Conservation of mulberry as silage. 1. Effect on nitrogenous compounds

F. Ojeda, I. Montejo and Guadalupe Pérez
Estación Experimental de Pastos y Forrajes Indio Hatuey
Central España Republicana, Matanzas, Cuba

INTRODUCTION

Because of its high edible biomass yield of 16-18 tonnes DM/ha/year; its high percentages of CP (15-25 percent) and IVDMD (75-85 percent); and its perennial nature and adaptation to various soil types (CATIE, 1986), mulberry is beginning to be used extensively for livestock and is likely to become a forage of excellence for feeding and supplementing ruminants.

Even if it is true that with the use of forage trees the seasonality of production is attenuated, in order to guarantee feeding in dry periods, it is indispensable that the ratio of production to unit of land be established on the basis of the periods with less yield.

In the case of mulberry, which has a cutting interval of three months, there is a surplus in the rainy season. If this additional forage is not harvested, there is an imbalance in the nutritional quality of the shoot through ageing, a decrease in edible biomass and a waste of productive potential (Martín, 1999, unpublished).

One way to avoid this situation is by conserving all the green material non-utilized as silage. However, it is known that tree forages have certain characteristics with regard to the conservation technologies established to date (Vallejo et al., 1994). In fact, tree forages contain much higher levels of CP (Oviedo et al., 1994) but, at the same time, this protein degrades during conservation and the animal performance decreases significantly in comparison with fresh forage (González et al., 1997).

To date, few studies have been conducted on the conservation of mulberry as silage, (Vallejo, 1995), and the dynamics of protein degradation during conservation have not been observed. In addition, research carried out in temperate forages has shown protein hydrolysis differs according to the type of forage, irrespective of whether silage is made with fresh or wilted material (Messman, Weiss and Koch, 1994). This explains why only with individual studies of forages species is possible to elucidate the changes in nitrogenous compounds during conservation.

The objective of this research was to conduct a study on the evolution of nitrogenous compounds in mulberry silages and on their transformation over time, taking into account the main indicators and how the inclusion of different doses of conserving agents and prewilting interact.

MATERIALS AND METHODS

Experiment 1. Fermentation dynamics in mulberry silage

Mulberry forage for this experiment was taken from a 3-year old plantation that had a homogenization cut in May, at the beginning of the rainy season and a fertilizer dose of 60 kg of N/ha. Forage was collected manually after 90 days of re growth, in the month of July. The green material was chopped to 1-2 cm and carefully mixed. Double nylon bags, with 3 kg capacity, were used as experimental units. Five bags per treatment were filled and sealed within two hours. Treatments were the opening times: 2, 8, 14, 30, 60, 90, 120 and 180 days.

Parameters measured were: DM, determined in an oven with forced ventilation at 70^ºC for 48 hours; total CP (TCP) determined by the methodology of AOAC (1965); soluble CP (SCP) and ammonia from silage juice extracted by a hydraulic press (Dulphy and Demanquilly, 1984).

Results were analysed with multiple regression equations using the Excel statistical analysis.

Experiment 2. Effect of additives and wilting

Mulberry utilized in this study was obtained from the same plantation as experiment 1, except that forage was collected in September, 60 days after the previous cut, and the fertilizer dose was 60 kg of N/ha. The procedure was similar to experiment 1, but the treatments were those shown in Table 1. Bags were opened after 60 days.

TABLE 1

Treatments for experiment 2

Control	Wilting
Final molasses 2 percent	Formic acid 0.1 percent
Final molasses 4 percent	Formic acid 0.2 percent
Final molasses 6 percent	Formic acid 0.3 percent

The experimental design was a complete randomized block and mean differences were determined by the Duncan (1955) test.

Because of the complexity of the indicators studied and their interactions, it was decided to weigh them. The method used was that of mean superindexes as a way to determine treatment differences for weighing purposes. In cases where two superindexes were similar, the mean of the respective values was taken. The established system for the indicators was set as shown in Table 2.

TABLE 2

Weighing of indicators from significant differences expressed by superindexes

DM	TCP	SCP/TCP %	N-NH3/N4 %	pH	Maximum weighing
a-3	a-3	d-3	c-3	c-3	15
b-2	b-2	c-2	2	b-2
c-1	c-1	b-1	1	a-1
		a-0		a-1

RESULTS

Experiment 1

DM showed a tendency to decrease during the whole measured period, adjusting well to a quadratic regression (Figure 1). TCP was maintained without major fluctuations (Table 3).

The percentage of SPC oscillated but was always above the initial value, with marked increases at the end. The best fit was a grade 4 polynomial equation (Figure 2).

TABLE 3

Changes in total crude protein (%) in mulberry silages

	Days										SD
	0	2	8	14	20	30	60	90	120	180
TCP	18.9	18.4	18.4	19.3	18.9	18.0	19.9	18.0	18.5	19.1	4.6

Figure 1. DM content in mulberry silages over time

Figure 2. Changes in the percentages of soluble protein in mulberry silages

Figure 3. Changes in N-NH3/total N [ percent] in mulberry silages

The percentage of ammonia N from total N showed an increase from 14 days onwards, with maximum values at 180 days. The best fit was a cubic polynomial equation (Figure 3).

The pH dropped rapidly during the first 8 days, but at 30 days started to rise, showed a slight decrease at 60 days and then a constant value until the end. The best fit was a cubic polynomial equation but the R² values were low (Figure 4).

Figure 4. Changes in pH in mulberry silages.

Experiment 2

The results of the indicators evaluated are shown in Table 4. The best DM contents were found in the wilted silages. The addition of final molasses to 4 and 6 percent of formic acid at 0.2-0.3 percent produced higher DM contents than the control, but nothing at lower doses. Formic acid favoured better total CP conservation, the same as wilting with 4 percent molasses compared to the control. Other treatments were different.

The treatment with the best SCP/TCP ratio was wilting, followed by 2 and 4 percent molasses and the control. The highest values were those of formic acid.

The lowest N-NH³/total N were obtained with wilting and with 6 percent molasses, not different from formic acid at 0.1 percent. Other treatments, with higher values, were no different.

The lowest pH was obtained with 6 and 4 percent of molasses. The later was no different from 2 percent molasses, formic acid at 0.1 and 0.2 percent and the control. The highest values were with 0.3 percent formic acid and wilting.

An analysis of the relative weighing of the results considering the significance index (Table 5), shows that wilted silage reaches 80 percent of the possible points, followed by 6 percent molasses, but with a difference of 13 points between them.

Silages with 0.1 and 0.2 percent of formic acid were of better quality than 0.3 percent formic acid, and this was similar to the control silages.

DISCUSSION

Experiment 1

DM losses were high, approximately 25 percent. The explanation for this should be studied from two points of view. First, it is known that during oven drying, silages lose volatile components which underestimate DM values, for this reason, values and, obtained should be taken with caution (Dulphy and Demanquilly (1981). Second, the duration of the trial should be considered. In practice, silages are not stored for more than six months.

Although Vallejo (1995) found similar losses in silages made from tree foliages, losses with mulberry in his experiment were lower, resulting from the high fermentative quality of the silage. For this reason additives enhance DM conservation and, in the case of final molasses, there is an additional supply of solids (De la Fuente, 1990).

TABLE 4

Effect of additives and wilting in fermentative quality of mulberry silages

Treatments	DM	CP	SCP/TCP (%)	pH	NH3/total N (%)
Control	31.82c	22.5c	39.9c	5.0b	11.2a
Wilting	40.20a	24.7b	12.3d	5.4ª	6.2c
Molasses 2 percent	33.68bc	21.8c	38.6c	4.9b	12.1ª
4 percent	34.76b	24.2b	38.6c	4.8bc	10.5ª
6 percent	35.67b	23.0bc	43.7b	4.6c	7.5bc
Formic acid: 0.1 percent	32.67c	26.4ª	63.8ª	5.0b	9.3b
0.2 percent	34.67b	27.3ª	61.6ª	5.0b	13.0a
0.3 percent	34.60b	26.7ª	62.2ª	5.3ª	14.0a
ES ±	1.36	2.4	0.8	0.1	2.6
Sig percent	0.5	0.5	0.1	0.1	0.5

TABLE 5

Weighing of indicators in mulberry silages wilted or with additives

Treatments	DM (%)	CP (%)	SCP/TCP (%)	pH	N-NH3/NT %	Total	Weight %
Control	1	1	2	2	1	7	47
Wilting	3	2	3	1	3	12	80
Molasses
2 %	1.5	2	2	2	1	8.5	57
4 %	2	2	2	2.5	1	9.5	63
6 %	2	1.5	1	3	2.5	10	67
Formic acid
0.1 %	1	3	0	2	2	8	53
0.2 %	2	3	0	2	1	8	53
0.3 %	2	3	0	1	1	7	47

It was clear that under the conditions in which these silages were prepared, there was a permanent degradation. There were no signs of stabilization.

Although TCP values were maintained in all treatments of experiment 1, formic acid preserves quality and being this is one of the main advantages in using it. (Ready and Murphy, 1996).

Mulberry proteins also suffer quality changes, with fermentative and nutritional implications. Forage protein hydrolysis is an inherent process in silage making (Ohshima and McDonald, 1978). The fermentation of mulberry in this experiment, without pretreatments or additives, could not control this process, since pH 4.3, considered the minimum necessary to stop proteases (McDonald, Henderson and Heron, 1991), was never reached. The SCP/TCP increased constantly.

The magnitude of the process was reflected in the fluctuations occurring between 30 and 120 days, which indicate condensation and rearranging of soluble N compounds. In this period almost all the soluble N was in the form of ammonia.

Formic acid addition induced high SCP/TCP ratios. The same effect was found by Carpintero, Henderson and McDonald, (1979), while studying increasing doses of formic and sulphuric acids in temperate grass-legumes mixes, where acidification promoted higher soluble nitrogen. This was seen as the result of a non-enzymatic hydrolysis. However, in the current experiment, high ammonia percentages were not found. This contradiction should be interpreted as the result of the microbe predominating in this silage, since it is mainly responsible for deamination.

In a study conducted by González et al., (1997) with micro-silos of mulberry, there was a direct relationship between pH and lactic acid, which allowed adequate ammonia values in relation to total N.

From these results it can be inferred that formic acid at 0.3 percent did not control the undesirable fermentations (Luis et al., 1991), agreeing a low quality index. This is not the situation with wilted silages, in which pH increases are due to a less intensive but higher quality fermentation (Narsh, 1979).

This line of thought agrees with the results obtained with final molasses. The addition of soluble carbohydrate facilitates the rise in acidity by promoting more vigorous lactic fermentation (Ojeda, 1993).

Experiment 2

The above action was detected in silages with 6 percent molasses. However, the response to the indicators was an increment in the SCP/TCP ratios compared to other doses, confirming that acidification promoted the presence of soluble N compounds, but substantially improving ammonia percentages. Vallejo (1995) also found decreases in pH and ammonia percentages in mulberry silages with 5 percent molasses. This effect was attributed to a better quality of fermentation with almost double lactic acid concentration when using molasses compared with silages with no additives.

In this study, the most effective treatment was wilting, since it gave the best indicators and ammonia contents. Although Narsh (1979) only found positive aspects of wilted silages, Ojeda et al., (1998) found that during sun drying of mulberry, the leaves lose water more quicly and thus proteases should be rapidly inactivated, restricting their action during fermentation.

Research on temperate forages has shown that there is different behaviour in protein hydrolysis depending on forage type, irrespective of whether there was wilting (Messman, Weiss and Koch, 1994).

From the results of this research, it can be concluded that mulberry silages should receive adequate attention not only in relation to initial crude protein content but also to the ways in which nitrogen is transformed. Wilting appears to be the most suitable technology for reducing protein degradation during conservation.

BIBLIOGRAPHY

AOAC. 1965. Official methods of analysis. 9^th ed. Washington, DC, Association of Official Analytical Chemists.

Carpintero, C.M., Henderson, A.R. and Mc. Donald. P. 1979. The effect of some pre-treatments on proteolisis during the ensiling of herbage. Grass and Forage Sci. 34(4): 311-316.

CATIE. 1986. Resumen de las investigaciones realizadas en rumiantes menores, cabras y ovejas, por el proyecto de sistemas de producción animal. Technical Report No. 67. Turrialba, Costa Rica, CATIE.

De la Fuente, B.A. 1990. Estudio de aditivos y cinética del ensilaje de madero negro (Gliricidia sepium). Turrialba, Costa Rica, CATIE. 97 pp. (thesis).

Dulphy, J.P. & Demanquilly, C. 1981. Problemes particuliers aux ensilages. In Previsión de la valeur nutritive des aliments des ruminants. France, INRA. p. 81

Duncan, D.B. 1955. Biometrics 11: 1.

González, J., Benavides, J., Kass, M., Olivo, R. & Esperance, M. 1997. Evaluación de la calidad nutricional de la Morera (Morus sp) fresca y ensilada, con bovinos de engorde. Actas de la III semana científica celebrada del 3 al 5 de febrero de 1997. Programa de Investigación. Turrialba, Costa Rica, CATIE.

Luis, L., Esperance, M. & Ramírez, M. 1991. Utilización de aditivos en la conservación de forrajes en forma de ensilaje. I. Aditivos biológicos. Pastos y Forrajes. 14(3): 185-198.

McDonald, P., Henderson, A.R. & Heron, S.J.E. 1991. The biochemistry of silage. Marlow, Bucks, Chalcombe Publications.

Messman, M.A, Weiss, W.P. & Koch, M.E. 1994. Changes in total and individual proteins during drying, ensiling and ruminal fermentation of forages. J. Dairy Sci., 77: 492-500.

Narsh, R. 1979. The effects of wilting on fermentation in the silo and on the nutritive value of silage. Grass and Forage Sci. 34(1): 1-10.

Ohshima, M and McDonald, P. 1978. A review of the changes in nitrogenous compounds of herbage during ensilage. J. Sci. Food and Agriculture, 29: 497-505

Ojeda, F. 1993. Conservantes químicos en la preservación de ensilajes tropicales. Pastos y Forrajes. 16(3): 193-200.

Ojeda, F., Martí, J., Martínez, N. & Lajonchene, G. 1998. Harina de morera: un concentrado tropical. In III Taller Internacional Silvopastoril "Los árboles y arbustos en la ganadería. Memorias. p. 202. Estación Experimental de Pastos y Forrajes Indio Hatuey Matanzas, Cuba.

Ready, T. W. J. & Murphy. J. 1996. Effects of inoculant treatment on ryegrass silage fermentation, digestibility, rumen fermentation, intake and performance of lactating dairy cattle. Grass and Forage Sci. 51: 232-241.

Vallejo, M. E. 1995. Efecto del premarchitamiento y la adición de melaza sobre la calidad del ensilaje de diferentes follajes de árboles y arbustos tropicales. p.115. Turrialba, Costa Rica, CATIE. (thesis).

Vallejo, M., Benavides, J., Kass M., Jiménez, C. & Ruiz. A. 1994. Evaluación preliminar de la calidad y el consumo de ensilajes de leñosas forrajeras. In Taller Internacional Sistemas Silvopastoriles en la producción ganadera. Resúmenes. p.25. Matanzas, Cuba, Estación Experimental de Pastos y Forrajes. "Indio Hatuey".