THE NUTRITIONAL VALUE OF MULBERRY LEAVES AND THEIR USE AS SUPPLEMENT TO GROWING SHEEP FED AMMONIATED RICE STRAW Liu, J.X.1*, Jun Yao1, Yan, B.1 J.Q.Yu2, Z.Q.Shi1 and X.Q.Wang1 1 College of Animal Sciences, Zhejiang University (Huajiachi Campus), Hangzhou, China 2 Animal Production and Health Station, Tongxiang, Zhejiang, China INTRODUCTION The use of crop residues as basal diets for fattening cattle and lambs has been promoted in China during the last decade with a lot of success (Dolberg and Finlayson 1995; Guo and Yang 1996). Farmers still generally use as supplement high levels of concentrates, including cereal grains and oilseed meals. It is important to find alternative supplements (Preston, 1995). With growing lambs, Liu et al. (1998) observed that the growth rate was dramatically increased when the ammonia bicarbonate rice straw (ABRS) diet was supplemented with small amount of rape seed meal (RSM), and that the benefits of ammoniation were least when high level of RSM was used. Sericulture, based on mulberry (M. alba) leaves, is an important farming activity in China, with over 106ha planted in China and 105ha in Zhejiang Province. Yield of fresh leaves is in the order of 15 to 22 t/ha/year. Silkworm production is not always profitable, since depends on silk price and world trade. Alternative ways of using mulberry foliage would be welcomed by farmers when income from sericulture is low. Mulberry leaves are relished by sheep and goats and have high nutritive value with protein content of about 20 % of dry matter (DM) (FAO, 1998). Roothaert (1999) observed that dairy heifers had higher voluntary intake, and thus higher potential of milk production, when consuming mulberry fodder than with cassava tree (Manihot glaziovii) and leucaena (Leucaena diversifolia). Mulberry leaves could be considered an appropriate supplement for sheep fed a basal diet of ammoniated straw, replacing partially or totally the oilseed meals, which could then be used in monogastric diets. However, there is little information on this subject. The objectives of the present study were: (1) to evaluate the nutritional value of mulberry leaves collected from clones at different stages of maturity (Experiment1); and (2) to determine the effect of mulberry leaves as supplement, substituting RSM, to growing lambs fed ABRS (Experiment2). The results of Experiment1 has been published in Livestock Research for Rural Development (Yao et al, 2000). MATERIALS AND METHODS Experiment 1. Evaluation of the nutritional value of mulberry leaves. Sampling of mulberry leaves. Mulberry clones were: Tuantou Heyebai (TH), Husang No.9 (HS), Tongxiangqing (TX) and Nongsang No.8 (NS). Leaves were sampled six times in 1998, three in the spring: 28 April, 14 May and 29 May; and three in autumn: 28 August, 30 September and 30 October. Samples were taken in the morning, weighed immediately and oven-dried at 650C. The sub-samples were milled to pass a 2mm-sieve for further nutritional evaluation. Nutritional evaluation All samples were analysed for crude (CP) and true protein (TP) following AOAC (1990); for neutral detergent fibre (NDF) of Van Soest et al. (1992). Amino acid (AA) contents were determined using an AA analyser (Knauer, Germany). The nutritional value of mulberry leaves was evaluated with in vitro gas production (GP) of Menke and Steingass (1988) with calibrated glass syringes (Model Fortuna, Häberle Labortechnik, Lonsee-Ettlenschie, Germany). Samples for GP determination were ground with a hammer mill to pass a 1mm-screen. About 200mg (DM) samples were introduced into syringes with rumen liquor collected from two rumen fistulated Huzhou sheep, fed an ammoniated rice straw diet (75% ABRS, 5% RSM and 25% concentrate mixture). The GP data were then fitted to the equation GP=a+b(1-exp(- ct)) (Ørskov, 1985), where a, b and c are constants and GP is the gas production from the substrate at time t. Statistical analysis Results for each season were analysed according to a 3 x 4 factorial design. The difference of means was tested using Duncan's new multiple range test (Steel and Torrie, 1980). Experiment 2. Effect of mulberry leaves to replace rapeseed meal on performance of growing sheep fed ammoniated rice straw. Experimental feeds The ABRS was prepared by the ‘stack method’. Fertiliser grade ammonium bicarbonate (17% N) was used and the weight proportion of straw:bicarbonate:water was 100:10:30 (Liu et al., 1991). The straw was treated under the ambient temperature (15-200C) for 30 days. Treated straw was exposed to the air for a maximum 24h before feeding it to allow free ammonia to escape. Mulberry leaves (M. alba) were collected at Tongxiang Silkwork Breeding Farm. All leaves were harvested in Autumn (late October) and air-dried before storage. Animals and design Forty five growing lambs of the Huzhou breed (a local prolific breed) with initial weight of 16-18kg, were divided into 5 equal groups, considering sex and weight, and randomly allocated to the following treatments: A: 100g RSM B: 75g RSM + 60g mulberry leaves C: 50g RSM + 120g mulberry leaves D: 25g RSM + 180g mulberry leaves E: 240g mulberry leaves Within each group lambs were housed in three pens (male, females and mixed). Supplementation with RSM at 100 g/d was based on the result in a previous study (Liu et al., 1998). All five supplementary treatments were calculated to have similar CP content. All animals had ABRS, mineralized salt block and water ad libitum and 100g/head/d of ground corn. Feeding trial The experimental period lasted 75d (15d for adaptation). The lambs were weighed, before the morning feeding, every 15d.Feed intake was recorded daily. Feed samples were periodically taken for CP and NDF analysis. The ruminal degradation of DM and CP for RSM and mulberry leaves was measured using the nylon bag technique of Ørskov (1985) as by Liu et al. (1997). Data of disappearance rate were fitted to the model of Ørskov (1985): p=a+b(1-exp(-ct)), where p is the disappearance rate at time t (hr), a is the rapidly digestible fraction in the rumen, and b is the fraction slowly digested at rate c (c>0). In vitro gas production test GP, as described above, was measured to compare the nutritional value of different diets. In order to analyze the associate effect on GP parameters, only the mixtures of RSM and mulberry leaves at different ratio were also incubated. The proportions of both supplements for different combinations (PA, PB, PC, PD and PE) were based on their ratios in the corresponding diets (A, B, C, D and E) in feeding trial. Statistical analysis The results were analyzed by one way analysis of variance. The difference of means for the five treatments was tested by using Duncan's new multiple range test (Steel and Torrie, 1980). RESULTS AND DISCUSSION Composition of mulberry leaves The chemical composition of mulberry leaves is shown in Table 1.There were no significant differences (P>0.05) among mulberry clones in DM, CP, TP and NDF (for spring leaves). Only the clone Tuantou Heyebai had lower NDF (P<0.05) in the autumn. Sampling time (maturation stage) had great effects on chemical compositions. For spring leaves, contents of CP and TP were slightly higher at mid stage than at early or late stage, whereas the CP content of autumn leaves decreased significantly (P<0.05) with maturation stage. The NDF content increased with maturation stage (P<0.05) in both seasons. Table 1. Chemical composition of mulberry leaves in spring and autumn ===================================================================== Sample Dry Crude True NDF Matter Protein Protein (%DM) (%) (%DM) (%CP) --------------------------------------------------------------------- Spring leaves: Date 28 April 98 25.1 21.1ab 87.5 34.6Bc 14 May 98 25.2 21.9a 90.4 38.9Aa 29 May 98 26.6 20.0b 86.3 42.9Aa Clone Tuantou Heyebai 24.4 21.6 86.7 39.5 Husang No. 9 24.1 20.9 89.6 37.5 Tongxiangqing 23.7 20.9 88.3 39.7 Nongsang No. 8 23.6 20.8 88.5 38.5 SE 0.10 0.17 1.16 0.59 Autumn leaves Date 28 August 98 25.9C 22.3A 86.9 36.7Bc 30 September 98 29.9B 21.4A 85.7 40.4Bb 30 October 98 33.8 18.9B 84.7 47.2Aa Clone Tuantou Heyebai 30.4 21.9 84.4 38.9b Husang No. 9 29.7 20.3 86.4 40.8ab Tongxiangqing 29.6 19.6 87.5 42.6a Nongsang No. 8 29.8 21.7 84.9 43.4a SE 0.42 0.23 0.57 0.46 --------------------------------------------------------------------- A,B,C Means with different superscripts within mulberry trains or sampling times differ (P<0.01) a,b,c Means with different superscripts within mulberry trains or sampling times differ (P<0.05) Little seasonal differences were found in contents of CP and TP of mulberry leaves. Average CP contents were 21.1 and 20.9% of DM, and the TP accounted for 88.2 and 85.8% of CP in spring and autumn, respectively. However the NDF content was lower for mulberry leaves in spring (38.8%DM) than for those in autumn (41.4%). Excepting few amino acids, no significant difference (P>0.05) was observed in individual AA content among clones (data no included). There were little differences in total, essential or non-essential AA among clones. AA content tended to increase with the time, but the differences showed no statistical significance. Nutritional value of mulberry leaves The nutritional value of mulberry leaves based on the GP test is presented in Table 2. In spring, clone Nongsang No.8 had higher nutritive value (P<0.05). There was little difference between clones in the GP24 and potential GP for autumn leaves, though rate of GP was slightly higher for clones Nongsang and Tongxiangqing. The estimated organic matter digestibility (OMD) (Menke and Steingass, 1988) showed a similar tendency as GP parameters (Table 2). Similar to CP content (Table 1), mid-spring samples tended to have a higher GP (P<0.05) than early or late sampling, while for autumn leaves the GP in late season was lower (P<0.05), with little difference between those at early and middle season. Table 2. In vitro gas production (GP) parameters and estimated organic matter digestibility (OMD) of mulberry leaves ======================================================================= Sample GP at Potential Rate of GP OMD (%) 24h (ml) GP (ml) (%h-1) ----------------------------------------------------------------------- Spring leaves: Date 28 April 98 43.7ab 47.8ab 9.70 69.2ab 14 May 98 46.9a 52.2a 8.79 71.3a 29 May 98 38.5b 43.3b 9.25ab 65.6b Clone Tuantou Heyebai 41.9ab 45.7ab 7.26 69.1ab Husang No. 9 43.1ab 47.4ab 9.02 68.8ab Tongxiangqing 39.6b 43.9b 8.90 65.9b Nongsang No. 8 47.6a 52.7a 9.50 71.9a SE 0.94 1.04 0.11 0.74 Autumn leaves Date 28 August 98 32.5a 38.7 7.15A 61.4Aa 30 September 98 31.7ab 38.3 6.69AB 60.3ABa 30 October 98 28.7b 35.4 6.18B 56.3Bb Clone Tuantou Heyebai 31.0 37.8 6.57ab 60.0 Husang No. 9 30.8 38.0 6.16b 58.8 Tongxiangqing 30.8 36.7 7.01a 58.4 Nongsang No. 8 31.3 37.4 6.95a 60.1 SE 0.55 0.60 0.11 0.47 ----------------------------------------------------------------------- A,B,C Means with different superscripts within mulberry trains or sampling times differ (P<0.01) a,b,c Means with different superscripts within mulberry trains or sampling times differ (P<0.05) In General the nutritional value of spring leaves was much higher than autumn leaves. The spring mulberry leaves with OMD of 65.6~71.3% are comparable to some leguminous hays such as alfalfa and vetch (FAO, 1998). According to the farmers’ practice, the twigs of mulberry trees must be cut and modified in late May or early June in order assure autumn leaves for the silkworm. These twigs and leaves may be dried and stored for winter use. Dry matter intake CP and NDF contents for mulberry leaves used in feeding trial were 23.0 and 43.7% DM, and the corresponding values for RSM were 42.3 and 51.1% DM, and for ABRS, 13.3 and 63.9% DM, respectively. The rumen degradation results are shown in Table 3. The rates of disappearance of DM were higher for mulberry leaves than for RSM, but those of CP were lower. CP fractions degrading rapidly (a) or slowly (b) were much lower for mulberry leaves than for RSM. Rumen escape protein was higher in mulberry leaves than in RSM. Table 3. Constants of dry matter and crude protein of the equation p=a+b(1-exp(-ct)) together with 48h rumen degradability (D48) of the rapeseed meal and mulberry leaves ======================================================================= Rapeseed meal Mulberry leaves ----------------- ----------------- Dry Crude Dry Crude matter protein matter protein ----------------------------------------------------------------------- a (%) 19.2 32.6 20.5 19.5 b (%) 47.9 67.4 53.9 50.2 c (%/hr) 2.70 2.07 3.10 2.57 se 2.07 2.50 0.65 0.84 a+b (%) 67.1 86.5 74.4 69.7 EDa with passage rate at 2.00 %/hr 46.7 66.9 53.3 47.7 4.00 %/hr 38.5 55.6 44.0 39.1 D48 (%) 54.5 76.2 62.1 54.9 ----------------------------------------------------------------------- a ED = effective degradability Animals consumed all rapeseed meal, ground corn and mulberry leaves offered. The intake of ABRS was slightly increased when supplementing with of mulberry leaves, and hence total intake increased with the increases in mulberry leaves (Table 4). Total DM intakes were 451, 455, 495, 540 and 590 g/d for lambs on diets A, B, C, D and E, respectively. Basal ammoniated straw accounted for more than 50% of total diets in all treatments, and intake from forage exceeded 85 % of total diets when the RSM was fully substituted by mulberry leaves (diet E). Intake of the basal diet is usually decreased due to a substitution effect when forage is supplemented to a straw-based diet. Tharmaraj et al. (1989) observed a decline in the DM intake of both ammoniated and untreated RS when supplemented with Gliricidia, a leguminous tree. Liu et al. (1997) found that inclusion of milk vetch silage at a level higher than 23 % of diets reduced the intake of ammoniated rice straw by growing heifers. In the present study the mulberry leaves accounted for 11, 21, 29 and 35 % of dietary DM intake in diets B, C, D and E, but the ABRS intake was even slightly increased with the rising levels of mulberry leaves. Table 4. Dry matter intake and growth performance of lambs offered different diets. ======================================================================= Parameter Experimental diets (g Mulberry : g RSM) ----------------------------------- 0:100 60:75 120:50 180:25 240:0 ----------------------------------------------------------------------- No. of animals (head) 9 9 9 9 9 Feed intake (g DM/d) Rapeseed meal 88 66 44 22 0 Mulberry leave 0 52.3 104.5 156.6 209.0 Ground corn 86 86 86 86 86 Ammoniated rice straw 277.1 250.5 260.6 275.8 295.6 Total intake 451.1 454.8 495.1 540.4 590.6 Liveweight Initial weight (kg) 18.0 17.7 17.8 16.2 18.4 0.5 Growth rate (g/d) 584 a 478 b 406 c 465 b 553 a 1 Feed efficiency (kg/kg) Total intake/gain 7.78 9.67 12.37 11.75 10.74 Concentrate/gain 3.00 3.23 3.25 2.35 1.56 Feed cost/kg gain (Yuan) # 4.56 5.39 6.25 5.38 4.47 ----------------------------------------------------------------------- # Price (Yuan/kg): Ammoniated rice straw 0.20; RSM 1.20; corn 1.20, mulberry leaves 0.40 1 USD = 8.25 Yuan. a,b,c Means with different superscripts differ (P<0.05). Performance of lambs Lamb growth rates are shown in Table 4. The growth rate in diet with only RSM was comparable to that obtained in a previous trial (63g/d) by Liu et al. (1998), who supplemented ABRS diet with 100g RSM and 100g rice bran. The animals supplemented with mulberry leaves only grew the same as those with RSM, but the growth rates were lower (P<0.05) when both supplements were given together. There was not difference among sex and groupings. Among the limited information on mulberry leaves as animal feeds, Leng (1997) mentioned that dairy cows achieved up to 18 liters of milk/day on forage supplemented with fresh mulberry foliage. In his perspective, the production rate on high intakes of tree foliages such as mulberry may be as good as those of cattle on ammoniated straw and supplemented with 1-1.5 kg/d of cottonseed meal. While feed efficiency was higher when receiving only RSM, concentrate consumption per kg of weight gain was lower when higher level of mulberry leaves was supplemented. Compared to other treatments, feed cost per kg gain was lower in the diets with only RSM or mulberry, the lowest in the latter one. When mulberry leaves was used as the supplement to substitute for RSM, there was a benefit of Yuan 0.09 /day (1 US$=8.25 Yuan), equivalent to Yuan 9.00 for the usual fattening period of 100d. Relationship between growth performance of lambs and diet GP The results of in vitro GP test for different diets are summarized in Table 5. The GP for diets with low levels of mulberry (B and C) was lower than other diets. The potential GP (a+b) was significantly higher in diet with only RSM (P<0.05). The GP48 value showed a similar trend to the potential GP, although the difference between diets was not statistically significant. These results suggest a negative associative effect between rapeseed and mulberry leaves. Table 5. Parameters of in vitro gas production (GP) for different experimental diets ======================================================================= Parameters Experimental diets SE (g Mulberry : g RSM) ----------------------------------------- 0:100 60:75 120:50 180:25 240:0 ----------------------------------------------------------------------- a (ml) 2.5 3.0 2.0 1.7 2.9 0.3 b (ml) 36.4 33.3 32.9 33.6 34.4 3.6 a+b, ml 38.9 a 36.3 bc 34.5 c 35.6 bc 37.7 ab 1.4 Rate of GP, %/h 3.56 4.01 4.38 5.33 4.43 0.07 GP48, ml 31.2 30.0 29.4 31.5 31.5 4.5 ----------------------------------------------------------------------- a,b,c Means with different superscripts differ (P<0.05). Table 6 presents the result of GP test when only mixtures of mulberry leaves and RSM were incubated. The potential GP and rate of GP for mulberry leaves (PE) were higher than for RSM (PA), but the GP parameters was not improved proportionally when mulberry leaves were increased (PB and PC). There was apparently negative associative effect between mulberry leaves and RSM where the estimated GP values were much lower than the measured ones. This may partially account for the growth rate of lambs offered different diets (Table 4). There may exist some secondary plant compounds in mulberry leaves that exert a detrimental effects on ruminal microbes or reduce the utilization of the dietary nutrients (Leng, 1997). Further study is needed to clarify these aspects. Table 6. Parameters of in vitro gas production of mixtures of mulberry leaves and rapeseed meal ======================================================================= Treatment PA PB PC PD PE Mulberry:RSM 0:100 41:59 68:32 86:14 100:0 ----------------------------------------------------------------------- GP parameters a(ml) 3.3 2.9 2.7 1.1 -0.2 b(ml) 29.1 29.5 32.4 38.0 41.7 a+b(ml) 32.4 32.4 35.1 40.1 41.5 c(%/h) 4.22 4.41 5.92 6.15 6.10 GP48 (ml) 27.7 28.3 32.7 37.3 39.2 ----------------------------------------------------------------------- IMPLICATIONS Mulberry leaves have a high nutritional, in spring higher than in autumn. When used as supplement to ammoniated rice straw diet, mulberry berry may fully substitute for rapeseed meal, but attention should be paid to the negative associate effect between rapeseed meal and mulberry leaves when supplemented together. The benefits resulting from supplementation with mulberry leaves included an increased intake of basal diet, less consumption of concentrate and an increased income. However the growth rate of lambs on the ammoniated straw diets in the present study were not very high regardless of the supplement. The one of the reasons may be that the straw intakes were not high. Further study is needed to investigate the response to the increasing percentage of mulberry leaves in diets for lambs. ACKNOWLEDGEMENTS This work was funded by the International Foundation for Science (Grant Agreement No: B/2720-1). The authors express their sincere gratitude to T.R. Preston and Rene Sansoucy for their kind advise, to Dr. C.F. Lou for his permission to sample the mulberry leaves from his experimental trees, and to Ms.Jinmei Zhou for her assistance of mulberry leaves sampling. 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