J Yao, B Yan, X Q Wang and J X Liu
College of Animal Sciences, Zhejiang University (Huajiachi Campus),
Hangzhou 310029, P.R.Chinajunyao@mail.hz.zj.cn or jxliu@email.com
Abstract
The leaves from four mulberry (Morus alba) strains cultivated in China were sampled at three stages of maturity in spring and autumn, respectively. Their nutritional value was evaluated in terms of chemical composition, amino acid (AA) content and in vitro gas production (GP). Excepting one strain which was slightly inferior to other strains, there was no great difference in chemical composition between different strains regardless of season. There was little seasonal difference in content of crude protein (CP) and true protein (TP) of mulberry leaves. Average CP contents were 21.1 and 20.9 (% in DM), and TP accounted for 88.2 and 85.8% of CP in spring and autumn, respectively. Content of neutral detergent fiber (NDF) was higher in autumn (41.1%) than in spring (38.8%). Except for a few amino acids, no difference (P>0.05) was observed in AA contents among the four strains. Total and essential amino acids were 70.0 to 81.6 and 24.7 to 31.6% of the CP for spring leaves, and 75.6 to 78.0 and 29.0 to 30.8% of the CP for autumn leaves, respectively. Potential GP and organic matter digestibility estimated from GP were 43.3 to 52.2 ml and 65.6 to 71.3% for spring leaves, and 35.4 to 38.7 ml and 56.3 to 61.4% for autumn leaves, respectively, indicating that the nutritional value of spring leaves is superior to that in autumn. Chemical composition of mulberry leaves was influenced by stage of maturuty, though the extent of the effect was different between spring and autumn leaves. For spring leaves, content of CP and TP was superior at mid-stage to that in early or late stage, whereas the CP content in autumn decreased significantly (P<0 .05="" acids="" amino="" and="" content="" differences="" essential="" increase="" increased="" individual="" maturity.="" maturity="" ndf="" non-essential="" not="" of="" regardless="" season.="" significant="" stage="" tended="" the="" though="" to="" total="" were="" with="">0.05). Parameters of in vitro GP showed a similar trend to the CP content, suggesting that the nutritional value of mulberry leaves was higher at mid-stage in spring and at early stage in autumn. From these results, it is inferred that mulberry leaves, with their high protein and low fibre content and their high digestibility, may be used as supplementary protein source for ruminants.Key words: mulberry leaves, chemical composition, amino acids, in vitro gas production
Introduction
Great progress has been made in China in utilization of crop residues to develop ruminant production during the last decade (Guo and Yang 1996; Dolberg and Finlayson 1995). However, a large amount of roughage remains underutilized and current efforts are still directed at increasing straw utilization in animal feeding. Through the projects supported by FAO and the Government of China, the use of rice straw as basal diet for fattening lambs and steers has been promoted in our region, while generally farmer practice is still the use of high levels of concentrate mixture including cereal grains and oilseed meals. Therefore it is important to find adequate types and levels of supplements to overcome the dependence on conventional feeds much of which are imported (Preston 1995; Liu 1995).Due to their high nutritive value, and positive effects on rumen function, microbial yields and body metabolism, tree foliage is being increasing recognized as a potentially high quality feed resource for ruminants, particularly to supply crude protein (Leng 1997). The developing countries such as China have little land for ruminant production per se, as crop production for human consumption is given the first priority. It is inconceivable that large tracts of land could be set aside for fodder tree planting. The animal production system should be integrated with the whole agricultural system.
Silkworm cultivation on the leaves of the mulberry tree (Morus alba) is an important farming activity in southeast China, with over 106 and 105 ha planted in China and Zhejiang Province, respectively. The biomass yield of fresh leaves is of the order of 15 to 22 tonnes/ha/year. Silkworm production is not always profitable, as it is dependent upon the price of silk and world market trends in this commodity. Alternative ways of using the foliage of mulberry tree would be welcomed by farmers for the occasions when income from silkworm is low. The leaves of mulberry are relished by sheep and goats and have high nutritive value with protein content at about 20 % of dry matter (FAO 1998). They could be appropriate protein supplements for sheep fed a basal diet of ammoniated rice straw as partial or total replacement for oilseed meals, which could then be used in the diets for mono-gastric animals. However, little information is available on the nutritional values of mulberry leaves as animal feed.
The present study was conducted to evaluate the nutritive value of mulberry leaves, which were sampled from different strains at different stages of maturity. Chemical composition and amino acid contents were analyzed, and in vitro gas production (GP) was measured, as indices of nutritional value.
Materials and Methods
Sampling of mulberry leaves
Mulberry strains used included Tuantou Heyebai (TH), Husang No.9 (HS), Tongxiangqing (TX) and Nongsang No.8 (NS), which are the main mulberry strains in China. Mulberry leaves were sampled at the Experimental Farm, Zhejiang Agricultural University. Samples of mulberry leaves were taken in two seasons: spring and autumn. Mulberry leaves were sampled three times in every season: 28 April, 14 May and 29 May 1998 in spring, and 28 August, 30 September and 30 October in autumn, respectively. The different sampling interval in two seasons was due to the availability of the mulberry leaves, which is dependent on the silkworm cultivation activity. The sampling at different times within one season was conducted in one mulberry tree of each strain. Samples taken in the morning were weighed immediately, and oven-dried at 65ºC. The sub-samples were ground to pass a sieve size of 2mm for chemical analysis and further GP test.Chemical analysis
All samples were analysed for dry matter (DM). Content of crude protein (CP) was determined using the macro-Kjeldahl method (AOAC 1990). True protein (TP) was analysed according to the method of Ning (1993). Neutral detergent fibre (NDF) was analysed as outlined by Van Soest et al (1992). Amino acid (AA) contents were determined using an AA analyser (Knauer, Germany).In vitro gas production studies
The GP was measured using the technique of Menke et al (1979), and Menke and Steingass (1988), with calibrated glass syringes (Model Fortuna, Häberle Labortechnik, Lonsee-Ettlenschie, Germany) of 32 mm internal diameter and 100 ml volume. Samples for GP determination were ground with a hammer mill to pass a 1 mm screen. About 200 mg DM of sample was introduced into a syringe with rumen liquor collected from two rumen-fistulated Huzhou sheep, that were fed on an ammoniated rice straw (ARS) diet (75% ARS, 5% Rape seed meal and 25% concentrate mixture) at 1.3 times maintenance. Following the introduction of the sample in the syringe pre-warmed to 39ºC, 30 ml of the rumen liquor and buffer solution were added, and the syringes were placed in a water bath at 39ºC. The GP was then recorded after 2, 4, 6, 9, 12, 24, 36, 48, 72 and 96 h incubation.The GP data were then fitted to the equation GP=a+b(1-e-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 spring and autumn mulberry leaves 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).Results and Discussion
The chemical composition and content of AA are shown in Table 1 and Figure 1 for the mulberry leaves in spring, and Table 2 and Figure 2 for those in autumn, respectively.| Table 1. Chemical composition and amino acid content of mulberry leaves in spring | ||||||||
Mulberry strains#
| Sampling time## | |||||||
| TH | HS | TX | NS | SE | SM | SL | SEM | |
| Composition, % | ||||||||
| DM,% | 24.4 | 24.1 | 23.7 | 23.6 | 25.1 | 25.2 | 26.6 | 0.10 |
| CP, % DM | 21.6 | 20.9 | 20.9 | 20.8 | 21.1ab | 21.9a | 20.0b | 0.17 |
| TP, % CP | 86.7 | 89.6 | 88.3 | 88.5 | 87.5 | 90.4 | 86.3 | 1.16 |
| NDF, % DM | 39.5 | 37.5 | 39.7 | 38.5 | 34.6c | 38.9b | 42.9a | 0.59 |
| Amino acid concentration, g/100 g protein | ||||||||
| Lysine |
5.3
|
5.02
|
5.05
|
5.21
|
5.16
|
5.14
|
5.13
|
0.13
|
| Methionine |
1.78
|
1.58
|
1.89
|
2.03
|
1.39
|
1.87
|
2.28
|
0.21
|
| Phenylalanine |
4.34
|
4.83
|
5.15
|
4.38
|
4.53
|
4.58
|
4.87
|
0.14
|
| Leucine |
6.89
|
7.19
|
7.45
|
7
|
7.17
|
7.43
|
6.77
|
0.27
|
| Isoleucine |
4.74
|
3.99
|
4.29
|
4.29
|
4.04
|
4.53
|
4.18
|
0.15
|
| Threonine |
4.25 a
|
3.79 b
|
3.98 b
|
3.69 b
|
3.95 a
|
3.97 a
|
3.86 b
|
0.08
|
| Histidine |
3.56
|
2.22
|
3.32
|
2.86
|
2.78
|
3.27
|
2.86
|
0.2
|
| EAA |
30.86
|
28.62
|
31.13
|
29.46
|
29.02
|
30.79
|
29.95
|
*
|
| Valine |
5.16
|
5.12
|
4.9
|
4.52
|
4.62
|
4.81
|
5.4
|
0.16
|
| Arginine |
4.52
|
4.19
|
4.59
|
4.24
|
4.66 a
|
4.20 b
|
4.29 b
|
0.14
|
| Asparanin |
8.26
|
8.08
|
7.76
|
7.19
|
7.53
|
8.18
|
7.78
|
0.25
|
| Glutamine |
11.46
|
10.1
|
9.13
|
9.22
|
10.31
|
9.39
|
10.32
|
0.63
|
| Serine |
4.2
|
3.55
|
3.27
|
3.18
|
3.32
|
3.27
|
2.86
|
0.25
|
| Glycine |
5.94 a
|
5.32 b
|
5.51 b
|
4.93 b
|
5.38
|
5.28
|
5.66
|
0.14
|
| Ala+Tyr |
11.42
|
10.83
|
11.79
|
10.74
|
10.72
|
11.21
|
11.69
|
0.29
|
| Non-EAA |
50.96
|
47.19
|
46.95
|
44.02
|
46.54
|
46.34
|
48
| |
| Total AA |
81.82
|
75.81
|
77.63
|
73.48
|
75.56
|
77.13
|
77.95
| |
| EAA/NEAA, % |
61
|
61
|
66
|
67
|
62
|
66
|
62
| |
| # TH - Tuantou Heyebai; HS - Husang No.9; TX - Tongxiangqing; NS - Nongsang No.8 ## Sampling time: SE - 28 April, SM - 14 May, and SL - 29 May 1998.### DM: dry matter; CP: crude protein; TP: true protein; NDF: neutral detergent fibrea,b,c Means with different superscripts within mulberry strains or sampling times differ (P<0 .05="" em=""> | ||||||||
| Table 2. Chemical composition and amino acid content of mulberry leaves in autumn | ||||||||
Mulberry strains#
| Sampling time## | |||||||
| TH | HS | TX | NS | AE | AM | AL | SEM | |
| Chemical composition, % | ||||||||
| DM (%) | 30.4 | 29.7 | 29.6 | 29.8 | 25.9c | 29.9b | 33.8a | 0.42 |
| CP (% DM) |
21.9
|
20.3
|
19.6
|
21.7
|
22.3a
|
21.4a
|
18.9b B
|
0.23
|
| TP (% f CP) |
84.4
|
86.4
|
87.5
|
84.9
|
86.9
|
85.7
|
84.7
|
0.57
|
| NDF (% DM) |
38.9 b
|
40.8 ab
|
42.6 a
|
43.4 a
|
36.7 c
|
40.4b
|
47.2a
|
0.46
|
| Amino acid content, % of crude protein | ||||||||
| Lysine |
5.3
|
5.02
|
5.05
|
5.21
|
5.16
|
5.14
|
5.13
|
0.13
|
| Methionine |
1.78
|
1.58
|
1.89
|
2.03
|
1.39
|
1.87
|
2.28
|
0.21
|
| Phenylalanine |
4.34
|
4.83
|
5.15
|
4.38
|
4.53
|
4.58
|
4.87
|
0.14
|
| Leucine |
6.89
|
7.19
|
7.45
|
7
|
7.17
|
7.43
|
6.77
|
0.27
|
| Isoleucine |
4.74
|
3.99
|
4.29
|
4.29
|
4.04
|
4.53
|
4.18
|
0.15
|
| Threonine |
4.25 a
|
3.79 b
|
3.98 b
|
3.69 b
|
3.95 a
|
3.97 a
|
3.86 b
|
0.08
|
| Histidine |
3.56
|
2.22
|
3.32
|
2.86
|
2.78
|
3.27
|
2.86
|
0.2
|
| EAA |
30.86
|
28.62
|
31.13
|
29.46
|
29.02
|
30.79
|
29.95
|
*
|
| Valine |
5.16
|
5.12
|
4.9
|
4.52
|
4.62
|
4.81
|
5.4
|
0.16
|
| Arginine |
4.52
|
4.19
|
4.59
|
4.24
|
4.66 a
|
4.20 b
|
4.29 b
|
0.14
|
| Asparanin |
8.26
|
8.08
|
7.76
|
7.19
|
7.53
|
8.18
|
7.78
|
0.25
|
| Glutamine |
11.46
|
10.1
|
9.13
|
9.22
|
10.31
|
9.39
|
10.32
|
0.63
|
| Serine |
4.2
|
3.55
|
3.27
|
3.18
|
3.32
|
3.27
|
2.86
|
0.25
|
| Glycine |
5.94 a
|
5.32 b
|
5.51 b
|
4.93 b
|
5.38
|
5.28
|
5.66
|
0.14
|
| Ala+Tyr |
11.42
|
10.83
|
11.79
|
10.74
|
10.72
|
11.21
|
11.69
|
0.29
|
| Non-EAA |
50.96
|
47.19
|
46.95
|
44.02
|
46.54
|
46.34
|
48
| |
| Total AA |
81.82
|
75.81
|
77.63
|
73.48
|
75.56
|
77.13
|
77.95
| |
| EAA/NEAA, % |
61
|
61
|
66
|
67
|
62
|
66
|
62
| |
| # TH - Tuantou Heyebai; HS - Husang No.9; TX - Tongxiangqing; NS - Nongsang No.8 ## Sampling time: AE - 28 August, AM - 30 September, and AL - 30 October 1998.### DM: dry matter; CP: crude protein; TP: true protein; NDF: neutral detergent fibrea,b,c Means with different superscripts within mulberry strains or sampling times differ (P<0 .05="" em=""> | ||||||||
 |  |
Regardless of the seasons, there were no differences (P>0.05) in content of CP and TP among the different strains. Little seasonal differences were found in content of CP and TP. Average CP content was 21.1 and 20.9 (% of DM), and the TP accounted for 88.2 and 85.8% of the CP in spring and autumn, respectively. The CP content in both seasons was higher than those cited in the Tropical Feed Database (FAO 1998). The NDF was higher for strain TX at middle stage of maturity, and higher for TH at late stage in spring. Strain NS had a higher NDF content at all three stages in autumn leaves (Figure 2). No significant differences were found in the NDF content between different strains, with the exception that in autumn strain TH had lower NDF content (P<0 .05="" 1994="" 1998="" alfalfa="" and="" as="" autumn="" chemical="" comparable="" composition="" content="" dm="" fao="" for="" forages="" in="" is="" leaves="" leguminous="" leucaena="" lower="" mith="" mulberry="" ndf="" ns.="" of="" p="" spring="" such="" than="" the="" those="" trees="" tx="" was="" with="">Except for higher contents of threonine and glycine (P<0 .05="" autumn="" difference="" in="" no="" other="" sampled="" strains="" th="" than="" those="">0.05) was observed in AA contents among the four strains (Tables 1 and 2). There were little differences in total, essential or non-essential AA among four strains. Total and essential amino acids were 70.0 to 81.6 and 24.7 to 31.6% of the CP for spring leaves, and 75.6 to 78.0 and 29.0 to 30.8% of the CP for autumn leaves, respectively.
The chemical composition of the mulberry leaves was influenced by stage of maturity, though the extent to which this was affected was different between spring and autumn leaves. While there was little difference in DM content of spring leaves between different stages of maturity (Table 1), DM content significantly increased with the maturity of mulberry tree in autumn (Table 2, P<0 .01="" 1="" aa="" and="" at="" autumn="" average="" content="" contents="" cp="" decreased="" differences="" early="" essential="" for="" higher="" igure="" increase="" increased="" individual="" late="" leaves="" maturity.="" maturity="" mid="" ndf="" non-essential="" not="" of="" or="" p="" regardless="" season.="" significant.="" significantly="" slightly="" spring="" stage="" tended="" than="" the="" those="" though="" to="" total="" tp="" were="" whereas="" with="">The GP of mulberry leaves sampled in both season is presented in Figure 3. All the spring leaves had higher nutritional values than the autumn leaves. Mulberry leaves sampled at mid season tended to have a higher GP (P<0 .05="" 15="" 30="" about="" and="" at="" autumn="" between="" cause="" days="" difference="" different="" early="" for="" gp="" in="" interval="" late="" leaves="" little="" lower="" mid="" middle="" might="" nutritive="" or="" p="" respectively.="" sampling="" seasons.="" spring="" stage.="" stage="" stages="" than="" that="" the="" this="" those="" two="" value="" was="" while="" with="">
 |  |
OMD (%)=0.76*GP24 + 0.6365*CP + 22.5, where GP24 (ml) and CP (%) is the GP at 24 hr incubation and CP content, respectively.
| Table 3. Parameters of in vitro gas production (GP) and estimated organic matter digestibility (OMD) for mulberry leaves with four strains at three different stages in spring | ||||||||
Mulberry strains #
|
Sampling time ##
|
SE
| ||||||
TH
|
HS
|
TX
|
NS
|
SE
|
SM
|
SL
| ||
| GP at 24 h (ml) | 41.9 ab | 43.1 ab | 39.6 b | 47.6 a | 43.7 ab | 46.9 a | 38.5 b |
0.94
|
| Potential GP (ml) | 45.7 ab | 47.4 ab | 43.9 b | 52.7 a | 47.8 ab | 52.2 a | 43.3 b |
1.04
|
| Rate of GP (% h-1) |
7.26
|
9.02
|
8.9
|
9.5
|
9.70 a
|
8.79 b
|
9.25 ab
|
0.11
|
| OMD (%) | 69.1 ab | 68.8 ab | 65.9 b | 71.9 a | 69.2 ab | 71.3 a | 65.6 b |
0.74
|
| # TH - Tuantou Heyebai; HS - Husang No.9; TX - Tongxiangqing; NS - Nongsang No.8 ## Sampling time: SE - 28 April, SM - 14 May, and SL - 29 May 1998. ,a,b Means with different superscripts within mulberry strains or sampling times differ (P<0 .05="" em=""> | ||||||||
| Table 4. Parameters of in vitro gas production (GP) and estimated organic matter digestibility (OMD) for mulberry leaves with four strains at three different stages in autumn | ||||||||
Mulberry strains #
|
Sampling time ##
|
SE
| ||||||
TH
|
HS
|
TX
|
NS
|
AE
|
AM
|
AL
| ||
| GP at 24 h (ml) |
31
|
30.8
|
30.8
|
31.3
|
32.5 a
|
31.7 ab
|
28.7 b
|
0.55
|
| Potential GP (ml) |
37.8
|
38
|
36.7
|
37.4
|
38.7
|
38.3
|
35.4
|
0.6
|
| Rate of GP (% h-1) |
6.57 ab
|
6.16 b
|
7.01 a
|
6.95 a
|
7.15a
|
6.69ab
|
6.18b
|
0.11
|
| OMD (%) |
60
|
58.8
|
58.4
|
60.1
|
61.4 a
|
60.3 ab
|
56.3 b
|
0.47
|
| # TH - Tuantou Heyebai; HS - Husang No.9; TX - Tongxiangqing; NS - Nongsang No.8 ## Sampling time: AE - 28 August, AM - 30 September, and AL - 30 October 1998. ,a,b Means with different superscripts within mulberry strains or sampling times differ (P<0 .05="" em=""> | ||||||||
In summary, mulberry leaves collected at different stages in both spring and autumn had a high nutritional value in term of chemical composition and in vitro gas production. There was little difference in crude protein content between spring and autumn leaves, but the spring leaves were of higher nutritional value than those in autumn with lower fibre content and higher in vitro gas production (digestibility). Mulberry leaves may thus be used as a supplementary protein source in straw diets for ruminants.
Acknowledgements
This work was funded by the International Foundation for Science (Grant Agreement No: B/2720-1). The authors express their sincere gratitude to Dr.T R Preston for his advice and suggestions, 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 in sampling of the mulberry leaves.References
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