INTRODUCTION
Forages, fed either as hay or silage constitute about 40 to 50% of dry matter intake for lactating dairy cows (Bolsen et al., 1993; Coblentz et al., 1996). Ensiling is increasing in popularity as an effective method to preserve forages in Turkey due to increased maize plant production as a second crop. Leguminous and cereal plants are the main forages which are ensiled in most parts of world.
Alfalfa is one of the leguminous forages which are very difficult to ensile due to their low dry matter (DM), water soluble carbohydrate content and high buffering capacity, and difficulty of wilting (Singh et al., 1996; Davies et al., 1998; McAllister et al., 1998). Ensiling a low DM green forage with chemical or biological additives (Strockey, 1990; Henderson, 1993) does not overcome the problem of low DM intake of the wet silage, and the problem of nutrient losses through effluent production (Bodine et al., 1983). Wilting and mixing low DM forage with other material such as straw is used to increase the DM content (Phillips and Penlum, 1984). Wilting is weather dependent.
Furthermore, excessive wilting results in losses of nutrient and reduction in the nutritive value of herbage (Muck, 1988). Therefore, direct ensiling of fresh legume plants is desirable and can be attained by increasing the dry matter content and rapid acidification of forage mass (Henderson, 1993; Miron and Ben-Gheddalia, 1997) using acids such as formic acid.
Maize (Zea mays) is the most popular cereal crop conserved as silage in many parts of the world (McDonald et al., 1991) due to its relatively high dry matter content, low buffering capacity and high water soluble carbohydrate for fermentation to lactic acid which is responsible for the reduction of pH to the required level (Meeske and Basson, 1998), although it has a low protein content. Ensiling of alfalfa forage with whole-crop maize forage with a high dry matter and water soluble carbohydrate content can be used to ensure reasonable ensiling of alfalfa forage. The other objective of ensiling alfalfa with maize is to complement the positive characteristics of two crops and thus to produce a silage which is a more complete feed.
However, there is little information regarding the nutritive value of alfalfa-maize silage mixtures and interaction between alfalfa and maize when ensiled together.
In vitro gas production technique was widely used to evaluate the nutritive value of legume forages (Evitayani et al., 2004) and tannin containing tree leaves (Rubanza et al., 2003; Kamalak et al., 2005).
The aim of this study was to evaluate the chemical
Effects of Ensiling Alfalfa with Whole-crop Maize on the Chemical Composition and Nutritive Value of Silage Mixtures
Durmus Ozturk, Mustafa Kizilsimsek1, Adem Kamalak*, Onder Canbolat2 and Cagrı Ozgur Ozkan Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Animal Science
Kahramananmaras, Turkey
ABSTRACT : The aim of this study was to evaluate the chemical composition, in vitro DM degradability, ME and OMD of alfalfa- maize silage mixtures in comparison to pure maize and alfalfa silages, and to test the existence of associative effects of ensiling alfalfa forage with whole-crop maize using the in vitro gas production technique. Ensiling alfalfa with whole-crop maize had a significant (p<0.001) effect on chemical composition, pH, in vitro DM degradability, OMD and estimated ME values of mixtures. DM content of the resultant silages significantly increased with increasing proportion of whole-crop maize in the mixtures, whereas the pH value, crude protein (CP), acid detergent fibre (ADF) and ash contents of mixtures decreased due to the dilution effect of whole-crop maize which was low in CP, ADF and ash. The pH values of all alfalfa-maize silage mixtures were at the desired level for quality silage. Gas production of alfalfa-maize silage mixtures at all incubation times except 12 h increased with increasing proportion of whole-crop maize. When alfalfa was mixed with whole-crop maize in the ratio 40:60, ME and OMD values were significantly (p<0.001) higher than other silages. Maximum gas production (Agas) ranged from 65.7 to 78.1 with alfalfa silage showing the lowest maximum gas production.
The results obtained in this study clearly showed that maximum gas production increased with increased percentage of whole-crop maize in the silage mixtures (r = 0.940, p<0.001). It was concluded that ensiling alfalfa with whole-crop maize improved the pH, OMD and ME values. However, trials with animals are required to see how these differences in silage mixtures affect animal performance.
(Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 4 : 526-532)
Key Words : Alfalfa-maize Silage Mixtures, Organic Matter Digestibility, Gas Production, Metabolizable Energy
* Corresponding Author: Adem Kamalak. Tel: +90344 2210568, Fax: +903442230048, E-mail: akamalak@ksu.edu.tr
1 Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Crop Science, Kahramanmaras, Turkey.
2 Bursa Uludag University, Faculty of Agriculture, Department of Animal Science, Bursa, Turkey.
Received May 3, 2005; Accepted September 30, 2005
composition, in vitro DM degradability, ME and OMD of alfalfa-maize silage mixtures in comparison with pure maize and alfalfa silages, and to test the existence of associative effects of ensiling alfalfa forage with whole- crop maize using the in vitro gas production technique.
MATERIALS AND METHODS
Silages
A commercial alfalfa variety (Elci) adapted to the region in Kahramanmaras, Turkey was sown in October, 2002 at the seed rate of 30 kg ha-1 with N (50 kg/ha-1) and P2O5 (150 kg/ha-1) fertilizer application. A commercial maize hybrid (32K61) was sown in July 2003 also with N (180 kg/ha-1) and P2O5 (100 kg/ha-1) fertilizer as a second crop after wheat was harvested. In this region, the harvesting time of second crop maize for silage production coincides with the 7-8th cut of alfalfa sown in the previous year. The second-year crop of alfalfa was harvested in October, 2003 at the beginning of flowering (~10%) for silage production. Th maize crop was harvested at the stage of half milk line in October, 2003. Representative maize and alfalfa plants were chopped to about 2-3 cm in length.
Chopped plant materials were ensiled in a plastic experimental silo with a capacity of 5 kg. The silage combinations used in this experiment are given in Table 1.
After two months storage, silos were opened and representative samples of silages were taken and dried at 60°C by force air drier. Dry samples were ground to pass a 1 mm screen for use in the subsequent chemical analysis.
Chemical analysis
Dry matter content was determined by drying the samples at 105°C overnight and the ash by igniting the samples in a muffle furnace at 525°C for 8 h. Nitrogen (N) content was measured by the Kjeldahl method (AOAC, 1990). The CP was calculated as N×6.25. Neutral detergent fiber (NDF) and ADF were determined by the method of Goering and Van Soest (1975). The pH of silages was determined using a combination electrode of a pH meter (SensION HACH, USA). All chemical analyses were carried out in triplicate. Fleigh Points of the silages were calculated according to Kilic (1984).
Fleigh points = 220+(2×DM%-15)-(40×pH)
Where Fleigh Points denote values between 85 and 100, very good quality; 60and 80, good quality; 55 and 60, moderate quality; 25 and 40, satisfying quality; <20, worthless.
In vitro gas production
Rumen fluid was obtained from two fistulated sheep fed twice daily with a diet containing alfalfa hay (60%) and concentrate (40%). The silage samples were incubated in vitro with rumen fluid in calibrated glass syringes following the procedures of Menke and Steingass (1989). The 0.200 g dry samples were weighed in triplicate into the calibrated 100 ml glass syringes. Three syringes containing only buffered rumen fluid were incubated and considered as the blank. Each incubation was completed in triplicate. The syringes were prewarmed to 39°C before the injection of 30 ml rumen fluid-buffer mixture into each syringe followed by incubation in a water bath at 39°C. The syringes were gently shaken 30 min after the start of incubation and every hour for the first 10 h of incubation. Readings of gas production were recorded before incubation (0) and 3, 6, 12, 24, 48, 72 and 96 h after incubation. Total gas values were corrected for blank and hay standards with known gas production. Cumulative gas production data were fitted to the model of France et al. (1993) using the MLP (Most Likelihood Program), (Ross, 1987).
Where y represents the cumulative gas production (ml), t the incubation time (h), Agas the asymptote (total gas, ml) T the lag time (h), b and c are the rate constants (h-1) and (h-
1/2). Estimated values of four parameters, Agas, T, b and c were determined from a time-course experiment of 96 h incubation.
The ME (MJ/kg DM) of silages was calculated using equations of Menke et al. (1979) as follows:
ME (MJ/kg DM)
= 2.20+0.136 GP+0.057 CP+0.0029CP2
Where,
GP is 24 h net gas production (ml/200 mg), CP = Crude protein (%)
The OMD of silages was calculated using equations of Menke et al. (1979) as follows:
OMD (%) = 14.88+0.889 GP+0.45 CP+XA
Where, GP is 24 h net gas production (ml/200 mg), Table 1. Silages combinations
Raw materials Silages
Alfalfa Maize
A 100 0
B 0 100
C 70 30
D 55 45
E 40 60
F 25 75
)]}
T - t c(
- T) - [-b(t exp - {1 A y = gas
CP = Crude protein (%) XA = Ash content (%)
Associative effect
To study associative effects it was necessary to test additivity. Hence, the observed gas productions of mixtures were compared against the expected values calculated from the value obtained from the fermentation of maize and alfalfa ensiled alone (Rosales et al., 1998).
Statistical analysis
One-way analysis of variance (ANOVA) was carried out to compare the mean chemical composition, pH, gas production, estimated parameters, in vitro DMD and ME values using the General Linear Model (GLM) of Statistica for windows (Stastica, 1993). Significance between individual means was identified using the Tukey’s multiple range test (Pearse and Hartley, 1966). Mean differences were considered significant at p<0.05. Standard errors of
means were calculated from the residual mean square in the analysis of variance. A simple correlation analysis was carried out to establish the relationship between chemical composition and the percentage of maize in the silage mixtures.
RESULTS AND DISCUSSION
Chemical compositions of the silages are given in Table 2. There was considerable variation between forages in terms of chemical composition. The crude protein content of forages ranged from 7.93 to 19.25%. Silage A was very rich in crude protein and higher than that of the other silages. The crude protein content of silage A and B was similar to that reported by Ots and Kart (2003), Fonseca et al. (2000) and Anil et al. (2000).
As can be seen from Table 2, NDF content varied with silage type in the range 43.89 to 45.43% and did not differ (p>0.05) between silages. There were significant Table 2. The chemical composition (%) and pH of silages
Silages Constituent
A B C D E F SEM Sig.
DM 20.44a 28.78d 23.61b 26.64 c 26.03 c 26.13 c 0.441 ***
CP 19.25f 7.93 a 14.75 e 11.95 d 11.34 c 10.36 b 0.080 ***
NDF 45.43 44.47 43.89 43.27 44.03 45.15 0.566 NS
ADF 34.56d 23.90ab 28.53c 25.55bc 26.02bc 26.52c 0.480 ***
Ash 11.64e 4.71 a 9.43d 7.63 c 7.42c 6.54 b 0.161 ***
pH 5.43e 3.14 a 4.11 d 3.49 c 3.46c 3.35 b 0.021 ***
FP 13.67 a 108.89c 72.83 b 99.23 c 102.99 c 107.51 c 1.300 ***
A: 100% alfalfa, B: 100% maize, C: 70% alfalfa+30% maize, D: 55% alfalfa+45% maize, E: 40% alfalfa+60% maize, F: 25% alfalfa+75% maize, DM:
dry matter (%), CP: crude protein (%), NDF: Neutral detergent fibre (%), ADF: acid detergent fibre (%), FP: fleigh point, row means with different superscripts differ significantly (p<0.05).
SEM: standard error mean, Sig: level of Significance. *** p<0.001.
0 10 20 30 40 50
DM CP NDF ADF Ash
Chemical constituents Proportion of nutrients in the silage mixture (%)
0% maize+100 alfalfa 30% maize+70% alfalfa 45% maize+55% alfalfa 60% maize+40% alfalfa 75%maize+25% alfalfa 100% maize 0% alfalfa
0 10 20 30 40 50
DM CP NDF ADF Ash
Chemical constituents Proportion of nutrients in the silage mixture (%)
0% maize+100 alfalfa 30% maize+70% alfalfa 45% maize+55% alfalfa 60% maize+40% alfalfa 75%maize+25% alfalfa 100% maize 0% alfalfa
Figure 1. The effect of ensiling alfalfa with whole crop maize silage on the chemical composition.
differences between silages in terms of ADF, with silage A higher than the others. The values were in agreement with those reported by Anil et al. (2000), De Boever et al. (1997), Haddad and Grant (2000). The ash content ranged from 4.71 to 11.64%. The ash content of silage A and B was similar to that reported by Ots and Kart (2003), Fonseca et al. (2000) and Anil et al. (2000).
As can be seen from Figure 1 there were significant negative correlations between the maize proportion of the silage mixture and CP (r = -0.976, p<0.001), ash content (r
= -0.983, p<0.001) or ADF (r = 0.868, p<0.001). The CP, ash and ADF content of silage mixtures significantly decreased with increasing proportion of whole-crop maize due to the dilution effect of whole-crop maize which was low in CP, ash and ADF when compared with alfalfa silage.
On the other hand, there was a significant positive correlation (r = 0.910, p<0.001) between the maize proportion of mixtures and DM content. DM content of silage mixtures increased with increasing proportion of whole-crop maize due its high DM content compared with alfalfa.
The pH of silages ranged from 3.14 to 5.43. The pH of silage A was significantly higher than that of other silages.
It was about 5.43 which is above the pH level required for quality silage. The pH values of silage A and B were similar to those reported by Ghedalia and Miron (2001) and Fonseca et al. (2000). The pH of silage from wilted alfalfa was 5.91 whereas the pH of silage from alfalfa treated with SO2 was 4.51 (Ghedalia and Miron, 2001). On the other hand Fonseca et al. (2000) found that the pH values of 37 maize silages produced in North-West Portugal ranged from 3.36 to 4.33.
There are several reasons why silage A had a high pH value. First may have been a low level of water soluble carbohydrate in silage A from which lactic acid was
produced. Lactic acid is responsible for reducing the pH of ensiled fresh forage. The second reason may have been a high level of soluble protein in silage A which can degrade rapidly and yield ammonia. The ammonia combines with H+ to form NH4+
which prevents the pH of silage from reaching the required pH level. The third reason may have been the high buffering capacity of the alfalfa silage.
Buffering capacity of alfalfa silage is higher than that of maize silage, as reported by Davies et al. (1998), Singh et al.
(1996) and Meeske and Basson (1998).
There was also a negative correlation between the proportion of whole-crop maize in the silage mixture and pH (r = -0.715, p<0.001) (Table 2). The pH values decreased with increased percentage of maize silage in the mixtures possibly due to whole-crop maize having a high water soluble carbohydrate content suitable for lactic acid production. All silages except silage A and C were very good quality according to the Fleigh Point Scale. The silage quality increased with increased level of maize crop in the silage mixtures (Table 2).
It would be essential to present sufficient chemical composition details of silages (and mixtures) to allow adequate interpretation of the results and comparison with other research. This is particularly important in the current experiment since the silage pH data suggest that alfalfa ensiled alone was poorly preserved. In order to confirm if this was indeed poorly preserved and to fully interpret the potential implications of this outcome, it would have been necessary to quantify the concentration of fermentation products such as lactic, acetic and butyric acids, and the extent of protein degradation using ammonia-N as an index.
This is one of the limitations of the current experiment.
The data on gas production during the fermentation period are shown in Table 3. The cumulative volume of gas production increased with increasing time of incubation.
Table 3. Gas production (ml) and estimated parameters of different silages when incubated with rumen buffered liquid in vitro Silages
IT (h)
A B C D E F SEM Sig.
3 20.97a 26.00c 22.70 b 23.50 b 23.50 b 25.83 c 0.247 ***
6 33.83 a 37.50 b 34.17 a 35.17 ab 35.17 ab 36.33 ab 0.548 ***
12 48.17 49.33 48.00 48.17 48.83 48.67 0.838 NS
24 56.33 a 60.83 c 57.00 a 58.00 ab 64.33 d 59.50bc 0.513 ***
48 61.17 a 69.67 c 61.83 a 64.75 b 69.17 c 68.00 c 0.584 ***
72 65.17 a 74.17 b 67.33 a 67.33 a 73.77 b 72.00 b 0.6475 ***
96 68.17 a 78.17 d 71.00 b 71.33 b 79.83d 74.83c 0.548 ***
Estimated parameters
b 0.93 a 0.98 bc 0.97abc 0.96 ab 0.96 ab 0.98 bc 0.006 ***
c 0.88b 0.80 a 0.81 a 0.82 a 0.85ab 0.80 a 0.015 ***
Agas 65.72a 78.09c 69.93ab 69.48 ab 76.43bc 75.23 bc 0.836 ***
ME 10.95 b 10.92 b 10.79 b 10.24 a 11.66 c 10.87 c 0.095 ***
OMD 73.42 b 72.50 b 72.20 b 68.42 a 77.71 c 72.36 b 0.638 ***
A: 100% alfalfa, B: 100% maize, C: 70% alfalfa+30% maize, D: 55% alfalfa+45% maize, E: 40% alfalfa+60% maize.
F: 25% alfalfa+75% maize, Row means with different superscripts differ significantly (p<0.05).
SEM: standard error mean, Sig: level of significance.
*** p<0.001, IT: incubation time, Agas: total gas production (ml/incubated DM), b: fermentation rate (h-1), c: fermentation rate (h-1/2).
Gas produced after 96 h incubation ranged between 68.17 and 79.83 ml per 0.200 g of substrate. There were significant (p<0.001) differences between silages in terms of gas production at all incubation times except 12 h. Gas production at 3 h incubation of silages B and F was significantly (p<0.001) higher than the others, possibly due to a higher water soluble carbohydrate content, but at 6 h incubation gas production of silage B was significantly (p<0.001) higher than only silage A. At 24 h incubation, gas production of silage B was significantly (p<0.001) higher than the others. At 72 h incubation, gas production values of silage B, E and F were significantly (p<0.001) higher than the others whereas the gas production of silage E significantly higher than B and F.
All incubation times except 12 h, showed that generally silage A produced less gas than silages B, D, E and F since silage A had a higher protein content than all other silages.
Protein in alfalfa silage is poorly utilised by ruminants, especially when high-forage diets are fed with relatively low available energy (Buxton, 1996). Extensive degradation occurs during harvest and storage of silage (Albrecht and Muck, 1991) followed by further microbial degradation in the rumen (Buxton, 1996). Under in vitro conditions, gas is produced both directly as a result of fermentation (CO2 and
CH4) and indirectly, from the acidifying effect of VFAs on CO2 released from the bicarbonate buffer solution (Getachew et al., 1998). The degradation of protein yields ammonia, which combines with H+ from the buffer to form NH4+
, which remains in solution so inhibiting the indirect gas production. This may be one of the reasons why silage A had generally a lower gas production value than the other silages.
The estimated parameters are also given in Table 3.
There were significant (p<0.001) differences between silages in terms of estimated parameters. Although silage B had a significantly higher initial gas production rate (b) than silage A, the later gas production rate (c) for silage A was significantly higher than for the other silages. Maximum gas production (Agas) of silage B was significantly higher than that of silages A, C and D. The lag time for all silages was very low and very close to zero. Therefore, lag time was ignored and not included in Table 3. The results obtained in this study clearly showed that maximum gas production increased with increased percentage of whole- crop maize in the silage mixtures (r = 0.940, p<0.001).
The ME values ranged from 10.24 to 11.66. The OMD values ranged from 68.42 to 77.71. The OMD and ME values for silage B were similar to those reported by De Boever et al. (1997). The ME and OMD of silage E were significantly higher than the others. This result is very interesting and indicates that there was a positive interaction effect of ensiling alfalfa with maize silage in the ratio 40- 60%. The concept of interaction effects refers to non- additive interactions among ingredients in mixed diets.
Interaction effects of mixtures of alfalfa-maize silage may have an important impact on animal nutrition. The magnitude of the interaction effects found in this study varied from 4.38 to 9.04% (Table 4). It is likely that the silage mixture which resulted in the associative effect may provide more digestible organic matter and ME for ruminant animals. However, it is very difficult to predict accurately what consequences an associative effect of the Table 4. Relative differences (%) between observed gas
production and parameters predicted from maize and alfalfa silages fermented alone
Mixtures Incubation
time (h) C D E F
3 0.75 0.51 -2.85 3.36
6 -2.17 -0.87 -2.38 -0.68
12 -1.07 -1.06 -0.08 -0.75
24 -1.17 -0.61 9.04*** -0.35
48 -2.81 -0.13 4.69* 1.05
72 -1.05 -1.67 4.38* 0.11
96 0.14 -2.24 7.08** -1.70
C: 70% alfalfa+30% maize, D: 55% alfalfa+45% maize.
E: 40% alfalfa+60% maize, F: 25% alfalfa+75% maize.
*** p<0.001, ** p<0.01, * p<0.05.
0 1 2 3 4 5 6
0% maize +100 alfalfa
30% maize+
70% alfalfa
45% maize+
55% alfalfa
60% maize+
40% alfalfa
75% maize+
25% alfalfa
100% maize+
0% alfalfa
Silage mixtures
pH
0 1 2 3 4 5 6
0% maize +100 alfalfa
30% maize+
70% alfalfa
45% maize+
55% alfalfa
60% maize+
40% alfalfa
75% maize+
25% alfalfa
100% maize+
0% alfalfa
Silage mixtures
pH
Figure 2. The effect of ensiling alfalfa with whole crop maize silage on the pH of silage mixture.
highest magnitude would have on animal production. If effects of the same magnitude obtained in this study are reflected in vivo, the consequences of an associative effect would be important.
Interaction effects may be seen with forage based diets when supplemented with a protein or energy rich supplement. The reason for obtaining interaction effects from a mixture of alfalfa-maize silage may be due to providing energy and protein for rumen micro-organisms in the required ratio. Energy and protein are the key limiting factors for microbial growth. Interaction effects did not occur in all alfalfa-maize silage combination. This result is in agreement with the findings of Rosales et al. (1998) who found an interaction effect of mixing tropical fodder tree leaves in vitro which varied with time and level of nitrogen in the medium.
Although in some parts of the world it is not possible to ensile whole alfalfa and whole-crop maize in mixtures since the two crops mature at different times of the year, climatic conditions make it possible to do so in Turkey. The fact that in Turkey, both crops can be synchronised offers a unique advantage.
IMPLICATIONS
Ensiling of alfalfa with whole-crop maize had a significant effect on the chemical composition, pH, in vitro DM degradability, in vitro OMD and ME of the resultant silage mixtures. Ensiling of alfalfa with whole-crop maize plants overcame some of the drawbacks related to low DM and water soluble carbohydrate contents and high buffering capacity of alfalfa plants. The pH of all alfalfa-maize silage mixtures was lower than pH 4 except for silage C. The results have good practical implications. This result indicated that direct ensilage of alfalfa can be attained by mixing with crop maize. There is no other requirement for additives to obtain the required pH level for high quality silage. Furthermore, positive associative effects occurred when alfalfa forage was mixed with whole-crop maize in the ratio of 40:60%, which increased the OMD and ME of silage E. The idea of combining two different forages and ensiling them together is not altogether new; this study shed more light on the idea and can provide useful data not only for fellow scientists, but also for those grappling with practical application of ruminant feeding. However, trials with animals are required to determine how these differences in silage mixtures affect animal production.
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