production. References Anttila, V. & Kankare, V. 1983. The fatty acid composi- tion of milk lipids. Milchwissenschaft 38: 478-481. AOAC. 1990. Official methods ofanalysis. Fat (crude) or ether extract in animal feed (920.39). Association of Official Analytical Chemists. 15th Edition. Bergman, E.N. & Pell, J.M. 1985. Intergration of amino acid metabolism in the ruminant. In: Gilchrist, F.M.C. & Mackie, R.J. (eds.). Herbivore nutrition in the sub- tropics and tropics. Johannesburg: The Science Press, p. 613-628. Broster, W.H., Sutton, J.D., Smith, T., Broster, V.J. & Balch, C.C. 1970. The effect of supplements of su- crose and of glucose monohydrate on the milk pro- duction and live weight of dairy cows. Journal of Ag- ricultural Science Cambridge 74: 217-225. Chalupa, W., Rickabaugh, 8., Kronfeld, D.S. & Sklan, D. 1984. Rumen fermentation in vitro as influenced by long-chain fatty acids. Journal of Dairy Science 67: 1439-1444. Chamberlain, D.G., Thomas, P.C., Wilson, W., Newbold, C.J. & MacDonald, J.C. 1985. The effects of carbo- hydrate supplements on ruminal concentrations of ammonia in animals given diets of grass silage. Jour- nal ofAgricultural Science Cambridge 104: 331-340. Choi, B.R. & Palmquist, D.L. 1996. High tat diets increase plasma cholecystokinin and pancreatic polypeptide, and decrease plasma insulin and feed intake in lac- tating cows. Journal of Nutrition 126: 2913-2919. Coppock, C.E. & Wilks, D.L. 1991. Supplemental fat in high-energy rations for lactating cows: effects on in- take, digestion, milk yield, and composition. Journal of Animal Science 69: 3826-3837. Dhiman, T.R., Klaas, V.Z. & Sätter, L.D. 1995. Effect of dietary fat source on fatty acid composition of cow’s milk. Journalof the Science of Food and Agriculture 69: 101-107. Doreau, M. & Ferlay, A. 1994.Digestion and utilisation of fatty acids by ruminants. Animal Feed Science and Technology45: 379-396. - & Ferlay, A. 1995. Effect of dietary lipids on nitrogen metabolism in the rumen: a review. Livestock Pro- duction Science 43: 97-110. Grummer, R.R. & Carroll, D.J. 1991. Effects of dietary fat on metabolic disorders and reproductive perform- ance of dairy cattle. Journal of Animal Science 69: 3838-3852. Gutmann, I. & Bergmeyer, H.U. 1974. Determination of urea with glutamate dehydrogenase as indicator en- zyme. In: Bergmeyer, H.U. (eds.). Methods of enzy- matic analysis. 2nd English ed. Vol 4. Academic Press, New York. p. 1794-1798. Huhtanen, P. 1988. The effects of supplementationof si- lage diet with barley, unmolassed sugar beet pulp and molasses on organic matter, nitrogen and fibre digestion in the rumen of cattle. 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AGRICULTURAL AND FOOD SCIENCE IN FINLAND SELOSTUS Väkirehuun lisätyn glyserolin tai vapaiden rasvahappojen tai näiden yhdistelmän vaikutus maidontuotantoon ja pötsifermentaatioon ruokittaessa lypsylehmiä säilörehuun perustuvalla ruokinnalla Hannele Khalili, Tuomo Varvikko, Vesa Toivonen, Kari Hissa ja Marjatta Suvitie Maatalouden tutkimuskeskus ja Suomen Rehu Oy Tutkimuksessa selvitettiin lypsylehmille annettavaan väkirehuun lisätyn glyserolin tai suojaamattomien va- paiden rasvahappojen tai näiden yhdistelmän vaiku- tuksia rehun syöntiin, maidontuotantoon, maidon koostumukseen, pötsifermentaatioon, veriarvoihin ja ruokinnan sulavuuteen. Koejärjestelyssä mukana ol- leita 12 friisiläislehmääruokittiin säilörehuun perus- tuvallaruokinnalla. Koeruokintoja oli neljä. Kontrol- liruokinnassa lehmille annettiin säilörehua ja perus- väkirehua. Säilörehua lehmät saivat vapaasti ja perus- väkirehua 7 kiloa päivässä. Kilogrammassa perusvä- kirehua oli 600 g ohraa, 80 g kauraa, 150 g leikettä, 95 g soijarouhetta, 20 g melassia ja muita 55 g. Kah- dessa koeväkirehussa 36 g/kg ohraa oli korvattu joko glyserolilla tai rasvahapposeoksella. Yhdessä koevä- kirehussa 72 g/kg ohraa oli korvattu glyserolin (36 g/kg) ja rasvahappojen (36 g/kg) yhdistelmällä. Ras- vahappolisä koostui palmitiini- C |6o (126 g/kg), stea- riini- C, g;o (22 g/kg), öljy- C |g;| (376 g/kg), linoli- C |g2 (274 g/kg), linoleenihaposta C |g .3 (32 g/kg) ja vapai- ta rasvahappoja triglyseridimuodossa (170 g/kg). Rasvahapot heikensivät ruokinnan sulavuutta ja säilörehun syöntiä. Rasvahapot eivät kuitenkaan muuttaneet pötsifermentaatiotyyppiä, pötsin ammo- niakkipitoisuutta eikä alkueläinten lukumäärää pöt- sissä. Maitotuotos lisääntyi ja samalla maidon val- kuaispitoisuus pieneni hieman, mutta valkuaistuotos ei vähentynyt. Maidon rasvahappokoostumus parani siten, että steariini- ja öljyhapon suhteelliset osuudet lisääntyivät. Glyseroli lisä muutti pötsifermentaatiotyyppiä vä- hentäen etikkahapon suhteellista osuutta ja lisäten propioni- ja voihapon osuuksia. Muutokset fermen- taatiossa olivat kuitenkin niin pieniä, etteivät beta- hydroksivoihapon ja glukoosin pitoisuudet veressä, maidontuotanto eikä maidon koostumus muuttuneet. Tämän kokeen tulosten perusteella glyserolilisäykses- tä ei ollut hyötyä ruokittaessa lypsylehmiä säilöre- huun perustuvalla ruokinnalla. Kuitenkin on todetta- va, että maidontuotanto oli suurinta annettaessa gly- serolia ja rasvahappoja yhdessä. 362 Khalili, H. et al. Glyceroland unprotectedfreefatty acids in the diets ofdairy cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND