Effects of and interactions between the extent of silage fermentation and protein supplementation in lactating dairy cows Terttu Heikkilä, Vesa Toivonen and Pekka Huhtanen Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-3 1600 Jokioinen, Finland, e -mail: terttu.heikkila@mtt.fi Twelve Ayrshire cows were used to study the effects of and interactions between extent of silage fermentation and level of protein supplementation on silage intake and milk production. Experimen- tal design was a cyclic change-over with six dietary treatments, with two replicate blocks of six cows and four 3-week periods. Dietary treatments in a 2 x 3 factorial arrangement consisted of two wilted silages treated with either a formic acid-based (FA) 5 1/t or a bacterial inoculant additive (I) and three levels of protein supplementation obtained by fish meal (EM) inclusion (0, 60 and 120 g/kg concen- trate). Grass silage was given ad libitum and concentrates were offered at a rate of 10 kg/d. Both silages were well-preserved but I-silage was more extensively fermented than FA-silage. Feeding FA-silage decreased diet organic matter digestibility, particularly that ofneutral detergent fibre, com- pared with I-silage. Inclusion ofFM increased the digestibility of all dietary constituents. Dry matter (DM) intake of I-silage was lower than that ofFA-silage but milk yield was not significantly affected. Restricting silage fermentation increased milk fat content, fat yield and energy corrected milk yield. FM inclusion increased silage DM intake, milk yield, milk protein content and yield. FM 120 inclusion increased milk fat yield more with FA-silage than with I-silage, whereas the response in milk protein yield was greater with I-silage. Plasma glucose, plasma urea and milk ureaconcentrations were lower and blood (3-hydroxybutyrate higher in cows given FA-diets than those given I-diets. Key words: bacterial inoculant, digestibility, fish meal, formic acid, milk composition, milk produc- tion, silage intake, wilted silage ntroduction Extent of silage fermentation can be modified by additives and wilting. Restriction of fermen- tation can be achieved by using formic acid or other acids at high levels ofapplication. Fermen- tation is stimulated by inoculants of lactic acid bacteria, enzymes or by fermentable carbohy- drates added during ensiling. Wilting herbage generally reduces the extent offermentation (van Vuuren et al. 1995). Modifications in the chem- ical composition during ensilage can alter the supply of nutrients from the digestive tract both quantitatively and qualitatively, which affect both milk yield and composition (Chamberlain © Agricultural and Food Science in Finland Manuscript received September 1997 329 Vol. 7(1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND and Choung 1993, van Vuuren et al. 1995).Lac- tic acid in silage increases the molarproportion of propionate in the rumen, whereas water sol- uble carbohydrates (WSC) in silage increase lipogenic volatile fatty acids (VFA, Jaakkola et al. 1991, 1993, Robertson et al. 1993, Martin et al. 1994). Silage fermentation acids have limit- ed value as an energy source for rumen microbes (Chamberlain 1987),and increased fermentation in the silo has resulted in a reduced efficiency of rumen microbial protein synthesis (Jaakkola et al. 1991, 1993, Robertson et al. 1993). In many of our studies where enzymes or enzymes and bacterial inoculants have been used for direct cut material, extensive fermentation has markedly reduced silage DM intake, and consequently production (e.g. Heikkilä et al. 1989, 1993) compared with formic acid-treated silages. The purpose of this study was to inves- tigate the effectiveness of an inoculant additive in wilted silage compared with formic acid and, in addition study interactions between the ex- tent of silage fermentation and protein supple- mentation, since the protein value of extensive- ly fermented silages have been shown to be less than thatof restrictively fermented silages (Jaak- kola et al. 1991, 1993). Different levels of fish meal were used as a protein supplement. Material and methods Silages Silages were made on 7 June 1993 from the pri- mary growth of a timothy (Phleum pratense) and meadow fescue (Festuca pratensis) sward, ferti- lized with 90-18-36 kg N-P-K/ha. Grass was cut with a mower-conditioner and harvested, after wilting for 4-6 h, using a precision-chop forage harvester. Grass was ensiled in two roofed bun- ker silos of 50 t capacity with either a formic acid-based additive (FA, 800 g/kg formic acid and 20 g/kgorthophosphoric acid) at 5 1/t or with a bacterial inoculant (I), which was a mixture of Lactobacillus rhamnosus and Propionibacteri- um freudenreichii ssp. shermanii (Valio Ltd), at 5 x 106 colony forming units/g grass. During harvesting, temperature, wind and relative hu- midity were between 11-17°C, 3.1-5.7 m/s and 45-74%, respectively. Silos were opened after 266 days. Aerobic stability of silages was as- sessed by loosely packing duplicate 5 kg sam- ples, with four repetitions per treatment, in pol- ythene-lined styrox boxes (40 x 28 x 28 cm, 2.5 cm thick) fitted with partly open lids. Silage boxes were incubated for 10 days at 16°C. Tem- perature was measured twice daily. Animals and their management Twelve high-yielding Finnish Ayrshire cows in their 2nd-4th lactation were used. Cows had calved 48 days (SE 4.5) before the start of the experiment and their average daily milk yield was 36.5 kg (SE 0.4). Cows were housed in in- dividual stalls. Grass silages were offered ad li- bitum ensuring a refusal of at least 50 g/kg in- take. Concentrate mixtures were given at a rate of 10 kg/d throughout the experiment three times daily at 0100, 1300and 1630. Cows were milked twice daily at 0645 and 1530. Animals were weighed on two consecutive days at the begin- ning of the experiment and at the end of each period. Experimental design The study was conducted according to a cyclic change-over design with six treatments in a 2 x 3 factorial arrangement, with two replicate blocks of six cows and four 3-week experimen- tal periods (Davis and Hall 1969). Treatments consisted of two silages (FA-treated and I-treat- ed) each fed with three concentrate supplements differing in their crude protein content (CP. 129, 169, and 211 g/kg DM). The basal concentrate (FM () ) contained (g/kg) barley (382), oats (382), molassed sugar beet pulp (200) and mineral mix- 330 Heikkilä, T. et al. Extent of silage fermentation andprotein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND ture (36), of which either 60 (FM 6(| ) or 120 (FM 120 ) g/kg was replaced with fish meal (FM). Experimental procedures, chemical and statistical analyses Feed intake and milk yield of individual cows were recorded daily. The results of the last sev- en days of each period were used for statistical analysis, and feed samples were collected dur- ing this period for chemical analysis. Fresh si- lage samples were stored frozen at -20°C prior to analysis. Milk samples, in proportion to yield, were taken on four consecutive milkings on the last week of each period. Diet digestibility was determinedusing acid insoluble ash (AIA) as an internal marker. Faecal samples were taken from all cows twice daily at 0730 and 1600. Blood samples were taken prior to feeding and 4 h af- ter feeding on the last day of each period from the coccygeal vessel of each cow. Each sample was analysed for glucose, (i-hydroxybutyrate (BHB) and urea. Chemical analyses of milk, feed and faecal samples were made as described previously by Huhtanen and Heikkilä (1996). Silage ethanol content was determined according to Huida (1982). Blood samples were treated and analysed as described by Miettinen and Huhtanen (1997). Intake of metabolizable energy (ME) was estimated from organic matter (OM) intake and digestibility determined using AIA as a marker and assuming a ME content of 16 MJ/kg digest- ible OM (MAFF 1984). ME contents of silages were also estimated from D-values (digestible organic matter in DM) determined in three sheep fed at a maintenance level. ME of concentrates were derived from chemical composition and digestibility coefficients documented in Finnish feed tables (Tuori et al. 1996). Utilisation ofME for milk production was calculated ignoring the effects of live weight change. The supply of amino acids absorbed from the small intestine (AAT) was calculated based on feed table val- ues (Tuori et al. 1996) for each feed or feed in- gredient. Data was analysed using the general linear model directive of the Statistical Analysis Sys- tem (SAS Institute Inc. 1989). The model includ- ed block, cow(block), period, treatment and car- ry-over. Results for production parameters are adjusted for carry-over effects. Data of one cow was removed due to mastitis and gastrointesti- nal disorders. Sum of squares of the treatment effect was further separated into orthogonal com- parisons of the effects of silage type (FA vs. I), linear and quadratic effects of the protein con- tent of concentrates (FM inclusion) and corre- sponding interactions. Results Both silages were well-preserved as indicated by low concentrations ofVFA and ammonia N (Ta- ble 1). Propionate was not found in I-silage, al- though the inoculant contained Propionibacte- rium. Inoculatedsilage was more fermented than FA-silage in terms of a lower pH and higher lac- tic acid content. Lactate to acetate ratio was high- er in I-silage than in FA-silage (28 vs. 2.7) indi- cating the fermentation being almost homofer- mentative. Neutral detergent fibre (NDF) con- tent was slightly higher in I-treated silage com- pared with FA-treated silage. The aerobic sta- bility of FA-silage compared with I-silage was better as indicated by a slower temperature in- crease (Figure I). The intake ofsilage DM was higher (P<0.05) in cows given FA-treated silage than in those given I-treated silage (Table 2). Increasing die- tary CP content by FM inclusion increased si- lage DM intake linearly (P<0.01). However, be- cause the amount of concentrate refusals in- creased at the same time, differences in total DM intake only approached significance (P=0.09). Silage type had no effect on calculated ME in- take, whereas both ME and AAT supply in- creased linearly (at least P<0.01) with supple- ment protein content. 331 Vol. 7 (1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 1.Chemical composition (g/kg dry matter) and calculated feeding values of experimental feeds. Silages Supplements FA-treated I-treated FM (| FM N| FM |20 Dry matter(g/kg) 313 322 889 889 889 pH 4.34 3.85 ND ND ND In dry matter Ash 77 75 65 66 69 Crude protein 160 157 129 169 211 Ether extract 43 57 33 37 39 NDF 1 458 477 278 285 291 ADF2 259 260 103 102 97 WSC3 160 61 ND ND ND Lactic acid 35 132 ND ND ND Acetic acid 13.2 4.8 ND ND ND Butyric acid 0.2 0.1 ND ND ND Isovaleric acid 0.7 0.3 ND ND ND Ethanol 6.8 5.2 ND ND ND In total N (g/kg) Ammonia N 38 38 ND ND ND Soluble N 635 680 ND ND ND ME4 (MJ/kg DM) 11.4 11.4 12.6 12.7 12.7 AAT 5 (g/kg DM) 87 85 101 116 130 PBV6 (g/kg DM) 12 II -35 -16 5 FA = formic acid, I = inoculant, FM = fish meal, ND = not determined. 1 Neutral detergent fibre. 2 Acid detergent fibre. 3 Water soluble carbohydrates. 4 Calculated from D-value determined in sheep for silage and from feed table digestibility coefficients for concentrates (Tuori et al. 1996). 5 Amino acids absorbed from the small intestine 6 Protein balance value. Silage type had no significant effect on milk yield or feed efficiency (Table 3). However, due to the higher (PcO.OOl) milk fat content in cows receiving FA-treated silage compared with those given I-silage, yields of fat (P<0.01) and energy corrected milk (ECM, P=0.051) were higher. Milk protein content and yield were not signifi- cantly influencedby the type of silage. Milk lac- tose content was lower (PcO.OOl) withFA-silage than with I-silage resulting in a higher (P=0.054) lactose yield in favour of I-silage. The response to FM depended on the fermen- tation of silage, indicated by a silage x fish meal interaction (P<0.05) in most production param- Figure 1.Effects of formic acid and inoculant additives on aerobic stability of silages. 332 Heikkilä, T. et al. Extent ofsilage fermentation and protein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 2. Feed intake and calculated nutrient intake of cows receiving formic acid-treated (FA) or inoculated (I) silages supplemented with different levels of fish meal (FM). Supplement FM0 FM WJ FM, 2O Significance of effect 1 SEM Silage FA I FA 1 FA 1 Silage FM hn Silage x Silage x FM,, FM .lm quad Intake (kg DM/d) Silage 13.01 12.41 13.26 12.80 13.78 13.25 0.237 * ** NS NS NS Concentrate 8.858.99 8.738.81 8.338.66 Total 21.86 21,40 21.9921.61 22.1121.91 0.159 * o NS NS NS Crude protein (kg/d) 3.32 3.27 3.70 3.66 4.05 4,06 0.032 NS *** NS NS NS NDF2 (kg/d) 7.79 7.83 7.97 8.14 8.05 8.21 0.093 NS �* NS NS NS ME (MJ/d)3 237 237 241 241 242 248 2.2 NS ** NS NS NS AAT (g/d) 4 2009 1976 2070 2042 2175 2174 12.7 o *** o NS NS 1 Significance: NS (P>0.10), o (P<0.10), * (P<0.05), ** (P<0.01), *** (PcO.001) 2 Neutral detergent fibre 3 Calculated from the intake of digestible OM determined in cows usingAIA as an internal marker 4 Calculated using for all feeds EPD-values from feed tables (Tuori et al. 1996) Table 3. Milk production, live weight and feed efficiency of cowsreceiving formic acid-treated (FA) or inoculated (I) silages supplemented with different levels of fish meal (FM). Supplement FM0 FMm FM 120 Significance of effect 1 SEM Silage FA I FA I FA I Silage FMta Silage x Silage x FM, FM „lin quad Milk (kg/d) 33.3 32.8 31.9 33.7 34.3 34.0 0.37 NS * * NS * ECM 2 (kg/d) 36.0 34,9 35.0 35.2 38.1 36.1 0.50 o * * * NS Milk composition (g/kg) Fat 46.2 44.9 46.7 43.1 48.3 44.0 0.87 *** NS NS * * Protein 32.2 31.5 33.7 32.7 33.1 33.4 0.33 NS ** o NS NS Lactose 50.3 50.7 49.5 50.8 49.7 50.0 0.19 *** ** NS NS ** Milk urea (mmol/1) 2.96 3.56 4.08 4.66 4.88 5.81 0.100 *** *** NS NS NS Milk constituents (g/d) Fat 1530 1468 1492 1437 1652 1491 33.3 ** o o ** NS Protein 1071 1033 1071 1094 1131 1133 10.6 NS *** NS NS o Lactose 1676 1665 1580 1719 1704 1700 22.3 o NS NS NS ** Feed efficiency ECM(kg/kgDM) 1.64 1.63 1.59 1.63 1.72 1.65 0.026 NS NS o NS NS Milk protein/CP intake (g/kg) 323 317 290 298 282 279 4.0 NS *** o NS o Live weight Mean (kg) 590 583 590 591 593 591 2.2 NS * NS NS o change (kg/d) -0.01 -0.33 -0.070.24 0.530.56 0.164 NS *** NS NS NS 1 Significance: NS (P>0.10), o (P<0.10), * (P<0,05), ** (PcO.Ol), *** (P<0.001) 2 ECM = energy corrected milk (Sjaunja et al. 1990). 3 CP = crude protein 333 Vol. 7 (1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 4. Milk fatty acid composition (g/kg fatty acids) in cows receiving formic acid-treated (FA) or inoculated (I) silages supplemented with different levels of fish meal (FM). Supplement FM, FM, FM,a Significance of effect'V). 60 SEM Silage FA I FA I FA I Silage FMlin Silage x Silage x FM, FM .Iin quad C 4 57 55 55 51 57 C 6 31 31 31 30 31 C 8 17 18 18 18 18 ClO 37 41 40 42 38 Cl 2 42 47 46 49 44 C 133 141 141 143 13514 C , 18 19 18 19 1814:1 C 4 C l 4 | total 335 352 349 352 338 Cl 6 322 314 295 317 313322 314 295 317 313 Cl6;| 24 24 25 24 24 Clgo 106 102 106 100 101 Clg:1 180 176 184 170 182 C 18;2 23 22 24 22 24 54 1.2 * NS * NS NS 31 0.6 NS NS NS NS NS 18 0.4 NS NS NS NS NS 44 1.3 ** NS NS NS NS 52 1.6 *** NS NS NS NS 149 2.3 *** NS NS NS o 19 0.3 ** NS NS NS NS 368 6.1 ** NS NS NS o 306 7.0 NS NS NS NS * 25 0.5 NS NS NS NS NS 90 2.7 * * NS NS NS 169 5.4 * NS NS NS NS 22 0.7 ** NS NS NS NS 1 Significance: NS (P>0.10), o (P<0.10), * (P<0.05), ** (P<0.01), *** (PcO.OOl) Table 5.Whole tract nutrient digestibility of formic acid-treated (FA) or inoculated (I) silage based diets supplemented with different levels of fish meal (FM). Supplement FM 0 FM m FM |20 Significance of effect' ” SEM Silage FA I FA 1 FA I Silage FMlm FM . Silage x Silage x FM, FM .jm quad Dry matter 0.716 0.732 0.725 0.736 0.726 0.750 0.0035 *** *** NS NS o Organic matter 0.729 0.745 0.740 0.749 0.742 0.765 0.0035 *** *** NS NS o Crude protein 0.676 0,693 0.699 0,719 0.715 0.747 0.0060 *** *** NS NS NS Neutral detergent 0.575 0.621 0.618 0.649 0.623 0.671 0.0083 *** *** NS NS NS fibre 1 Significance: NS (P>0.10), o (P<0.10), * (P<0.05), ** (PcO.OI), *** (P<0,001) Table 6. Concentrations of blood metabolites (mmol/1) ofcows receiving formic acid-treated (FA) or inoculated (I) silages supplemented with different levels of fish meal (FM). Supplement FM n FM W) FM | ,(| Significance of effect 1 SEM Silage FA I FA I FA I Silage FM|in Silage x Silage x FM,. FM ,nn quad Plasma Glucose 2.77 3.27 2.95 3.09 2.86 3.14 0.090 *** NS NS NS NS Urea 3.063.53 4.284.71 5.115.69 0.212 o *** NS NS NS Blood BHB: 1.270.83 1.390.94 1.371.02 0.113 *** NS NS NS NS 1 Significance; NS (P>0.10), o (PcO.10), * (P<0.05), ** (PcO.Ol), *** (PcO.OOl) 2 P- hydroxybutyrate 334 Heikkilä, T. et al. Extent ofsilage fermentation and protein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 7. Utilisation of ME and amino acids absorbed from the small intestine (AAT) of cows receiving formic acid-treated (FA) or inoculated (I) silages supplemented with different levels of fish meal (FM). Supplement FM0 FM W) FMpf| Significance of effect 1 SEM Silage FA I FA I FA I Silage FM hn FM quad Silage x Silage x FM, FM . lm quad Milk energy (ME/d) 113.0 109.5 109.9 110.7 119.5 113.3 1.6 * ** NS o NS Utilisation of ME 0.645 0.624 0,612 0.618 0.664 0.608 0.010 * NS o ** NS Utilisation of AAT 0.6600.651 0.6370.660 0.6380.634 0.007 NS * NS NS o AAT g/kg ECM2 45.145.7 48.347.1 47.049.6 0.71 NS ** NS NS o 1 Significance: NS (P>0.10), o (P<0.10), * (P<0.05), ** (P<0.01), *** (P<0.001). 2 ECM = energy corrected milk (Sjaunja et al. 1990). eters. FM supplementation increased linearly milk yield (P<0.05), milk protein content (PcO.Ol) and yield (PcO.001). The efficiency of the utilizationof dietary CP for milk protein pro- duction decreased with the level of FM. Protein supplementation (FM0 to FM |20 ) tended to in- crease milk protein yield more with I-silage than with FA-silage (100 vs. 60 g/d), whereas it in- creased milk fat yield more with FA-silage than 1-silage (122 vs. 23 g/d). The effect of silage type on milk fat content also depended on the level of FM supplementation (interactions P<0.05); differences between FA- and I-silages increased from 1.3 g/kg (FM 0 ) to 3.6 (FM 60 ) and 4.3 g/kg (FM 120 ). FM decreased (P<0.01) milk lactose content but as a result of increased milk yield, FM had no effect on lactose yield. The response in lactose yield to FM was slightly better with I- silage than with FA-silage, especially between the two lowest levels ofFM. Milk urea content was higher (P<0.001) with 1-silage than with FA- silage and increased linearly (PcO.001) with FM supplementation. Live weight gain was not sig- nificantly different between silages but FM in- creased it linearly and there was a tendency for a greater increase with I-silage (interaction P=0.115). Differences in milk fatty acid composition were small, although in many cases statistically significant (Table 4). Of the short chain fatty acids, the proportion of butyric acid was higher (P<0.05) in cows given FA-silage compared with those given I-silage while the reverse was true for C |o C l 4 fatty acids. FA-silage significantly (at least P<0.05) increased the proportions ofall C |B fatty acids. FM had very little effect on milk fatty acid composition and neither did it modify differences between silage types. Apparent digestibilities of OM, N and NDF were all significantly higher (P<0.001) with I- silage compared with FA-silage (Table 5). The difference was largest in NDF digestibility (0.605 vs. 0.647). Inclusion of FM significantly (PcO.001) increased digestibility of all dietary components. Plasma glucose concentration was signifi- cantly higher (PcO.001) and that of blood BHB lower (P<0.001) with I-silage compared with FA- silage (Table 6). FM had no effect on either plas- ma glucose or blood BFIB but plasma urea con- tent increased linearly (PcO.001) with the level ofFM inclusion. Plasma urea content tended to be higher (P<0.10) with I-silage than FA-silage. Milk energy yield was higher (P<0.05) with FA-silage than with I-silage (Table 7) and in- creased linearly (P<0.01) with the level of pro- tein supplementation. Efficiency ofutilization of energy for milk production averaged 0.628 ig- 335 Vol. 7(1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND noring the effects of live weight change. It was significantly (P<0.05) higher in cows given FA- silage compared with those receiving I-silage (0.640 vs. 0.617). Differences between the si- lages in ME utilisation increased with protein supplementation (interaction P<0.01). Both con- version ofAAT for ECM production and utilisa- tion of AAT for milk protein production de- creased (at least P<0.05) with the level of pro- tein supplementation. Discussion Effects of silage fermentation Due to wilting coupled with high WSC content (158 g/kg DM), it would be expected that the material was relatively easy to ensile. Formic acid restricted silage fermentation efficiently as indicated by high residual WSC and lower lac- tic acid contents compared with I-treated silage. Very high lactate to acetate ratio of the I-treated silage suggests that the inoculant dominated fer- mentation over epiphytic lactic acid bacteria which are often more heterofermentative. For- mic acid inhibits the growth of Lactobacilli in the initial phase of fermentation (Chamberlain and Quig 1987) as was also shown with the present silages by Rauramaa et al. (1996). A low- er lactate to acetate ratio in FA-silage compared with I-silage indicated heterofermentative char- acteristics of surviving epiphytic lactic acid bac- teria. The higher acetate concentration in FA-si- lage than in I-silage is in accord with previous data (Gordon 1989, Mayne 1993). The absence of propionic acid indicates that Propionibacte- rium was not active in the acidic conditions of I- silage. The effects of formic acid on aerobic stabil- ity of silage have been inconsistent. The better aerobic stability ofFA-silage compared with in- oculated silage is in agreement with results of Mayne (1993) and Keady and Murphy (1996, 1997), who used lower levels ofFA application. In contrast, Keady and Murphy (1997) found reduced aerobic stability with a high level ofFA. In agreement with the study of Huhtanen et al. (1997b), in which digestibility of diets based on the same silages was determined by the total collection method in dairy cows, feeding I-diets improved digestibility compared with FA-diets. Differences in OM digestibility can almost en- tirely be attributed to the lower NDF digestibil- ity ofFA-silage. Also in the study of Keady and Murphy (1997), NDF digestibility was decreased with high levels ofFA application compared with inoculated silage. On the other hand, Gordon (1989) working with sheep reported that the D- value of inoculated silage was higher compared to that of untreated and FA-treated silage in the absence of differences in silage fermentation. The reason for the lower digestibility ofFA-di- ets is not clear. There were no differences in ru- men pH (Huhtanen et al. 1997b) which could explain differences in NDF digestibility. The lower NDF digestibility of FA-diets may be at- tributed to a high WSC content, which may de- press cellulolytic activity in the rumen. Intraru- minal sugar infusions have depressed ruminal fibre digestibility without affecting rumen pH (Rooke et al. 1987, Huhtanen 1987), whereas lactic acid had no effect on fibre digestion (Jaak- kola and Huhtanen 1992). However, an in vitro study with continuous fermentors (Nousiainen et al. 1996) suggested that FA treatment can also depress fibre digestibility when silage soluble fractions were removed by washing. Lower NDF content of FA-silage and as high WSC content in silage as in grass before ensiling indicate hy- drolysis ofmore readily digestible hemicellulose in the silo, such that the residue might be ex- pected to be of a lower digestibility. However, assuming a similar NDF content in FA-silage to that in I-silage would only slightly reduce dif- ferences in NDF digestibility (from 0.605 vs. 0.647 to 0.618 vs. 0.647). Restricted fermentation of FA-silage in- creased silage DM intake by 0.53 kg/d compared with I-silage. No significant difference in silage DM intake was observed between wilted FA- treated and enzyme/inoculant-treated silages 336 Heikkilä, T. et al. Extent ofsilage fermentation and protein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 7 (1998): 329 when the difference in the extent of the fermen- tation between the silages was smaller than in the present study (Jaakkola et al. 1996). The de- crease in silage DM intake in the present study can be attributed to the higher lactic acid con- centration since other fermentation parameters were not affected. The adverse effect of silage lactic acid on intake is in line with observations of Thomas et al. (1980) and Choung and Cham- berlain (1993). However, our study and that of Choung and Chamberlain (1993) do not support the hypothesis of Thomas et al. (1980) that the adverse effect oflactic acid on silage DM intake can be overcome by feeding protein supplements, for silage intake increased similarly with FA- and I-silage with increasing FM. When extensively fermented unwilted silages had higher lactic acid content or also contained more ammonia N and VFA than their formic acid treated counterpart, the difference in silage DM intake was greater than in the present study (Heikkilä et al. 1989, 1991, 1993). However, fermentation character- istics do not always explain differences in silage DM intake. For example, Gordon (1989) and Mayne (1990) reported higher DM intakes with inoculated silage than with untreated or FA-si- lage in the absence of marked changes in fer- mentation pattern. Our results suggest that si- lage DM intake is limited by at least two fac- tors, lactic acid and ‘a protein responsive factor’ (Figure 2). An increase in silage DM intake re- sults from effects of FM on the protein respon- sive factor while lactic acid continues to con- strain the intake of I-silage below that of FA- silage. Increased NDF digestibility with FM in- clusion suggest that the mechanism by which protein responsive factor increases intake, is in- creased rate of digestion in the rumen. Howev- er, the higher NDF digestibility of dietbased on I-silage suggest that the lactic acid constraint is related to some other factors. When the AAT in- take was corrected for the lower efficiency of microbial synthesis with I-silage than with FA- silage (Huhtanen et al. 1997), the relationship between AAT content of the diet and silage DM intake was very close. This suggests that the mechanism of the lactic acid constraint is a re- -343. duced amino acid to energy ratio at tissue level. There were no significant differences in milk yield between the formic acid and inoculated silages, which is in agreement with other exper- iments (Chamberlain et al. 1992, Heikkilä et al. 1991, Mayne 1993, Jaakkola et al. 1996, Keady and Murphy 1996). However, Gordon (1989) and Mayne (1990) reported a higher milk yield in cows given inoculated silage than in those given FA-treated silage, even when differences in si- lage fermentation were small. It should, howev- er, be noted that in their studies the rate of for- mic acid application was 2.3-3.4 1/t, whereas in Finnish studies the application rate has been 4- 5 1/t. in agreement with the literature review of van Vuuren et al. (1995) restricting the extent of si- Figure 2. Relationship between uncorrected (above) and corrected dietary AAT content and silage DM intake. Si- lage DM intake was corrected for differences in concen- trate intake assuming a substitution rate of0.5. 337 AGRICULTURAL AND FOOD SCIENCE IN FINLAND lage fermentation by using formic acid increased milk fat content. Higher milk fat content in cows fed FA-silages can be attributed to the higher ratio of lipogenic (acetate and butyrate) to glu- cogenic (propionate) VFA in the rumen (Cham- berlain and Choung 1993, van Vuuren et al. 1995). This ratio was markedly higher with FA- silage than with I-silage (4.6 vs. 3.6) when the same silages used in the present study were used in another study (Huhtanen et al. 1997b). Silage lactic acid increases the proportion of propion- ate in rumen VFA (Robertson et al. 1993, Mar- tin et al. 1994, van Vuuren et al. 1995), whereas silage WSC increase either acetate (Martin et al. 1994, Huhtanen et al. 1997b) or butyrate (Jaak- kola et al. 1991, 1993, Smith et al. 1993). Slightly higher milk protein content (P=0.12) in cows given FA-treated silage than in those given I-silage is consistent with many other stud- ies examining the effects of silage fermentation on milk production (Heikkilä et al. 1989, 1991, Chamberlain et al. 1992, Smith et al. 1993). Higher milk protein content with restrictively compared with extensively fermented silage can partly be attributed to increased silage DM in- take and partly to increased protein supply from higher rumen microbial protein synthesis with FA-treated silage (see van Vuuren et al. 1995). With the present silages, microbial protein pro- duction estimated from urinary excretion of pu- rine derivatives was about 10% higher with FA- silage than with I-silage (Huhtanen et al. 1997b). Lower lactose content in milk with FA-treat- ed silage may be attributed to the higher lipo- genic to glucogenic ratio in rumen VFA. Increas- ing the proportion of butyrate in VFA-mixture infused into the rumen has consistently decreased milk lactose content (Miettinen and Huhtanen 1996, Huhtanen et al. 1997a). However, differ- ences in milk lactose content have been small, although statistically significant, and are proba- bly of minor practical importance. Differences in milk fatty acid composition between the silages were generally small. High- er proportions of short-chain fatty acids with inoculated silage than with FA-silage are large- ly similar to those reported by Chamberlain et al. (1992) between restrictively and extensively fermented silages fed with a barley supplement. In the present study FA treatment increased the proportions of all C lg fatty acids compared with I-treatment. In agreement, Chamberlain et al. (1992) reported higher proportions of stearic acid with FA-treated silage, but in their study oleic or linoleic acid were not affected by the extent of silage fermentation. Higher proportions of oleic and linoleic acid in milk fat in this study suggest that the fatty acids of grass were less influenced by ensiling in FA-treated silage than in I-silage. Lower plasma glucose and higher blood BHB in cows fed on FA-diets than in those fed on I- diets is in accord with the data of Smith et al. (1993), Mayne (1993), Miettinen and Huhtanen (1997), and can be attributed to differences in rumen fermentation pattern. The supply of the most important glucose precursor, propionate, was markedly lower from FA-diets than from I- diets as indicated by the lower molarproportion of propionate (Huhtanen et al. 1997b). These observations suggest that with restrictively fer- mented silages the supply of glucose precursors is less than with extensively fermented silages. Lower milk lactose content is in line with this hypothesis. Since feeding of FA-silage was not associated with reduced feed intake, rather vice versa, it is not likely that the increase in blood BHB resulted from endogenous ketogenesis. A more likely explanation is a greater proportion of lipogenic VFA in the rumen with FA-silage compared with I-silage. Increased milk and plas- ma urea content may be explained by increased absorption of ammonia N from the rumen with I-diet compared with FA-diet as indicated by the decrease in the efficiency of microbial protein synthesis (Huhtanen et al. 1997b). Also lower N digestibility with FA-diets may partly explain the lower milk urea content with FA-treated silage. Effects of protein supplementation The response to FM supplementation was rela- tively small. The mean response of 0.11 g ofmilk protein/g increase in CP intake was smaller than the corresponding response reported by Cham- 338 Heikkilä, T. et al. Extent ofsilage fermentation and protein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND berlain et al. (1989) for cows given FM supple- ments. Also in our recent study with rapeseed meal supplementation (Huhtanen and Heikkilä 1996) better marginal responses to protein were obtained than in the present study. The small response to protein supplementation despite high production level in the present study may be re- lated to the high quality offorages used and con- sequently, to efficient microbialprotein produc- tion in the rumen (FA- vs. I-silage, 22.7 vs. 20.3 g microbial N/kg DOM). Reduced milk lactose content in cows given FM supplementation is in agreement with the data ofBroderick (1992) and Huhtanen (1993). In contrast to earlier studies (Chamberlain et al. 1989, Huhtanen 1993), FM supplementation did not improve the utilisation of ME for milk production. This may be due to the high k ; value (0.635) with the basal diet without FM in the present study, whereas in the other studies the corresponding kl values were considerably low- er than the value of 0.62 proposed for the effi- ciency of ME utilisation for milk production. Therefore there was less scope to improve ME utilisation by protein supplementation. Interactions between the silage fermenta- tion and protein supplementation Production responses to FM supplementation tended to depend on the silage fermentation char- acteristics. The reasons for higher increases in milk fat content and yield withFA-silage than I- silage in response to FM supplementation are not clear. ECM yield tended to increase more and utilisation of ME for milk production improved more (0.019 vs. -0.016) in cows given FA-si- lage than in those given I-silage. This observa- tion is in accord with the suggestion of Cham- berlain and Choung (1993) that, with restrictively fermented silages, the responses to protein sup- plementation are more related to repartitioning between milk and body tissues than to increased silage DM intake. However, changes in blood BHB and milk fatty acid composition with in- creasing FM do not indicate any major differ- ences between silages in mobilisation of body tissues with increasing level ofFM. Repartition- ing may have occurred simply by less deposi- tion of body tissue. Milk protein yield responses to FM ]2() inclu- sion tended to be greater with I-silage than with FA-silage (100 vs. 60 g/d). This supports the hypothesis of increased supply of amino acids from restrictively fermented silage compared with high lactate silage. The efficiency of mi- crobial protein synthesis was higher with restric- tively fermented silage compared with high lac- tate silage (Huhtanen et al. 1997b), and there- fore less benefits may be expected from protein supplementation with restrictively fermented si- lage. The supply of glucose from ruminal propi- onate production is smaller with restrictively fermented silages than with high lactate silages as indicated by the lower plasma glucose con- centration in the present study. Greater glucose supply from silage lactate may reduce utilisa- tion of amino acids for gluconeogenesis, and more amino acids may be available for milk pro- tein synthesis. Reduced milk protein yield when propionate was replaced isoenergetically with butyrate (Miettinen and Huhtanen, 1996, Huh- tanen et al. 1997a) indicate that differences in ruminal fermentationpattern affect utilisationof dietary amino acids for milk protein synthesis. Propionate has also increased nitrogen retention in sheep (orskov et al. 1979). The lack of re- sponse to the first level ofFM with FA-silage is unclear. It would seem that, at the zero level of FM, there is no shortage of glucose precursors with FA-silage because, at the same ME intake as with I-silage, it supports a higher milk pro- tein yield. It may be that the supply of glucose precursors becomes limiting as FM inclusion begins, and the response to FM 60 is reduced cor- respondingly. Unbalanced amino acid composition of mi- crobial protein is another alternative explanation for limited effects of protein supplementation of restrictively fermented silage on milk protein yield. In this context, it is noteworthy that, with the same silages used in another experiment that allowed a comparison of the response to restric- tion of silage fermentation with the response to 339 Vol. 7(1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND postruminal infusion of casein, whereas both treatments increased plasma concentrations of lysine and branched-chain amino acids, but only casein infusion increased plasma concentrations of histidine (Miettinen and Huhtanen 1997) and milk protein yield (Huhtanen et al 1997b). With silage based diets histidine appears to be the first limiting amino-acid for milk protein production (Vanhatalo et al. 1997). With FA-silage a great- er proportion ofsupplementary protein was prob- ably used for gluconeogenesis than with I-silage (see Miettinen and Huhtanen 1997). It would appear that in our study the better protein value of restrictively fermented silage was partly off- set by increased utilisation of amino acids for gluconeogenesis. In other studies more distinct differences in the response to protein supplementation with different forages have been found. Choung and Chamberlain (1992) reported a significantly greater response to postruminal casein infusion with untreated silage than with enzyme-treated silage. Similarly, FM supplementation increased milk protein yield more with lucerne silage than with lucerne hay (Broderick 1995). Conclusions In the conditions of the present study, both for- mic acid and bacterial inoculant produced si- lages ofgood fermentation quality. FA-treatment decreased diet digestibility and especially that of NDF compared with inoculated silage. Feed- ing more extensively fermented I-silage de- creased silage DM intake, but had no effects on milk or milk protein yields. Milk fat content and fat yield were higher with FA-silage than with I- silage. Responses to protein supplementation would seem to be different between the silages. From the results of the present study and other studies conducted with the same silages it can be concluded that restriction of silage fermenta- tion increases the supply of lipogenic VFA and amino acids. High lactate silages provide more glucose precursors from rumen fermentation but are more limited in amino acids than restrictive- ly fermented silages. Since glucose supply was lower with FA-diets, improved protein value of restrictively fermented silage may not have been completely realized in terms of milk protein yield. The results of the present trial suggest that optimal supplementation of grass silage may depend on the extent of silage fermentation, but more work is needed to fully understand inter- actions between silage fermentation and concen- trate supplementation to optimise supplementa- tion of different silage types. More work is also needed to understand the mechanisms affecting cell wall digestibility of silages. Acknowledgements. 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Extent ofsilage fermentation and protein supplementation in cows AGRICULTURAL AND FOOD SCIENCE IN FINLAND SELOSTUS Säilörehun käymisasteen ja valkuaistäydennyksen vaikutus maidon tuotantoon Terttu Heikkilä, Vesa Toivonen jaPekka Huhtanen Maatalouden tutkimuskeskus Tutkimuksessa selvitettiin säilörehun käymisasteen ja väkirehun valkuaistason vaikutusta säilörehun syön- tiin, maidon tuotantoon, maidon koostumukseen, re- huannoksen sulavuuteen ja veriarvoihin. Kokeessa oli 12 Ayrshire-lehmää ja kuusi koeruokintaa. Tutkitta- vina oli kaksi esikuivattua säilörehua, jotka säilöttiin bakteeriympillä (Lactobacillus rhamnosus ja Propio- nibacterium freudenreichii ssp. shermanii), 5 x 106 pmy/g ruohoa (ymppi) tai AIV 2:11 a 5 1/tn (happo) sekä kolme väkirehun valkuaispitoisuutta (raakaval- kuaista 129, 169, 211 g/kg ka), jotka valmistettiin korvaamalla kalajauholla 0, 60 ja 120 g/kg kaura- ohra-melassileike-kivennäisseoksesta. Säilörehua an- nettiin vapaasti ja väkirehua 10 kg/pv. Molemmat säilörehut olivat laadultaan hyviä. Ymppirehu oli huomattavasti enemmän käynyttä kuin happorehu, mitä osoitti suurempi maitohappo- (132 vs 35 g/kg ka) ja pienempi sokeripitoisuus (61 vs 160). Happorehuannoksen orgaanisen aineen, raaka- valkuaisen ja neutraalidetergenttikuidun sulavuus oli huonompi kuin ymppirehuannoksen. Kalajauho lisä- si rehuannoksen sulavuutta. Lehmät söivät happore- hua enemmän kuin ymppirehua (13.35 vs 12.82 kg ka/pv), mutta maitotuotoksissa ei ollut eroa (33.2 vs 33.5 kg/pv). Energiakorjattu maito- (36.4 vs. 35.4 kg/ pv) ja rasvatuotos (1558 vs 1465 g/pv) olivat happo- rehulla suuremmat kuin ymppirehulla, sillä happore- hulla tuotetun maidon rasvapitoisuus oli suurempi kuin ymppirehulla tuotetun(47.1 vs 44.0 g/kg). Mai- don valkuaispitoisuudessa ja -tuotoksessa ei ollut eroa rehujen välillä (33.0 vs 32.5 g/kg ja 1091 vs 1087 g/ pv). Kalajauho lisäsi säilörehun syöntiä, maito- ja valkuaistuotosta sekä maidon valkuaispitoisuutta. Valkuaislisän vaikutus riippui säilörehun käymisas- teesta. Runsaan kilon (1.2 kg) kalajauholisä lisäsi ras- vatuotosta enemmän happo- kuin ymppirehulla (122 vs 23 g/pv), kun taas valkuaistuotos nousi enemmän ymppirehulla (100 vs 60 g/pv). Happorehulla ruoki- tun lehmän plasman glukoosi- sekä maidon ja veren ureapitoisuudet olivat pienemmät ja veren P-hydrok- sivoihappopitoisuus oli suurempi kuin ymppirehulla ruokitun lehmän. Tulokset viittaavat siihen, että op- timitäydennys riippuu säilörehun käymisasteesta, mutta lisätutkimuksia vaaditaan säilörehun käymis- asteen ja väkirehutäydennyksen välisten yhdysvaiku- tusten sekä säilörehun kuidun sulatukseen vaikutta- vien tekijöiden selvittämiseksi. 343 Vol. 7 (1998): 329-343. AGRICULTURAL AND FOOD SCIENCE IN FINLAND