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Moringa oleifera as an Alternative Fodder for Dairy Cows in Nicaragua Bryan Mendieta-Araica Faculty of Veterinary Medicine and Animal Science Department of Animal Nutrition and Management Uppsala Doctoral Thesis Swedish University of Agricultural Sciences Uppsala 2011

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Moringa oleifera as an Alternative Fodder for Dairy Cows in Nicaragua

Bryan Mendieta-Araica Faculty of Veterinary Medicine and Animal Science Department of Animal Nutrition and Management

Uppsala

Doctoral Thesis Swedish University of Agricultural Sciences

Uppsala 2011

Acta Universitatis agriculturae Sueciae

2011:34

ISBN 978-91-576-7569-9 ISSN 1652-6880

© 2011 Bryan Mendieta-Araica, Uppsala Print: SLU Service/Repro, Uppsala 2011

Cover: Blooming Moringa oleifera tree (Photo: B. Mendieta-Araica)

Moringa oleifera as an Alternative Fodder for Dairy Cows in Nicaragua

Abstract The four studies comprising this thesis characterised Moringa oleifera as a fodder for dairy cows under dry tropical conditions in Nicaragua. An agronomy study examined, two planting densities (D1=100,000 and D2=167,000 plants ha-1) and four fertilisation levels (N1=0, N2=261, N3=521 and N4=782 kg N ha-1). The D2 density gave significantly higher yields of total dry matter ha-1 (TDMY) and fine fraction dry matter ha-1 (FFDM) compared with D1. There were significant interactions between fertilisation level and the variables year and cut with regard to TDMY and FFDM. However, fertilisation levels N3 and N4 gave the highest yield in both years and among all cuts.

A study on Moringa leaf meal (MLM), as a protein source in concentrates to dairy cows found no significant difference in milk production when comparing isocaloric and isoproteinic concentrates with or without MLM. In an ensiling experiment, Moringa was ensiled alone with 10 g kg-1 fresh matter (FM) molasses and compared with several mixtures with Elephant grass and sugar cane. Pure Moringa biomass produced silage with a higher crude protein (CP) content and had a favourable effect on silage pH, with higher lactic acid concentrations, but the presence of Moringa decreased time to spoilage by 67 h (22%) compared with the Elephant grass silages.

Feeding Moringa as the sole roughage, either fresh or ensiled, compared with feeding Elephant grass resulted in higher digestibility of both CP and fibre but milk yield did not differ (13.7 kg cow day-1). No differences in milk composition were found between treatments but when fresh Moringa was fed a grassy flavour and aroma was detected in the milk.

In conclusion, to maintain high biomass yield of Moringa over time, the best planting density-fertiliser combination was D2 and N3. MLM can successfully replace commercial concentrate ingredients for dairy cows. Furthermore, Moringa ensiled alone, with only 10 or 50 g kg-1 FM molasses added, produces good quality silage that can be fed to dairy cows in large quantities while maintaining the same milk production level and milk quality as for cows fed conventional roughages.

Keywords: Moringa oleifera, dairy cows, milk yield, milk composition, organoleptic

characteristics, silage, leaf meal, biomass yield, planting density, fertilisation levels. Author’s address: Bryan Mendieta-Araica, Departamento de Sistemas Integrales de

Producción Animal, Facultad de Ciencia Animal, Universidad Nacional Agraria, UNA, Km 12 ½ carretera Norte, Managua, Nicaragua, Apdo 453. E-mail: [email protected]

Dedication

To the memory of my Mother Isabel Araica, the most wonderful woman ever;

And to my beloved family, My kids: Brianna Isabella

Bryan Giancarlo Bryan Jeanfranco

And my wife Ivania.

If I have seen further it is only by standing on the shoulders of giants. Sir Isaac Newton

Contents List of Publications 7 Abbreviations 9 

1  Introduction 11 

2  Aims 15 

3  Summary of Materials and Methods 17 3.1  Treatments 17 3.2  Locations 18 3.3  Planting Moringa 18 

3.3.1  Soil preparation and sowing 18 3.3.2  Fertilisation 18 

3.4  Feeds 19 3.4.1  Moringa leaf meal (MLM) 19 3.4.2  Concentrates 19 3.4.3  Ensiling 19 3.4.4  Fresh forages 20 

3.5  Animal Management 20 3.6  Sampling 21 

3.6.1  Biomass 21 3.6.2  Feed and faeces 21 3.6.3  Milk 22 

3.7  Chemical Analysis 22 3.8  Organoleptic Characteristics 23 3.9  Experimental Design and Statistical Analysis 24 

4  Summary of the Results 27 4.1  Moringa as a crop (Paper I) 27 4.2  Moringa leaf meal in concentrates (Paper II) 28 4.3  Moringa as silage (Paper III) 29 4.4  Moringa as roughage (Paper IV). 29 

5  General Discussion 31 5.1  Biomass production 31 5.2  Characterisation of Moringa as a feedstuff 33 

5.2.1  Chemical composition of Moringa foliage 33 5.2.2  Chemical composition of Moringa silage 37 

5.2.3  Labour requirement for silage making and leaf meal production 38 5.2.4  Practical implications of using different feed products from

Moringa 39 5.3  Effect of Moringa on milk yield and milk composition 40 5.4  Effect of Moringa on organoleptic characteristics of milk 41 

5.4.1  Flavour 41 5.4.2  Aroma 42 5.4.3  Colour and appearance 43 

6  Main Findings and Conclusions 45 

7  Future research 47 

References 49 

Acknowledgements 57 

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List of Publications

This thesis is based on the work contained in the following papers, referred to by Roman numerals in the text:

I Mendieta-Araica, B., Spörndly, E., Reyes-Sánchez, N., Salmerón-Miranda, F., Halling, M., (2011). Biomass production and chemical composition of Moringa oleifera under different planting densities and levels of nitrogen fertilization (Submitted to Agroforestry Systems).

II Mendieta-Araica, B., Spörndly, R., Reyes-Sánchez, N., Spörndly, E., (2011). Moringa (Moringa oleifera) leaf meal as a source of protein in locally produced concentrates for dairy cows fed low protein diets in tropical areas. Livestock Science 137, 10-17.

III Mendieta-Araica, B., Spörndly, E., Reyes-Sánchez, N., Norell, L., Spörndly, R., (2009). Silage quality when Moringa oleifera is ensiled in mixtures with Elephant grass, sugar cane and molasses. Grass and Forage Science 64, 364-373.

IV Mendieta-Araica, B., Spörndly, E., Reyes-Sánchez, N., Spörndly, R., (2011). Feeding Moringa oleifera fresh or ensiled to dairy cows- Effects on milk yield and milk flavor. Tropical Animal Health and Production 43,

1039-1047. Papers II-IV are reproduced with the permission of the publishers.

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Abbreviations

ADF Acid detergent fibre AOAC APHA BCN °C CFU CP CT CV DM ECM EG+C FAO FFDM FM GLM GR H IU LAB MAGFR ME MF MLM MPN MS NDF NRC

Association of official analytical chemists American public health association Central Bank of Nicaragua Degrees Celsius Colony-forming units Crude protein Tissue culture Cultivar Dry matter Energy corrected milk Elephant grass + concentrate Food and agriculture organization Fine fraction dry matter Fresh matter General linear model Growth rate Plant height International units Lactic acid bacteria Ministry of Agriculture and Forestry Metabolisable energy Moringa fresh Moringa leaf meal Most probable number Moringa silage Neutral detergent fibre National research council

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OM SAS SBM SIDA TDN TDMY UNA USD USDA WSC

Organic matter Statistical analysis system Soybean meal Swedish international development agency Total digestible nutrients Total yield of dry matter National University of Agriculture United States dollars United States Department of Agriculture Water soluble carbohydrates

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1 Introduction

Historically, the livestock sector has played an important role in Nicaragua, and the significant social and economic importance of the sector remains. The livestock sector accounts for 35% of agricultural gross domestic product and it employs about 200,000 people of an economically active population of 641,000 persons (BCN, 2009). At the farm level, livestock is seen as a source of wealth and insurance and has a high cultural significance establishing the status of the farmer.

Early in the 1990s, the dairy sector in Nicaragua started an extensive and rapid growth process. Dairy production grew from about 30 million litres of milk in 1990 to 730 million litres in 2009 (BCN, 2009; MAGFOR, 2009). Exports of dairy products, which generated revenue of USD 18.4 million in 2001, had grown to USD 116.1 million by 2009 (BCN, 2009). In less than two decades, Nicaragua became a country with net dairy exports. Nicaraguan farmers currently face opportunities and challenges. Nicaragua is well positioned to produce and expand dairy production for local and export markets because it has over three million head of cattle, the biggest herd in Central America (FAO, 2006) and good processing capacity. However, the challenge lies in many issues such as low milk yield, long calving intervals, low calving rate, underfeeding due to limitations in the quality and quantity of feed, mainly during the dry season, and high vulnerability to climate change. Furthermore, the livestock systems are mainly pasture-based and have been progressively moving towards marginal areas with less productive capacity (Holmann et al., 2004; Mendieta-Araica et al., 2000; Kaimowitz, 1995).

Livestock systems have been cited as the major cause of environmental degradation such as deforestation, soil degradation, production of greenhouse gas emissions and of rural poverty (Belli et al., 2009; Mauricio et al., 2008; Steinfeld et al., 2006). Therefore sustainable alternatives in animal production need to be developed and adopted to reduce the negative impact

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of livestock on the environment. Furthermore, there is an urgent need to enhance the efficiency of natural resource use in livestock production in order to overcome the strong seasonal effect on production and consequently improve the standard of living for farmers. There are many options that can be used to deal with the problems described above. However, the strategy that is finally adopted should take into account not only the technical aspect of the problem, but also the economic and social aspects. Interesting options can be found of silvopastoral systems that combine pasture with the production of shrubs and trees. According to some authors (Carvalho et al., 2001; Sánchez and Speed, 1999; Castro et al., 1998) these agro forestry systems offer sustainable management techniques for animal production in the tropics and provide a number of supplementary economic, social and environmental benefits.

In Nicaragua, forage trees such as Leucaena leucocephala, Gliricidia sepium, Erytrhina spp. and Guazuma ulmifolia have been studied for a long time and their role in silvopastoral systems and effects on animal production are well documented (Mosquera-Losada et al., 2005; Durr, 1992; Beer, 1989). However, many of these forage trees have not been widely used because they often contain anti-nutritional compounds that have deleterious effects on animal yield (Ghosh et al., 2007; Hammond, 1995). Therefore, researchers have increasingly been paying attention to Moringa (Moringa oleifera Lam synonym: M. pterygosperma Gaertn., M. moringa Mills), which is a widespread, drought-tolerant tree with negligible amounts of tannins, trypsin and amylase inhibitors (Gidamis et al., 2003; Makkar and Becker, 1997; Becker, 1995). It can have a total dry matter (DM) yield up to 24 ton ha-1 year-1 (Reyes-Sánchez et al., 2006a) and has a crude protein (CP) content in fresh leaves varying from 193 to 264 g kg-1 DM.

Moringa fresh foliage has been included into the diet of different animals. Positive effects on feeding behaviour in goats (Manh et al., 2005), growth rate in sheep (Ben Salem and Makkar, 2009) and milk yield in dual purpose cows (Reyes-Sánchez et al., 2006b) have been reported. Moringa can be also dried and used in the form of Moringa leaf meal (MLM). Promising results have been obtained on inclusion of MLM into the diet of fish (Richter et al., 2003), sheep (Murro et al., 2003), laying hens (Kakengi et al., 2007) and cross-bred dairy cows (Sarwatt et al., 2004). However, reports on feeding MLM to dairy cows are still few. No reports have been found about Moringa silage and its use, or the use of fresh Moringa foliage as the entire roughage component in the dairy cow ration. Interestingly, Moringa has been reported to be a valuable component in human food due to its adequate amino acid profile and CP content, its high level of vitamin A and its low

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level of anti nutritional compounds (Sánchez-Machado et al., 2010; Anhwange et al., 2004).

In order to be a suitable source of fodder, Moringa should be able to produce and maintain high biomass yields over the years. However, under dry tropical forest conditions and without fertilisation, an interannual reduction in yield was reported by Reyes-Sánchez et al. (2006a). Only a few studies on the mineral nutrition of this plant have been performed, all of them under laboratory conditions (Dash and Gupta, 2009; Oliveira et al., 2009; Pamo et al., 2005). Therefore studies of fertilisation strategies in Moringa are required.

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2 Aims

The overall aim of this thesis was to gain further knowledge on how to cultivate, preserve and feed Moringa oleifera to dairy cows in order to obtain more high quality feeds and higher milk yields, mainly during the dry season under dry tropical conditions in Nicaragua.

Specific objectives of the studies were:

To determine the effect of two plant densities and four levels of nitrogen fertilisation on the biomass production and chemical composition of leaves, petioles and stems of Moringa oleifera under field conditions.

To appraise how Moringa leaf meal (MLM) compares to commercial concentrate constituents with regard to milk yield, milk composition and ration digestibility.

To evaluate the effect on fermentation characteristics when Moringa is introduced for ensiling in various mixtures with at least one of the components of Elephant grass, sugar cane or sugar cane molasses.

To assess the effect of inclusion of Moringa on aerobic stability of silages in terms of CO2 production and time to spoilage.

To investigate the effect on milk yield, milk composition and digestibility of feeding fresh or ensiled Moringa compared with feeding a conventional diet of Elephant grass plus commercial concentrate.

To measure the effect of feeding Moringa, fresh, ensiled or as leaf meal, on the organoleptic characteristics of milk produced by the cows on these diets.

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3 Summary of Materials and Methods

3.1 Treatments

An agronomy experiment was performed (Paper I) with two different planting densities (D1= 100,000 and D2= 167,000 plant ha-1) and four levels of nitrogen (N) fertilisation as follows: N1=0, N2= 261, N3= 521 and N4=782 kg N ha-1 year-1. In Paper II a basal diet of Pennisetum purpureum was offered to the cows in the study and concentrate containing 20% soybean meal (SBM) as protein source was compared with a concentrate where SBM was replaced with the same amount of MLM. In a third diet, commercially available components were used to compose an ‘Iso’ concentrate with the same energy and protein content as the concentrate containing MLM.

To investigate the ensilability of Moringa, 14 different treatments were tested in Paper III. These were four different combinations of Moringa and Elephant grass with 10 or 50 g kg-1 fresh matter (FM) molasses, two combinations of Moringa and sugar cane, two treatments based on Moringa with 10 or 50 g kg-1 FM molasses, one treatment with a combination in equal parts of Moringa, Elephant grass and sugar cane, two combinations of Elephant grass and sugar cane, two treatments with Elephant grass with 10 or 50 g kg-1 FM molasses and one treatment based only on sugar cane.

In Paper IV, Moringa foliage, either fresh or ensiled, was compared with a conventional diet for dairy cows. A feed ration was planned to fulfil DM and ME requirements (NRC, 1988) for the control treatment in which 60% of the expected DM intake was given as roughage in the form of Elephant grass and the remaining 40% was given as commercial concentrate. Moringa treatments (fresh or ensiled) were planned to be isocaloric with the control treatment. As a promoter of palatability, 1 kg molasses was added to the Moringa treatments.

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3.2 Locations

The experiments described in Papers I, III and IV were performed at the farm of the National University of Agriculture (UNA) in Managua, Nicaragua, geographically located at 12°08’15”N; 86°09’36”W. The average annual rainfall is 1440 mm, with a marked dry season (November-May). The experiment described in Paper II was carried out at the Santa Ana farm in Masaya, Nicaragua, located at 13°29’16.5”N; 60°55’10”W.

All experiments were conducted under dry tropical forest conditions. The soil where Moringa oleifera was cultivated in Paper I belongs to the taxonomical order of Andosols (FAO, 1988) with clay loam textural class (USDA, 1995).

3.3 Planting Moringa

3.3.1 Soil preparation and sowing

For the agronomy experiment (Paper I), soil preparation consisted of conventional tillage using a tractor and mechanical tools to clean the land of plant debris, followed by disc ploughing, disc cultivation and two harrowings. Untreated seeds of Moringa were used for propagation. In June 2007, seeds were sown in 2 cm deep holes at the study site (2 seeds per hole). After 2 months of growth, the stand was thinned and only one healthy plant was kept. Irrigation was not applied. Weeds were controlled manually 30 days after germination and every second month throughout the experiment.

3.3.2 Fertilisation

In this long-term experiment, June and October were set as the fertilisation occasion. To establish suitable experimental levels of N-fertilisation, data from a production experiment performed in Nicaragua with Moringa oleifera (Reyes-Sánchez et al., 2006a) were used to estimate expected dry matter yields and chemical composition of Moringa foliage. The total crude protein yield on a DM basis reported by Reyes-Sánchez et al. (2006a) was divided by 6.25 to get the amount of nitrogen ‘required’ by the crop, and in the present experiment this was set as N3. Levels 50% below and above this level were set as N2 and N4, A control treatment with no nitrogen fertilisation was set as N1. The levels of N fertilisation used in this study were therefore N1=0, N2= 261, N3= 521 and N4=782 kg N ha-1 year-1. In cuts corresponding to October and June of each experimental year, N fertiliser was applied 2 weeks after pruning and was directly incorporated into the soil by manual hoeing.

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The aim of the fertilisation strategy was to allow the effect of different N levels without risking a shortage in phosphorus (P) and potassium (K) that could influence the results. Therefore, for calculation of P and K requirements the data from Reyes-Sánchez et al. (2006a) were also used as reference for estimating DM yields and chemical composition of Moringa foliage. Based on the P and K contents in DM, the total amount of those minerals was calculated and set as 100% of their requirements, but 150% of the total K and P required was applied at sowing in the entire experimental area to ensure the requirements would be met, which corresponded to 44 kg of P ha-1 and 731 kg K ha-1, respectively. Urea (46-0-0), triple super phosphate (0-45-0) and muriate of potash (0-0-60) were used as source of N, P and K, respectively.

3.4 Feeds

3.4.1 Moringa leaf meal (MLM)

The branches with leaves and soft twigs used for production of MLM for the experiment presented in Paper II were collected from Moringa trees in an experimental area by cutting every 45 days. The harvested material was sun-dried for 24 h before the partially dried leaves were removed by threshing and then sun-dried again for approximately 48 h on black plastic sheets. The dried leaves were finely ground in a hammer mill, packed in sacks and stored in a well-ventilated storeroom.

3.4.2 Concentrates

The experimental concentrate mixtures used in experiments with MLM to dairy cows (Paper II) and feeding either fresh or ensiled Moringa to dairy cows (Paper IV) were produced at the Feed Concentrate Plant of the UNA. All concentrate ingredients were purchased on the local market. Sugar cane molasses was purchased from an agricultural feed supplier.

3.4.3 Ensiling

Non-irrigated and unfertilised Moringa foliage, Elephant grass (Pennisetum purpureum) and sugar cane (Saccharum officinarum) were harvested from the experimental field of UNA and used together with molasses for silage making in Paper III. All forages were hand-cut by machete 45 days after pruning. Sugar cane was cut 9 months after the previous cut. All leaves and roots of the sugar cane were removed from the stems and only the stems were

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ensiled. After cutting, forages were chopped into pieces of approximately 2 cm in length.

Once the forages were chopped, the 14 treatments with different proportions of Moringa, Elephant grass and sugar cane were prepared for ensiling. In treatments without sugar cane, molasses without any water dilution was added at rates of 10 or 50 g kg-1 FM. Mixtures for each treatment were prepared and from these batches fresh material was taken to fill on average 1564 g FM in glass jars with a nominal volume of 1800 ml. The fresh material was pressed into the jar to remove as much air as possible. The glass jars (micro-silos) were fitted with water-locks on their lids to let fermentation gases escape.

Moringa silage used in Paper IV was prepared 120 days before the experiment by cutting a field of Moringa 45 days after a previous harvest. The material was chopped and molasses was added at a rate of 5% according to fresh weight. The material was then put into 55x97 cm polyethylene bags and compressed by hand. The bags were sealed to serve as silos. In total 180 bags, weighing approximately 45 kg each, were stored indoors on a concrete floor until opening.

3.4.4 Fresh forages

Fresh forages such as Moringa and Elephant grass were used as part of the experimental diets in Papers II and IV. For the basal diet of cows fed locally produced concentrates (Paper II), Elephant grass cv. Taiwan was used. However, P. purpureum cv. CT115 was used as part of the control treatment in Paper IV.

The fields with Elephant grass and Moringa were already regularly harvested in a harvesting system before the start of the experiments in order to enable approx. 45-day harvest intervals throughout the experiments. Each day, Elephant grass and Moringa from a new plot were harvested, chopped mechanically into 2 cm lengths and offered fresh to the cows.

3.5 Animal Management

Six dairy cows were used in Papers II and IV, each in their second or third lactation and weighing on average about 450 kg. The cows were in their fourth week of lactation at the start of the experiments. All cows were treated according to EC Directive 86/609/EEC for animal experiments.

The animals were weighed at the beginning of the trials and were kept loose in individual, well-ventilated stalls with a concrete floor. Before the start of the trials, all animals were injected with Vitamin A (625,000 IU) and

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Vitamin D3 (125,000 IU), treated against external and internal parasites and vaccinated against anthrax. Water and minerals were supplied ad libitum. Except during the weeks when faecal samples were taken, the cows were allowed to exercise daily in a common area while the individual stalls were being cleaned.

Digestibility studies were performed in the feeding experiments (Papers II and IV). During the last week of each period, rubber mats were placed in the stalls while all of the faeces from each cow were manually collected. During the faeces collection period, cows were monitored 24 h per day to ensure total collection of faeces.

3.6 Sampling

3.6.1 Biomass

At the start of the agronomy experiment (Paper I) in October 2007, the whole plantation was uniformly cut at a height of 30 cm above the ground and all foliage was removed but not weighed. The regrowth was harvested throughout the two subsequent years, starting from mid-November 2007. Every 45 days, the regrowth was harvested at 40 cm above the ground using a machete. The fresh biomass from each plot was weighed and recorded to estimate fresh matter yield. The material obtained from each plot was separated into two fractions: a fine fraction, which included leaves, petioles and soft stems 5 mm in diameter or smaller, and a coarse fraction, which included stems larger than 5 mm in diameter. The weight of each fraction was recorded and samples of the fine and coarse fraction were taken for subsequent chemical analysis.

Average height of the plants was estimated by measuring the heights of five randomly selected plants in each sub-plot of each treatment. The measurements were made between the plant base (ground) and the highest tip of the leaves. Mortality was calculated as percentage of plants that died at the end of the year, divided by the number of live plants at the beginning of the corresponding year, for each sub-plot.

3.6.2 Feed and faeces

In feeding experiments (Papers II and IV), the amounts of feed offered and occasional refusals were weighed daily and sampling of feed was performed as follows. One kilogram of offered roughage per cow and day was collected and immediately frozen at -18 °C. After every period the frozen samples of

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offered feed for each cow were thawed and pooled into one sample per period for chemical analysis.

The occasional refused feed was individually sampled and frozen immediately at -18 °C. At the end of each experimental period these individual samples of refusals were thawed and sent to a laboratory for chemical analysis. From each of the concentrates used in Papers II and IV, 1 kg from every delivered batch was collected for analysis.

Samples of ensiled Moringa (Paper III) were obtained when micro-silos were opened 120 days after being sealed. The contents from each micro-silo were transferred into a plastic bag, thoroughly mixed and then 800 g samples were taken for analysis. An identical procedure was performed with samples taken after the aerobic stability study.

During digestibility studies in the feeding experiments (Papers II and IV), whenever a cow adopted the defecation position, a shovel was put under her tail to collect the faeces, thereby minimising urine and dirt contamination. The faeces from each cow were put into a large container and covered with a lid to avoid evaporation. Once daily, the faeces from each container were weighed and thoroughly mixed. Five percent were taken as a sub-sample and frozen before the containers were emptied. When the collection was completed, the sub-samples from each cow were thawed and mixed together. Approximately 300 g of this mixture were then taken as a faecal sample for each animal and experimental period to be used in the chemical analysis.

3.6.3 Milk

The cows in feeding experiments (Papers II and IV) were hand-milked twice daily at approximately 12-h intervals. At each milking, the yield was weighed and recorded and 100-ml samples were taken in sterile vials. The milk samples were immediately refrigerated at 4 °C and then pooled into one sample per cow and data collection period for analysis of fat, total solids, CP and casein. The same milk sampling procedure was repeated during the last three days of each experimental period and samples were submitted to the laboratory for organoleptic testing on the day after collection.

3.7 Chemical Analysis

In all experiments, the analyses of DM, CP and ash in feed offered, refusals, faeces and silages were performed using standard AOAC methods. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin were determined by the methods of Van Soest et al. (1991) using sodium sulphite. In the

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ensiling Moringa experiment (Paper III), pH and concentration of lactic acid, propionic and acetic acid were determined by High Performance Liquid Chromatography and Clostridium perfringens, lactic acid bacteria, aerobic enterobacteria and fungal growth were determined according to APHA (2001). Water soluble carbohydrates in Paper III were determined as described by AOAC (2000).

The CO2 concentration in potassium hydroxide solution and pH in silages from Paper III were analysed according to Ashbell et al. (1990). Nitrogen content in milk was determined using the Kjeldahl method (AOAC, 1984) and milk protein using the Babcock method (Pereira, 1988), while total solids and casein were analysed according to AOAC procedures (Papers II and IV).

Organic matter digestibility and the metabolisable energy (ME) of roughages were determined by in vitro incubation in rumen liquid. The ME was then estimated using an equation presented by Lindgren (1979). The Weende analysis was used to determine the ME content of the concentrate, as described by McDonald et al. (1988) (Papers II and IV).

3.8 Organoleptic Characteristics

The organoleptic evaluation of milk in feeding trials (Papers II and IV) was performed by an experienced panel. A triangle difference test (Witting de Penna, 1981) was applied using a milk sample with normal sensory characteristics (flavour, aroma, colour and appearance) as standard.

Table 1. Descriptive terms and scores used in test of organoleptic characteristics of milk (adapted and abbreviated from Witting de Penna, 1981). Score for aroma and flavour 1-5, and for colour and appearance 1-3

Score Aroma Flavour Colour Appearance

5 Undoubted characteristic

Undoubted characteristic

4 Normal Normal

3 Subtly lack of freshness Subtly impure Yellowish white Homogeneous when shaken

2 Subtly grassy Subtly grassy Markedly white Presence of small lumps after shaking

1 Markedly impure Markedly grassy

Abnormal coloration

Presence of large lumps after shaking

Twenty judges were asked to rate the sensory characteristics of milk using

the score sheet presented in Table 1, where 5 was the maximum score for

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flavour and aroma and 3 was the maximum for colour and appearance. The total score of each quality trait was the sum of scores of all judges, while classification was the set of data that occurred most often.

3.9 Experimental Design and Statistical Analysis

The agronomy experiment (Paper I) had a split-plot design with four randomised complete blocks. The two planting densities were the main plot factor and the four levels of N fertilisation were the sub-plot factor. In total, 32 plots were established. A repeated measures analysis of variance in the Mixed procedure of SAS (SAS, 2004) was conducted to determine the effect of plant density and level of fertilisation on the variables measured in a series of harvests over two years (Paper I). Tukey’s pair-wise comparison was used. Observations from the same sub-plot were assumed to be correlated with a first order autoregressive structure. Degrees of freedom were estimated using the Kenward and Rogers method. The interactions between planting density-year, planting density-fertilisation level, planting density-cut and the higher order interactions were tested but then removed from the model due to lack of significance (P>0.10).

A changeover 3x3 Latin square, as described by Patterson and Lucas (1962), replicated in two orthogonal Latin squares was used in feeding experiments (Papers II and IV). Each experimental period consisted of 2 weeks for treatment adaptation and 2 weeks of data collection. Data from Papers II and IV were analysed using the GLM procedure in SAS version 9.12 (SAS, 2004), while Tukey’s pair-wise comparison procedure was used whenever the overall F-test of treatment means showed a significant result. Carry-over effects from previous periods as well the interaction between periods and treatments were tested initially, as described by Patterson and Lucas (1962), but were excluded from the final model because of lack of significance (P>0.10).

A completely randomised experimental design with 14 treatments and three replicates of each treatment was used in the experiment on the ensilability of Moringa (Paper III). The GLM procedure in SAS (2004), including the options MEANS, PDIFF and ESTIMATE, was used. First and second-order mixture experiment models as described by Atkinson and Donev (1992) were used for the data in Paper III. Goodness-of-fit tests were performed using the differences of the sums of squares of the one-way model and of the mixture experiment model as the numerator in an F-test. In cases where a second-order model was applied, the product effects were successively tested and removed until only significant products remained. In

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parallel with significance testing of mixture experiment models, descriptive correlation coefficients of the predicted values and the treatment means were calculated. Variables not showing sufficient goodness-of-fit with the mixture experiment models were analysed by the one-way model only.

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4 Summary of the Results

4.1 Moringa as a crop (Paper I)

The agronomy experiment was conducted as a field experiment from June 2007 to October 2009 in an area which was previously fallow land. Year one consisted of the dry season of 2007 and the rainy season of 2008, while year two was the dry season of 2008 and the rainy season of 2009. There were great variations in precipitation between years, with rainfall of 1411 and 1439 mm in 2007 and 2008, but with unusually low rainfall during May and June of 2008 and very dry conditions in 2009 (796 mm). There were no significant differences (P<0.05) between planting densities with regard to biomass production. The density 167,000 plant ha-1 (D2) produced the highest total dry matter yield (TDMY) and fine fraction dry matter yield (FFDM), with 21.2 and 19.2 ton ha-1 respectively, while 11.6 and 11.0 ton ha-1 were obtained for the same variables at the density 100,000 plant ha-1 (D1). Growth rate (GR) in D2 reached a maximum value of 0.06 compared with 0.03 ton ha-1 day-1 in D1. Plant height (H) was on average 119 cm and no differences were found between planting densities.

There were no significant differences between N3 and N4 in any year with regard to TDMY. On average, those levels produced 22.5 and 27.7 ton ha-1 year-1 during 2007-2008 and 2008-2009, respectively. Both were significantly higher (P<0.001) than levels N1 and N2. During 2007-2008, the TDMY for levels N1 and N2 were 8.9 and 13.9 ton ha-1 year-1, while the corresponding values for the same treatments in 2008-2009 were 4.7 and 10.0 ton ha-1 year-1. N2 showed no significant differences between years, while a significant (P<0.05) reduction in TDMY of 47% was observed in N1 during 2008-2009 compared with the previous year. There were significant interactions between year and fertilisation level in terms of FFDM and GR.

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Levels N1 and N2 gave higher yields of FFDM and higher GR in 2007-2008 than in the following year, while N3 and N4 showed an increase in 2008-2009 compared with the previous year for these variables. No interactions were found between year and fertilisation level for plant height. Plants were consistently taller in 2007-2008 than in the following year.

There were significant interactions between cuts and years with regard to biomass production. A maximum yield of well over 5 ton ha-1 TDMY was observed in Cut 6 in May of both years. Cut 6 also gave a significantly higher (P<0.05) FFDM in 2008-2009 than in 2007-2008. In addition, a significant interaction (P<0.01) was found between year and cut for GR, which was at least 25% lower in Cuts 4 and 6 in 2008-2009 compared with 2007-2008. There was a significant (P<0.05) interaction between year and cut for H, which was higher during the first three cuts in 2008-2009 compared with 2007-2008.

There were significant interactions between fertilisation level and cut with regard to FFDM, GR and H. The levels N1 and N2 consistently differed significantly (P<0.05) from the higher fertilisation levels on all cutting occasions. At Cut 6, levels N3 and N4 each produced just over 5 ton ha-1 FFDM, while 3 and 2 ton ha-1 were obtained on average for the same fraction with levels N1 and N2, respectively. The same pattern was observed in GR, while plant height (H) from different fertilisation levels followed more or less the same pattern.

No significant differences were found between planting densities with regard to the chemical composition of plant fractions. On average, the content of DM, CP, NDF, ADF and lignin in the fine fraction was 115 g kg-1

and 276, 352, 241 and 82 g kg-1 DM respectively, while 203 g kg-1 and 72, 670, 683 and 109 g kg-1 DM were found for the same variables in the coarse fraction. No significant differences were found among fertilisation levels with regard to CP content in the fine fraction. However, a significantly (P<0.05) low value (71.7 g kg-1 DM) of CP content was obtained in the coarse fraction in level N1.

4.2 Moringa leaf meal in concentrates (Paper II)

In an experiment where MLM was used as an alternative protein source to locally produced concentrates, no significant differences were found between the two isocaloric and isoproteinic treatments with regard to digestibility or milk yield. The SBM treatment displayed higher digestibility in CP and produced significantly (P<0.05) more milk (13.2 kg cow-1 day-1)

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compared with the MLM treatment (12.3 kg cow-1 day-1), in line with the higher allocation of CP, ME and starch.

There were no differences in milk composition. The milk contained 34.9 g fat, 34.5 g protein, 126.1 g total solids and 27.4 g casein per kg. No differences in colour, aroma and flavour were found between treatments and all of them were classified as normal.

4.3 Moringa as silage (Paper III)

The silages prepared with different combinations of Moringa, Elephant grass and sugar cane fell within a narrow DM range of 210-269 g kg-1, where Elephant grass treatments had the highest DM content regardless of their content of molasses. Furthermore, CP concentration increased markedly with increasing proportion of Moringa, with the highest CP concentration being found at the highest proportion of Moringa (treatment with 99% of Moringa and 1% molasses). The concentration of NDF in silage decreased when the proportion of Moringa increased.

The presence of Moringa in treatments decreased pH values by 0.8 (P<0.001). Correspondingly, the presence of Elephant grass increased pH values by 0.7 (P<0.001). No such effect was seen with the presence of sugar cane. Both Moringa and Elephant grass, as well as the proportion of molasses, affected the lactic acid concentration, by 16 g kg-1 DM (P<0.001), -21 g kg-1 DM (P<0.001) and -12 g kg-1 DM (P<0.05) respectively. The presence of sugar cane decreased acetic acid concentration (P<0.05).

A tendency for higher Clostridia numbers was observed in treatments with sugar cane rather than with molasses. Intermediate LAB numbers were found in treatments with Moringa and no differences were found among treatments with regard to fungal growth. All silages had Enterobacteria numbers below the detection limit of 0.5 most probable number (MPN) g-1. The presence of Moringa caused a significant (P<0.001) increase in CO2 production, decreased time to spoilage by 67 h (22%) (P<0.05) compared with the Elephant grass silages and increased pH after spoilage by 1.55 (P<0.001).

4.4 Moringa as roughage (Paper IV).

When Moringa, either fresh or ensiled, was tested as the sole roughage in the diet of dairy cows, intake of DM was approximately as planned. There were no refusal in the control treatment and feed refusals in the Moringa treatments were minor, representing only 0.97% of the daily amount

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offered. No differences between Moringa silage (MS) and Moringa fresh (MF) treatments were found in terms of apparent digestibility coefficient, with the exception of DM, where MS gave the highest value of 0.76 compared with 0.64 and 0.69 in the control diet and MF, respectively. Moringa silage treatment had significantly higher (P<0.05) values than the Elephant grass+concentrate treatment with regard to CP, OM, NDF and ADF digestibility, with values of 0.83, 0.77, 0.66 and 0.61, respectively.

There were no significant differences between treatments with regard to milk yield and energy corrected milk (ECM), which averaged 13.7 and 12.9 kg cow-1 day-1, respectively. Furthermore, there were no significant differences in milk composition between treatments and on average the milk contained 35.1 g fat, 34.5 g protein, 123 g total solids and 27.3 g casein per kg.

The colour and appearance of milk from all treatments were classified as normal and no differences in these traits were found between treatments. However, milk from the MF treatment was classified as subtly grassy and was significantly different (P<0.001) to the other treatments, which were classified as normal with regard to flavour and aroma.

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5 General Discussion

5.1 Biomass production

Significant levels of edible biomass production and tolerance to pruning are two important factors determining the suitability of fodder trees or shrubs as forage species (Benavidez, 1996). Moringa has proven to be tolerant to pruning under different cutting frequencies (Reyes-Sánchez et al., 2006a; Foidl et al., 2001). In terms of the biomass production factor, Paper I evaluated the effect of planting density and level of nitrogen fertilisation on biomass yield. The total biomass produced was divided into two categories (Figure 1), a fine fraction with leaves and stems less than or equal to 5 mm in diameter and considered easily edible for dairy cows, and a coarse fraction with stems of diameter over 5 mm and usually not considered edible. However, under Tropical Dry Forest conditions this coarse fraction has been observed to be consumed by steers and goats and could warrant further investigation.

Figure 1. Moringa oleifera foliage divided into (a) fine fraction and (b) coarse fraction. The ruler in the picture is 30 cm long.

a b

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A positive relationship between planting density and biomass yield in tropical tree legumes has been reported (Ella et al., 1989), although Reyes-Sánchez et al. (2006a) and Manh et al. (2005) observed no effect of planting density on biomass yield of Moringa. Paper I showed that of the two densities studied, the higher density (167, 000 plant ha-1) gave a higher yield of Moringa. The TDMY obtained at that density (21.2 ton ha-1) was higher than the 18.9 ton ha-1 reported by Reyes-Sanchez et al. (2006a) at a planting density of 750,000 plants ha-1.

The main reason for the difference is that at the high densities (250,000 to 750,000 plant ha-1) studied by Reyes-Sánchez et al. (2006a), the competition for nutrients and sunlight among plants was observed to be high. At lower planting densities such as those in the present experiment (100,000 and 167,000 plants ha-1), plants do not need to compete as much and therefore produce higher yield.

Fertilisation is a key point in cut-and-carry systems, where huge amounts of nutrients are removed from the areas where the crop is harvested. In a perennial crop such as Moringa this leads to a reduction in biomass yields over time, especially if high planting densities are used. The TDMY in 2007-2008 was 8.9, 13.9 and 22.5 ton ha-1 for N1, N2 and the average of N3 and N4

respectively, while in 2008-2009 the TDMY was 4.7, 10 and 27.7 ton ha-1 for the corresponding treatments. As can be seen, dry matter production increased yearly at the two higher levels of N (N3=521 and N4=782 kg N ha-1 year-1) but declined with N1=0 and N2=261 kg N ha-1 year-1. The response of plant growth to N is widely recognised in conditions where N is a limiting factor (Salmerón-Miranda et al., 2007).

There were important external factors besides the experimental conditions affecting the biomass production in Paper I. Biomass production followed the rainfall pattern and the sum of DM yield from the rainy season (May-October) was twice that harvested during the dry season (November-April). It is important to highlight that while the interaction between years and cuts was not very clear during the dry season (Cuts 1 to 4), the drier second year (2008-2009) produced larger yields with regard to FFDM compared with the first year (2007-2008). Plants tend to grow thinner in dry conditions compared with wet, where plants not only grow taller but also thicker, with a higher coarse fraction (Mommer et al., 2006). In general, higher levels of fertilisation generated higher yields in the rainy season. Furthermore, the physiological response of the plant itself to repeated cutting should be considered a factor that affects the proportion of fine or coarse fraction, as well the total dry matter yield. When Moringa was newly established it developed only one trunk, which was fairly thick. However, as

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soon as pruning started the regrowth became thinner and more branched. This can also be seen in data reported by Reyes-Sánchez et al. (2006a), where the coarse fraction was reduced by 60% in the second year compared with the first.

5.2 Characterisation of Moringa as a feedstuff

In tropical countries, including Nicaragua, forage quality is often too low to meet the nutritional requirement of animals. Furthermore, supplementation with conventional concentrates is generally too costly and the levels of concentrate feeding are therefore low. New low-cost alternatives to commercial concentrates are needed and Moringa has been shown to be one possible option. However, the first critical step in its general use in livestock diets is precise and reliable knowledge of its chemical composition, digestibility and nutritional value. Other practical issues in connection with the use of Moringa as a feedstuff are the labour requirement and how well it can be conserved. Such information is particularly vital in the current context, where farmers are trying to achieve more sustainable production throughout the year.

5.2.1 Chemical composition of Moringa foliage

Moringa foliage can be used either fresh or dry in animal diets depending on the species and production aim. Fresh foliage was used in the diet of dairy cows in Paper IV and the chemical composition of the fine and coarse fractions of Moringa foliage was determined in Paper I.

The DM in 45-day-old fresh foliage in this study was in the range 110.4-203.8 g kg-1. A variation in DM content is also reported in the literature, e.g. Reyes-Sánchez et al. (2006a and 2006 b) found a range of 164-228 g kg-1. This variation can partly be explained by the cutting frequency of the material used, as can be seen in Reyes-Sánchez et al. (2006a) where the DM content of fresh foliage increased from 164 to 228 g kg-1 in plants aged 45 and 75 days, respectively. The thickness of the material analysed can also be an important factor influencing the DM content. In this study fresh foliage was divided into two fractions, a fine fraction which included leaves, petioles and stems with diameter less than or equal to 5 mm, and a coarse fraction with diameter above 5 mm. When the fine fraction was analysed, the DM content was consistently between 110 and 120 g kg-1 (Paper I), while it was about 200 g kg-1 for the coarse fraction. A DM content of up to 460 g kg-1 has been reported for the coarse fraction with branches, stems, petioles and leaves analysed together (Aregheore, 2002).

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Different authors have cited Moringa as a high protein content fodder, with CP concentrations up to 290 g kg-1 DM (Aregheore, 2002; Foidl et al., 2001). In this study, the CP concentration in Moringa foliage fell within a narrow range of 241 and 277 g kg-1 DM in the fine fraction (Papers I and III). However, the CP concentration decreased to 70 g kg-1 DM when only the coarse fraction was analysed (Paper I). The relationship between plant fraction and CP concentration has been reported previously. Makkar and Becker (1997) reported 264 g kg-1 DM in leaves, 72 g kg-1 DM in twigs and 62 g kg-1 DM in stems. That CP value for leaves is similar to the CP content in the MLM (292 g kg-1 DM) used in Paper II.

The cell wall content in foliage is highly variable and influenced by certain factors, such as species, phenological stage at harvest and preservation method. The NDF content of Moringa foliage was between 510 and 521 g kg-1 DM (Papers III and IV) but when fraction differentiation was performed in the production trial, 348 g kg-1 DM was obtained for the fine fraction and 683 g kg-1 DM for the coarse fraction (Paper I). This is consistent with reports from other tropical fodder trees such as Morus alba, Gliricidia sepium, Guazuma ulmifolia and Sesbania grandiflora, which have an NDF content in leaves of between 281 and 570 g kg-1 DM and in stems of between 638 and 720 g kg-1 DM (Sultan et al., 2008; Solorio-Sánchez et al., 2000).

The in vitro dry matter digestibility (IVDMD) of Moringa foliage (0.69) did not differ greatly from published values in other tropical multipurpose trees (Sultan et al., 2008; El hassan et al., 2000; Solorio-Sánchez et al., 2000) and in other reports about Moringa (Reyes-Sánchez et al., 2006a). This digestibility is at the level of alfalfa hay or maize silage (Holden, 1999). This information, together with the CP and fibre content, is particularly interesting because Moringa fodder is intended to be used as a protein supplement for low-quality tropical fodders or even as the only source of roughage for dairy cows.

In Paper I, there were no obvious effects of N fertilisation level on CP, ADF and ash content of the fine fraction. The effect of N fertilisation on chemical composition of foliage seems to vary among species of fodder trees, but no other reports on this in Moringa have been found for comparison. When the shrub Manihot sculenta was studied, a significant effect of fertilisation on CP, NDF, ADF and ash content was reported (Phengvichith et al., 2006), but with the tree Morus spp., Rodriguez et al. (1994) reported no such effect. Different responses to N fertilisation can also be seen among tree species. Hartley et al. (1995) found significant differences in N content on foliage of Picea sitchensis depending on fertilisation, but no effect on chemical composition of Calluna vulgaris foliage in the same experiment.

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Moringa can be also dried in different ways. In Paper II it was sun-dried on plastic sheets in open sun in order to obtain Moringa leaf meal (MLM). This type of meal has been tested as a feed for different species in recent decades, as shown in Table 2. Based on the information in Table 2, some conclusions can be drawn. The first is that the nutritional effects obtained when feeding Moringa to different species are mixed; and the second is that experiments where MLM has been used as a feed for dairy cows are still few.

Table 2. Use of Moringa leaf meal (MLM) and its nutritional effects in different species.

Inclusion level Species Nutritional effect Source

10 % of total dietary protein

Nile tilapia Same growth rate as commercial concentrate

Richter et al., 2003

Substitution of 30 % of fishmeal

Nile tilapia Same growth rate as commercial concentrate

Afuang et al., 2003

4.3 to 42.5% of total diet

Nile tilapia Reduced growth compared with a commercial concentrate

Dongmeza et al., 2006

45.2 % of total diet

Abalone Improvement in yield Reyes and Fermin, 2003

Up to 20% of total diet

Rabbits Leaner carcass Nuhu, 2010

10 % of total diet

Laying hens Unclear effects on egg weight

Kakengi et al., 2007

Up to 5% of total diet

Broiler chickens

Reduction in performance in terms of weight gain, feed conversion ratio and final body weight at 8 weeks when above 5% of total diet

Olugbemi et al., 2010

20% of total diet

Growing sheep 20% improvement in growth rate but poorer feed conversion

Murro et al., 2003

1.65 kg DM Cross-breed cows

Same milk yield as cows fed 1.23 kg DM cottonseed meal

Sarwatt et al., 2004

Under the conditions in which MLM was produced in this study (Paper

II), the chemical composition obtained was 922 g kg-1 DM and 292 g CP, 161 g NDF, 151 g ADF, 68 g lignin, 94 g ash and 10.9 MJ EM kg-1 DM. A broad variation in nutrient content has been reported for MLM, with CP concentration varying from 120 to 349 g kg-1 DM (Madalla, 2008; Murro et

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al., 2003); NDF from 159 to 320 g kg-1 DM (Sarwatt et al., 2004; Richter et al., 2003); and ADF from 44 to 320 g kg-1 DM (Sarwatt et al., 2004; Afuang et al., 2003). Ash content has been reported to range from 71 to 194 g kg-1 DM (Nuhu, 2010; Sarwatt et al., 2004).

There are many different reasons for those variations. However, the main reason that chemical composition of MLM can differ considerably is that sometimes a certain amount of smaller branches and twigs is included along with the leaves in the leaf meal and the proportion of this non-leaf fraction has a large impact on the chemical composition. Fujihara et al. (2005) reported a decrease of 22% in CP concentration when soft twigs were included along with leaves in leaf meal compared with leaves alone. Other authors reported 254 g kg-1 DM when only leaves were used in leaf meal and 120 g kg-1 DM when leaves and branches were used (Afuang et al., 2003; Murro et al., 2003). The CP content of soft twigs alone is lower but this fraction can be used for animals with lower nutrient requirements such as dry cows, which readily consume this feed.

Reyes and Fermin (2003) have also suggested differences in agro-climatic conditions and different age of trees as a source of variation in the chemical composition when material is collected from uncultivated trees. Pok et al. (2005) performed an experiment where new leaves (recently open at the top of braches) and old (near to yellow colour at the bottom of branches) were compared with regard to protein concentration and N digestibility. No great variation was found in crude protein concentration between new and old leaves, which contained 32.7 and 30 g kg-1 DM, respectively. However, a 46% decrease in in vitro N digestibility (IVND) was reported. Based on basic plant physiology, a higher concentration of CP and a lower content of fibre can be expected in younger tissues than in older.

The drying method can also be considered a factor in variations in chemical composition. There are different procedures to obtain MLM; freeze-drying has mainly been used in aquaculture trials (Dongmeza et al., 2006; Afuang et al., 2003; Reyes and Fermin, 2003; Richter et al., 2003), while air and shade drying have been reported by other authors (Olugbemi et al., 2010; Kakengi et al., 2007; Murro et al., 2003). The sun-drying method used in Paper II allows dried leaves to be easily removed from the coarser fraction, giving a highly digestible product with a high nutrient content similar to that reported by other authors (Nouala et al., 2006; Nouala, 2004; Sarwatt et al., 2004). Even though CP concentration seemed not to be significantly affected by drying method (Olsson and Wilgert, 2007; Atega et al., 2003), oven and air-drying under a roof has been shown to increase the cell wall content significantly (Atega et al., 2003).

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Both the high CP concentration and the high rumen degradability reported (Fujihara et al., 2005; Soliva et al., 2005; Makkar and Becker, 1997) suggest that MLM can be used as a supplement for animals mainly in tropical areas, where basal diets are CP deficient. The rumen degradability of MLM has been reported to be similar to that of soybean and rapeseed meal and MLM also seems to promote rumen microbial protein synthesis due to the substantial contents of readily fermentable N and energy (Soliva et al., 2005). However, Fujihara et al. (2005) showed that the proportion potentially degraded in the lower digestive tract was lower than for Leucaena leucocephala.

5.2.2 Chemical composition of Moringa silage

Even though Moringa is not a legume, it shares characteristics with leguminous plants that might need to be taken into account in the silage making process, such as the high CP content in foliage and very low content of water soluble carbohydrates (WSC). Reported levels of WSC in Moringa vary from <50 to 110 g kg-1 DM (Mendieta-Araica et al., 2009; Mustapha and Babura, 2009) and differences could be mainly attributable to the different detection method used in those studies. In Paper III, 10 and 50 g kg-1 DM of molasses were used in pure Moringa silages as a way to increase the rapidly fermented carbohydrates.

Moringa ensiled either pure or in mixtures with Elephant grass or sugar cane substantially increased the CP content of the silages and produced good silage in general (Paper III). When Moringa was ensiled pure with only 50 g kg-1 DM molasses the DM varied from 212 to 267 g kg-1, which is within the adequate range for silages (Buxton et al., 2003). However, the CP

concentration varied from 144 to 226 g kg-1 DM and the NDF concentration from 397 to 435 g kg-1 DM (Papers III and IV). Those differences could be due to the use of only leaves and soft twigs in Paper IV compared with twigs and branches in Paper III.

In silages made from tropical grasses, a pH value of 4.2 has been reported as the maximum to consider silage well-preserved (Cárdenas et al., 2003; McDonald et al., 2002). Weissbach (1996) presented a critical limit for good quality silage depending on DM content in which pH should be no higher than 0.0257xDM content (expressed as percentage) + 3.71. Moringa silages with either 10 or 50 g kg-1 DM molasses were within the range of good silages according to these criteria. Moringa silages had higher lactic acid concentrations (93 to 106 g kg-1 DM) than silage made from Elephant grass or sugar cane. The highest concentration of acetic acid was obtained with a mixture of 0.99 Moringa and 0.01 molasses (31 g kg-1 DM). Even so,

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Moringa silages never reached 60 g acetic acid kg-1 DM, which is considered the upper recommended level (Cárdenas et al., 2003).

Microorganisms are always present in the fermentation process and depending on their final fermentation products and the way they degrade nutrients, they can be regarded as beneficial or detrimental. As main genera lactic acid bacteria (LAB), clostridia, enterobacteria and fungi were studied in Paper III. Intermediate LAB numbers (3.6-2.5 log CFU g-1) were found in Moringa silages. These microorganisms are necessary to preserve high-moisture forages as silage. Even though LAB levels of at least 3.9 log CFU g-1 are desirable for a good fermentation process in temperate grasses, lower values have been reported as normal in silages from tropical grasses (Pedroso et al., 2005; Tjandraatmadja et al., 1994). There was a discrepancy between the lactic acid concentration obtained in the mixture with 95% Moringa and 5% molasses and the LAB count (2.5 log CFU g-1), demonstrating that counts of viable bacteria and lactic acid concentrations do not always coincide. Clostridia can be regarded as unavoidable in raw material. However, when found in silages they can lead to undesirable characteristics in the final product, such as bad smell or increased DM losses. Under the conditions in which the silages in Paper III were produced Clostridium perfringens was identified in the silage. Even though the Clostridium levels in Moringa silages were 3.6-3.8 log CFU g-1 DM, these values were below the limit of 5 log CFU g-1 DM reported as a maximum permissible level for good silage (Lindgren, 1990). Fungal growth was very low and no differences were found between silages. Enterobacteria numbers were below the detection limit of log 0.5 MPN g-1 in all silages.

5.2.3 Labour requirement for silage making and leaf meal production

Nicaraguan farmers at the small and medium scale usually work under conditions where cash availability is limited and the main resource accessible is labour, either of the individual or as a family. Therefore the labour requirement for Moringa silage making and Moringa leaf meal (MLM) production is important information in helping such farmers plan their feeding systems. The figures presented here are based on the harvests that were carried out in Papers I-IV.

At Moringa planting densities of 100,000-167,000 plants ha-1 and a harvesting interval of 45 days, the work required to harvest 1 ton of Moringa foliage is about 19 man-hours. One common way to make silage under the conditions prevailing on small and medium-scale Nicaraguan farms is in 55x97 cm polyethylene bags with approximately 45 kg capacity. The material to be ensiled is put into the bags in layers of approximately 20

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cm and pressed by hand until the top of the bags is reached, whereupon it is sealed. Therefore, the labour calculations were based on this technique. To chop up 1 ton of Moringa foliage using a mechanical chopper requires 1 man-hour. To fill up, compress and seal one plastic bag as a silo using the technique described above, another 1 man-hour is needed. Therefore to prepare the approximately 22 plastic bag silos that can be obtained from one ton of fresh Moringa (about 260 kg DM) requires 42 man-hours.

For MLM production the harvesting labour requirement is the same as above (19 man-hours). The work involved in spreading out the ton of Moringa foliage, threshing and final drying on plastic sheets is 6.4 man-hours. The total amount of work required to produce dry Moringa leaves from 1 ton of fresh Moringa (about 120 kg DM) is therefore 25.4 man-hours.

5.2.4 Practical implications of using different feed products from Moringa

Even though Moringa is a good source of protein for dairy cows and can help farmers overcome the strong effect of dry season feed shortages on milk yield, there are several practical implications that should be kept in mind when using feed products from Moringa.

Fresh Moringa has good intake characteristics, but it is necessary to have an adaptation period to allow cows to get used to the feedstuff. Based on the experience of the author, this period is never longer than two or three days. A simple strategy to overwhelm the small distrust of animals facing the feed can be to offer small amounts of Moringa mixed with other forages and gradually increase the amount of Moringa and reduce the other forages. Another option can be to use a small amount of a palatable agent such as molasses. Once the animals start to eat Moringa, intake does not seem to be a problem and animals often consume considerable amounts, as seen when fresh and ensiled Moringa were offered in Paper IV.

Moringa can be used in a cut-and-carry system where the daily forage requirement can be harvested from the field every morning and offered to the cows. In contrast to Elephant grass, which needs to be cut and ensiled at the right stage of development to ensure silage quality (Reyes-Sanchez et al., 2008), Moringa does not need to be ensiled to ensure high CP content and high digestibility of the DM. However, it is practical to feed Moringa from a silo rather than harvesting and transporting the roughage daily. Furthermore, based on the pattern of biomass yield (Paper I), the fodder production is abundant in the rainy season and not preserving the foliage implies a complete loss of surplus production.

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5.3 Effect of Moringa on milk yield and milk composition

There are many factors affecting milk yield, genetics and management among them. However, in tropical areas shortage of feeds in terms of both quantity and quality is the most important constraint. This is particularly pronounced during the dry season. In countries like Nicaragua, dry season milk yield can decrease to 40% of the rainy season milk yield (Fujisaka et al., 2005).

Moringa has been reported to increase milk yield (Reyes-Sánchez et al., 2006b; Sarwatt et al., 2004). However, it is important to highlight that those studies were performed with low yielding creole cows (3 to 6 litres milk cow-1 day-1) and low quality basal diets. Papers II and IV were performed under medium-scale farm conditions, which can be summarised as follows: Specialist dairy breeds producing approximately 16 kg of milk d-1, two milkings per day, stall-feeding of planted forage; mainly Elephant grass (Pennisetum purpureum) as roughage and supplementary feeding with either molasses, locally available by-products or commercial concentrates at rates of 1 to 10 kg d-1 (de Leeuw et al., 1998). The main aim in Papers II and IV was to determine whether Moringa can support the same milk yield as a control diet representative of the typical diets used in the above-described milk production system using Elephant grass + commercial concentrate to cover the nutrient requirements of the cows.

Even though the soybean meal diet in Paper II gave a higher milk yield, the difference compared with the treatment using Moringa as a source of protein in concentrate was only 7%. The main reason for differences in milk yield in Paper II was probably the higher ME and CP intake when cows were fed soybean meal concentrate. On the other hand, when MLM was compared with another concentrate with the same nutritional concentration no differences were found between them, indicating that Moringa as a protein source has a similar value with regard to milk yield as the commercially available concentrate in Nicaragua. In this case these constituents were sorghum, peanut meal and soybean meal.

In Paper IV, where fresh and ensiled Moringa were compared with a conventional diet with Elephant grass and concentrates, there was no difference in milk yield between the treatments, although the Moringa treatments had higher CP and ME intake. This was probably mainly because no response to extra protein supplementation can be expected when the ME and CP requirements of cows are met (Broderick, 2003; Oldham, 1984).

Milk composition was not affected by any of the treatments where Moringa was fed (Papers II and IV). This is consistent with other studies

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where no relationship between milk protein and percentage of dietary CP has been observed when the energy concentration in the diet is similar (Reyes-Sánchez et al., 2006b; Schingoethe, 1996; Sutton, 1989).

5.4 Effect of Moringa on organoleptic characteristics of milk

There is some evidence in the literature that feeding fresh Moringa to dairy cows can cause off-flavour and aroma (Agrodesierto, 2010). This was fully confirmed in Paper IV when feeding fresh Moringa. Avoiding feeding fresh Moringa before the morning milking has been suggested to decrease the problem, but up to now this has not been confirmed experimentally. Furthermore, Paper IV showed that when Moringa silage was fed instead fresh Moringa, no problems in organoleptic characteristics were detected at all. The possibility of using surplus production during the rainy season by producing silage, combined with the finding that the milk of silage-fed cows was characterised by a good flavour and aroma, seems to indicate that there is potential in the production of Moringa silage for dairy cows.

5.4.1 Flavour

Quoting WHO & FAO (2007), milk is the normal mammary secretion of milking animals without either addition to it or extraction from it, intended for consumption as liquid milk or for further processing. However, to be consumed, organoleptic characteristics such as flavour, aroma, colour and appearance are taken into account by consumers.

Normal milk has a bland but characteristic milk flavour that is pleasing and slightly sweet, mainly due to the presence of fat globules, salts and lactose (Nursten, 1997). Flavour is also claimed to be the most important attribute for consumer acceptance and preference (Croissant et al., 2007; Thomas, 1981). Many studies have reported on the relationship between milk flavour and dietary factor or factors linked to animal management (Kalac, 2010; Martin et al., 2009; Croissant et al., 2007; Martin et al., 2005). The transmission mechanism by which flavour substances can be transmitted to the milk via either the digestive or respiratory route have been described earlier (Dougherty et al., 1962; Shipe et al., 1962).

Milk is very susceptible to off-flavour, which can originate from multiple causes. These causes can be divided into two main categories: those originating from secondary metabolites and those which are transferred to milk from the environment.

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A number of compounds are regarded to contribute to the off-flavour in milk, including metals, terpenes, linolenic acid oxidation products, esters, glucosinolates, phenolics, phytol derivates and nitrogen heterocycles (Bendall, 2001; Makkar and Becker, 1997; Walker and Gray, 1970; Shipe et al., 1962). Some authors have reported the presence of glucosinolates (4-(α-L-rhamnopyranosyloxy)-benzylglucosinolate) in Moringa foliage as the reason why Moringa gives off-flavour to milk (Bennet et al., 2003; Makkar and Becker, 1997; Walker and Gray, 1970). However, many authors have also reported that glucosinolates are susceptible to degradation or are highly reduced by heat or ensiling (Oerlemans et al., 2006; Panciera et al., 2003; Vipond et al., 1998; Fales et al., 1987; Nash, 1985). That is presumably the reason why no off-flavour was found in milk from MLM or Moringa silage treatments, whereas milk from fresh Moringa was classified as grassy.

5.4.2 Aroma

It is not easy to define milk aroma. It is commonly described as characteristic but bland, mainly due to the fact that the concentration of aroma compounds in fresh milk is very low (Bendall, 2001). Some even suggest that the real fresh milk aroma is a belief rather than a fact (Nursten, 1997). Due to its blandness, milk can easily get an off-odour.

A very complex combination of different compounds is needed to get the characteristic milk aroma and more than 70 compounds have been reported so far (Bendall, 2001). Compounds such as indole and skatole have been reported to give a faecal smell to milk (Brudzewski et al., 2004). However, neither of these has been found in Moringa leaves (Bennett et al., 2003).

The organoleptic characteristics of the milk in Paper IV was analysed through sensorial analysis, using the method of Witting de Penna (1981). Using the same methodology as in Paper IV, no effect on milk aroma was reported by Reyes-Sánchez et al. (2006b). This may be due to the small amount of Moringa fed to the cows (3 kg DM) in that experiment, and the long time interval between feeding and milking (24 h). In contrast, in the experiments reported in this thesis, the treatments containing Moringa constituted 100% of the roughage (Paper IV) or 8% of the total diet (Paper II) and cows were milked twice a day. The time interval between feeding Moringa and milking was 1 h and 12 hours after morning and afternoon milking, respectively.

Milk from cows fed fresh Moringa was classified as subtly grassy and this can be attributed to the presence of thiocarbamate glycosides in Moringa leaf tissue (Faizi et al., 1995). Thiocarbamate glycosides have been recognised as

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odour-active substances (Breme et al., 2007) associated with odours resembling garlic, onion tops, mushroom and cress-like, to fresh, spearmint-like in other vegetables from the orders of cruciferous and brassica plants (Breme et al., 2007; MacGregor, 2000; Walker and Gray, 1970).

The mechanism by which milk can gain a bad smell could either be through the animal (via digestive or respiratory route) or through the stall atmosphere. Due to their volatility, thiocarbamates are greatly reduced by silage making and in the dehydration process used to obtain leaf meal, as has been reported by many authors (Figueiredo et al., 2007; Oerlemans et al., 2006; MacGregor, 2000). This explains the absence of a negative effect on aroma of milk from cows fed Moringa silage or MLM (Papers IV and II).

5.4.3 Colour and appearance

Milk colour is due to a combination of many compounds such as casein, carotenoids and fat globules and the concentration of those compounds in milk is related to breed, parity, physiological stage, production level and sanity state. However, nutrition also plays a very important part, especially for milk fat content and the carotenoids, which can affect colour. Casein and fat are regarded as giving a white colour to milk, while carotenoids give a yellowish shade. The carotenoids are taken up from the blood by the mammary gland (Martin et al., 2005).

A relationship between more yellow milk and pasture-based diets compared with silage or concentrate-based diets has already been reported (Kalac, 2010; Martin et al., 2009; Croissant et al., 2007). However, the diets used in this study (Papers II and IV) showed no effect on milk colour, even though fresh forage, silage or a high proportion of concentrate were used. The reasons for this can be found in four main aspects: a) all the treatment diets used in Papers II and IV generated the same concentration of casein in the milk; b) the method used for colour determination was visual and has been proven not to be as accurate as a spectrocolorimeter (Nozière et al., 2006a; Nozière et al., 2006b); c) the effect of dietary carotenoid concentrations is generally observed after several weeks of adaptation (Calderón et al., 2007; Nozière et al., 2006b) and d) milk colour index alone is unable to provide complete discrimination of diet effects (Nozière et al., 2006a)

Even though no reports have been presented about the relationship between nutrition and milk appearance, this trait is an important factor when milk is evaluated by the industry and consumers. No effect on milk appearance was found among the treatments used in this study (Papers II and IV).

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6 Main Findings and Conclusions

Moringa can help small and medium-scale farmers overcome shortages of good quality feeds and therefore sustain and improve their livestock systems.

Under Tropical Dry Forest conditions and when phosphorus and potassium are available in the soil, Moringa can maintain high biomass yield over time but this requires nitrogen to be supplied in sufficient amounts to cover that removed at harvest. Moringa ensiled either alone with 10 or 50 g kg-1 fresh matter molasses added or in an mixture with Elephant grass can provide acceptable fermentation patterns and stability after silo opening, while still maintaining the nutritive value of the silage. Ensiled Moringa can be fed to dairy cows in large quantities without any negative effect on nutrient intake or digestibility. Cows fed large quantities of Moringa silage can produce the same quantity and quality of milk as cows fed conventional Elephant grass diets. Moringa leaf meal is a potential source of protein to supplement poor quality forage such as Elephant grass. It can successfully replace commercial concentrate constituents for dairy cows as long as the substitution is isocaloric and isoproteinic. While a fresh Moringa diet can lead to off-flavour and aroma in milk, a Moringa silage diet gives milk with good organoleptic characteristics.

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7 Future research

This study gives a good idea of how nitrogen fertilisation can affect biomass yield and chemical composition over a two-year period, but experiments over longer periods such as five years can be valuable to further verify whether high production levels can be maintained at the planting densities and fertilisation levels recommended here.

Urea was used as a source of nitrogen in this study. However, different nutrients such as phosphorus and potassium as well different fertiliser sources, either mineral or organic, should be studied in combination with different irrigation regimes.

Silage made from combinations of Moringa and Elephant grass or sugar cane are well documented here. However, combinations with other common tropical grasses and the use of silage as a supplement to grazing animal still need to be investigated.

More studies with MLM in other common tropical diets are needed to obtain detailed results with regard to how feeding Moringa affects dairy production under conditions where protein quantity and quality are limiting factors

Further studies are needed on the potential of MLM as an alternative protein source for milk production based not only on biological results but also including economic aspects.

It would be valuable to study protein availability of Moringa diets in vivo.

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57

Acknowledgements

This thesis is the result of great team work. There were so many persons involved that is impossible for me to remember all of them; however, they all will always have my gratitude.

I would like first to thank the Swedish International Development Agency SIDA for the financial support not only for this work but for the more than three decades of helping my country in so many different ways.

Special thanks should be given to Lars Ohlander, who was the pillar supporting our programme throughout the years.

In Sweden there are so many people who gave me selfless help, support, advice and friendship. Eva Spörndly at the head of them, my main supervisor and the person who guided me step by step in this amazing world of science. She also opened the doors of her home to me and together with her husband Rolf Spörndly, who is also my supervisor, gave me long and absolutely interesting conversations as well very delicious meals in our soirees in this home far away from home; they have been with me beyond any limit and for them my eternal gratitude. Magnus Halling, Börje Ericson, Tommy Pauly, Margarita Cuadra and her husband Peter Petersson, Gloria Gallardo, Cristian Alarcon, Edwin Briceño and his wife Petra and Jette Jacobsen were always there when I needed them, and this long and lonely walk was easier because of them.

To the staff of the Animal Nutrition and Management Department my endless gratitude for their permanent willingness to share knowledge, to Brian Ogle, Inger Ledin, Margareta Norinder, Jan Erik Lindberg, Peter Udén, Ana-Greta Haglund, Torbjörn Lundh, Karin Lyberg, Ragnar Tauson, Maria Neil, Hans Petterson, Ewa Wredle, Sigrid Agenäs and Gunnar Petterson.

Who knows better than my fellow PhD students all the endurance needed in this adventure, but together we managed to end up with success,

58

to Malavanh, Seuth, Vo Lam, Millogo, Martin, Markus, Emma, Hue, Haoyu, Lampheuy, Daovy, Thi Da, Maja, Ulises, Cicci, Mikaela, Roldan, Odum, Gerardo, Matilde, Laki, Vanthong, Teddy, Thieu, Salimata, Thuy, Tram, Salifou and so many more, thanks a lot for your sincere friendship. All my life, part of you will always stay with me.

In Nicaragua I would like to express my sincere gratitude to Nadir Reyes, my national supervisor, who has also been my friend for more than 15 years, his continued support and encouragement finally convinced me to take the decision to apply for PhD studies. My gratitude also to my brother Allan Báez, we share no blood but spirit and throughout more than 30 years he has been giving me his friendship. Friends, thank you.

This study would not have been possible without the tireless work and full dedication of my research team: Rosario Rodríguez, Zoila Romero, David Aráuz, Víctor Acevedo, Elio Zeledón, Marlon Aragón, Néstor Hurtado and his brother, Jasser García, Vera Argüello, Ivan Oliva, Perla Gutiérrez and Eliezer Pichardo. No matter whether the work was done under the strong tropical sun or under the heavy rain harvesting Moringa or during our endless, sleepless nights collecting faeces or measuring temperature in silages, they always kept their optimism and commitment generating useful knowledge to help our farmers. To all of you, thank you very much.

In Nicaragua there are also other persons I want to thank because they did everything in their power to support me; Pablo Valdivia put his cows and his farm in my hands to perform my research and helped me as much as possible; Miguel Ríos, Norlan Caldera, Víctor Aguilar, Edgardo Jiménez, Juan López and his staff, Leonardo García and Vidal Marín helped me either with animals, forage, analysis or advice any time I came to them. Lester Rocha and Francisco Salmerón continuously gave me advice and support with my experiments and papers.

Last but never least, I deeply want to thank my family, this achievement is also their achievement. I stole from them the time I dedicated to my studies but still they always give me their love, support and encouragement. If I have reached the end it is because they also sacrificed many things for me and for that there are no words in the entire universe to express how much I want to thank them. Ivania, Briannita, Bryan see dedication for you, all my love.

Ι

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15

Discussion�

Effect�of�planting�density�on�biomass�production�

��%�*�� ����%�����"�(���������!������� ����"%���������� �� �%����� ���� ��!'���� �'��� ���

Calliandra� calothyrsus�� Sesbania� grandiflora�� Leucaena� leucocephala� ���� Gliricidia� sepium� ���� "����

���%����>�����et�al��9;:;@�� ��%'!������������������������������������!������������������������

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02/�///�� 2//�///� ���� .2/�///� ������ ���9������� et� al�� >0//2@� ���%� ���%���� �%� �&&��� %&� ������ %��

"%����������(�����������%&�902�///����������9�%�� �%(���(����'����� %(�*���� ������'���%&� ���

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������%&�988�888����������9�!�*�����!����������������9:�;�%�����9����%����"����������������et�

al��>0//8"@���.2/�///����������9���%������%��"�(������������������!�������(���%"���*�����

���������� �'����C�%(�� ����(����%'"��� �� ����!���������!������� �%�������(�� ��� �%(����

(���������%���������%�������(�����%������������������C�%(������%&��%��!��������������

�'��� �� ��� (%������!� �������(��� /�/4�/�/8� %�� ���9� ����9��(���� �� !�������(��� �%������"���

�!���� ���� ��� /�//:� ����/�//2� %�� ���9� ����9� ���%���� &%�� ��� &%����� �����Leucaena� leucocephala�

>����%���������("�A���9;;:@�����Gliricidia�sepium�>��%���������(���9;;:@��������*������

Effect�of�nitrogen�fertilization�on�biomass�production�

I�� �'� ���� ������ �������� �'!�� ��%'��� %&� �'����� ���� ���%*��� &�%�� ��� �%�� (����� ��� ��%�� ��

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"�� �������������� et� al�� >0//8"@� &%�� '�&������� �%��!�� �� �� (%������ ����� =��� (��� %� A���� %��

�������� ����� �� ��������� ��%��� �� %� ������ ��� �%�� �'����� �� �� �'&&������ �!�� ��*��� "��

&�����%�����%!������������*%����%������������*��%���������"%��������������(�������&%���

��� &%�'�� %&� ��� �-�������� ,���� �'���� ��*�� ���%���� ��� �%�*�� �&&��� %&� �� &�����%�� %��

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(�������&%���������"%��%�����*�������������'�������%�"����������-���%�����%�&�����%��%����

=��� �'��� ��%(��� ��� �����%�� "�(���� ��*��� %&� &�����%�� ���� ������ =��� ��$�� ���� C�� (����

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16

���� �0�� =����� ���'��� �%'��� "�� �'�� %� ��� �%�"���� �&&��� %&� �� ����� ��*�%����� ���� �%(� �%�� ��

�%������%��� (���� �'���� �%��� *���"���� �� ��� ���%��� ������ �'�����%���� ��� �&&���� %&� �%(� ��

&�����%�������A����%�"��%����%�����%�%'�����%*�������������%��!���'�����"��%������������%&�

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17

Effect�of�planting�density�on�chemical�composition�of�Moringa�

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alba� ���� ���'"�� �'��� ���Manihot� sculenta� ���%� ���%���� �%� ����� �&&��� %&� �����!� ������ %�� 3+�

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18

Mortality�

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Moringa (Moringa oleifera) leaf meal as a source of protein in locallyproduced concentrates for dairy cows fed low protein diets in tropical areas

B. Mendieta-Araica b, R. Spörndly a, N. Reyes-Sánchez b, E. Spörndly a,⁎a Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, P.O. Box 7024, SE-750 07, Uppsala, Swedenb Facultad de Ciencia Animal, Universidad Nacional Agraria, P.O. Box 453, Managua, Nicaragua

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 June 2010Received in revised form 22 September 2010Accepted 22 September 2010

The effect on milk yield, milk composition and ration digestibility of using Moringa leaf meal asa protein source in concentrate given to six lactating dairy cows fed a basal Elephant grass dietwas tested using a changeover 3×3 Latin square design, replicated twice. The basal Elephantgrass diet and a concentrate containing 20% soybean meal was compared with a concentratewhere the soybean meal was replaced with the same amount of Moringa leaf meal. In the thirddiet commercially available components were used to compose an “Iso” concentrate with thesame energy and protein content as the concentrate containing Moringa leaf meal. The intakeof dry matter, organic matter, neutral detergent fibre and acid detergent fibre did not differsignificantly between treatments and averaged 15.4, 13.9, 7.2 and 5.9 kg day−1, respectively,while crude protein (CP) intake was higher (Pb0.001) for the soybean meal treatmentcompared to the other treatments, 1.7 and 1.2 kg CP day−1, respectively. The treatments didnot differ with regard to digestibility with the exception of CP digestibility, which wassignificantly higher in the soybeanmeal treatment compared with the Iso concentrate, 0.70 and0.62, respectively. Mean daily milk yield was significantly higher (Pb0.05) when cows weregiven soybean meal compared with both Moringa leaf meal and the optimized concentrate,13.2, 12.3 and 12.1 kg day−1, respectively. There was no significant difference betweentreatments in either the milk composition, or the organoleptic characteristics of the milk. Theconclusion is that locally produced Moringa leaf meal can, at the same protein and energylevels, successfully replace the commercial constituents in concentrate for dairy cows.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Moringa leaf mealMilk yieldMilk compositionOrganoleptic characteristics

1. Introduction

One of the most important constraints in tropical livestockproduction systems is underfeeding due to limitations in bothquantity and quality of feed. This has been recognized bymany authors (Franziska and Baccini, 2005; Olafadehan andAdewumi, 2009, 2010). These restrictions lead to low milkyields or growth rates which, in turn, gives low net incomesfor farmers (Olafadehan and Adewumi, 2008; de Leeuw et al.,1999). This is particularly pronounced during the dry season,when natural pastures are mature and dry, and therefore

have a low nutritive value. In many areas of the tropics,Elephant grass (Pennisetum purpureum) constitutes the basaldiet for dairy cows (Sarwatt et al., 2004; Shem et al., 2003).However, due to the relatively low quality of Elephant grass, itis essential to provide a protein-rich feed supplement.

Dairy production in tropical areas is a complex system andcannot be seen isolated from the economical and socialdimension in which the farmers live. Supplementation withconventional concentrates during the dry season is generallytoo costly and the levels of concentrate feeding are thereforelow (de Leeuw et al., 1999). The use of concentrate or otherby-products as feed supplements to dairy cows on smallfarms will depend on the access to cash and the price of thefeeds and also the cost of transportation and availability.Therefore there is a need to find alternative low-cost

Livestock Science 137 (2011) 10–17

⁎ Corresponding author. Kungsängen Research Centre, SE-753 23 Uppsala,Sweden. Tel.: +46 18 671632; fax: +46 18 672948.

E-mail address: [email protected] (E. Spörndly).

1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.livsci.2010.09.021

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supplements which can be cultivated by the dairy farmer andare available all-year around. This will allow farmers toimprove the nutritional level in dairy production in thetropics and step by step improve the economy in small scaledairy production.

An important concentrate ingredient in many countries issoybean meal (SBM) which has a high crude protein (CP)content varying from 437 to 480 g kg−1 dry matter (DM)(Broderick et al., 1990; Castillo et al., 2001) and provides acombination of amino acids that can support high milk yields(Broderick et al., 1990; McDonald et al., 2002). However, localconditions for soybean cultivation are not always favourableand in such areas it is expensive to import SBM. In tropicalcountries such as Nicaragua, SBM is one of themost expensivefeed ingredients in concentrate feeds. During the last fouryears, the price of SBM has increased almost 200% (USDA,2009) while the price of other protein-rich feeds such aspeanut meal has increased only 9% (MAGFOR, 2008). In sometropical regions urea has been used as an alternative nitrogensource in ruminant diets but in many countries such asNicaragua, urea is an imported market commodity withvariable availability and price. Small farmers are reluctant touse urea because they need to purchase it on the market,availability is uncertain and it is considered difficult to use asa feed. Faced with these facts, it would be beneficial for smallfarmers in to replace all or part of the SBM in theirconcentrate mixture with a more economical protein sourcethat could be grown locally with limited resources besidelabour.

Leaf meal is a good and cheap source of protein(Duckworth and Woodham, 1961; Paterson et al., 1998).Different forage trees and shrubs, such as Chromolaenaodorata (Fasuyi et al., 2005), Leucaena leucocephala(Kakengi et al., 2001), Morus alba, Azadirachta indica(Patra et al., 2003) and Acacia karroo (Mapiye et al.,2009) have been fed to goats, layers, steers and dairy cows,with good production results. However, the presence ofvarious anti-nutritional compounds in foliage from treesand their deleterious effects in animals has also beendiscussed (Ghosh et al., 2007; Hammond, 1995). Largelydue to the presence of anti-nutritional compounds such asmimosine, cyanogenic glycosides, condensed tannins andalkaloids, the use of forage trees and shrubs has beenlimited and ad libitum feeding of these forages is rarelyused in livestock feeding.

Another potential protein source for livestock productionis Moringa (Moringa oleifera). Although it is a widespread,drought tolerant tree with a high DM yield in the tropics(Reyes-Sánchez et al., 2006), the potential for using Moringaas animal feed is still underappreciated. It is a tree with a highCP content, varying from 179 to 268 g kg DM−1 (Reyes-Sánchez et al., 2006; Mendieta-Araica et al., 2009), and withnegligible amounts of tannins, trypsin and amylase inhibitors(Becker, 1995; Gidamis et al., 2003; Makkar and Becker,1997). Moringa has also been reported to be a valuablecomponent in human food due to its adequate amino acidprofile and CP content, its high level of vitamin A and its lowlevel of anti-nutritional compounds (Anhwange et al., 2004;Sánchez-Machado et al., 2009). Due to this recent interest inMoringa, feeding trials using fresh Moringa have beenperformed with many types of animals such as pigs, goats

and creole cows (Ly et al., 2001; Aregheore, 2002; Reyes-Sánchez et al., 2006). Feeding freshMoringa is convenient butthere is a large variation in production over the year.Therefore, Moringa leaf meal (MLM) is an interesting productas it can be produced during periods of high yields and laterused for feeding during the dry seasonwhen high quality feedresources are scarce. An important advantage with theproduction of Moringa leaf meal (MLM) is that the requiredtechnology is affordable and feasible even for small farmers.Moringa foliage (branches, twigs and leaves) can be obtainedeither from pure crop plots or live fences, cut with macheteand sun-dried on a black plastic sheet placed on the ground(Olsson andWilgert, 2007). Based on the authors' experience,the whole drying process can be completed in 72 h renderingapproximately 1 kg of MLM from 10 kg fresh material. Afterdrying, the leaves can be removed by simple threshing andthe remaining small dry leaves can be crushed or ground byhand to obtain MLM.

In recent years, the interest in MLM as a diet componentin animal production has received some attention byresearchers who have reported promising productionresults in fish (Richter et al., 2003), sheep (Murro et al.,2003) and laying hens (Kakengi et al., 2007). Furthermore,in an interesting experiment performed with cross-breddairy cows MLM was compared with cotton seed cake(CSC) as a concentrate component together with maizebran and minerals (Sarwatt et al., 2004). The cows were feda basal Elephant grass diet together with one of threeconcentrate mixtures. Moringa leaf meal substituted 43, 73and 100% of the CSC in these mixtures. The cows that werefed higher proportions of MLM yielded significantly moremilk indicating that MLM is an interesting feed resource indairy cow diets.

However, the number of dairy cow experiments withMLM is limited (Sarwatt et al., 2004) and it is thereforeinteresting to study the potential of MLM as an alternativeprotein source for milk production further, and explore thelabour requirements for small scale on-farm production ofMLM. Therefore, the aim of this study was to evaluate howMLM compares to commercial concentrate constituents withregard to milk yield, milk composition and ration digestibilityand to estimate the work needed to produce MLM undersmall scale farming conditions.

2. Materials and methods

2.1. Location

The experiment was carried out during the dry season atSanta Ana Farm in Masaya, Nicaragua, located at 13°29´16.5″N and 60°55´10″ W. The average annual temperature inMasaya is 26.6 °C and the mean annual rainfall is 1361.3 mm,with a marked dry season (November–May).

2.2. Treatments, experimental design and management

A basal Elephant grass diet with a concentrate containing20% SBM was tested against the same basal diet with aconcentrate where the SBM was replaced with the sameamount of MLM on weight basis. In the third experimentaldiet, commercially available components (including SBM)

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were used to compose an “Iso” concentrate with the sameenergy and protein content as the concentrate containingMLM.

The concentrate mixtures used in the three experimentaltreatments are presented in Table 1. The SBM concentrate hadthe same composition as concentrates commonly used in thedairy industry of Central America. In the MLM concentrate,the SBM was replaced by MLM on weight basis. The Isoconcentrate had the same energy and protein content as theMLM concentrate but contained the cheapest availablecommercially ingredients instead.

The experiment was designed as a Changeover 3×3Latin Square, as described by Patterson and Lucas (1962),replicated in two orthogonal Latin squares. Each experi-mental period consisted of 2 weeks for treatment adapta-tion and 2 weeks of data collection with regard to milkyield and feed intake. The last week of each period wasused to estimate digestibility.

Six dairy cows from the farm herd, in their second or thirdlactation andweighing 467±22 kgwere used in the trial. Thecows were in their fourth week of lactation at the start of theexperiment. All of the cows were treated according to ECDirective 86/609/EEC for animal experiments. The animalswere loose confined in individual, well-ventilated stalls withconcrete floor which was covered with rubber mats duringfaecal collection periods. Before the start of the trial, the cowswere injected with Vitamin A (625,000 IU), Vitamin D3

(125,000 IU) and Vitamin E (125 IU); treated against externaland internal parasites and vaccinated against anthrax. Waterwas provided ad libitum and the cows had access to acommercial mineral supplement with Ca, P, Mg and traceelements.

The feed allotment was planned to fulfil the proteinrequirement (NRC, 2001) of CP for the SBM diet whenadopting the DM intake of 3.2 of body weight recom-mended in the region (Hazard, 1990; Romero and González,2004). Sixty percent of the expected DM intake was givenas roughage in the form of Elephant grass and theremaining 40% was given as one of the three concentrates.These proportions represent approximately those usuallyused by dairy farmers in the Central American region(Castro Ramírez, 2002; Vélez, 1997). Roughages wereoffered individually in separate feed troughs twice perday, at 07:00 h and 17:00 h. The concentrates were fedindividually during milking, at 05:00 h and 16:00 h. The DM

content of Elephant grass was determined twice per weekusing a microwave oven according to the proceduredescribed by Undersander et al. (1993).

The offered amounts of feed and occasional refusals wereweighed daily and sampling of feeds was performed asfollows. One kilogram of offered roughage per cow per daywas collected and immediately frozen at −18 °C. After everyperiod the frozen samples of offered feed for each cow werethawed and pooled into one sample per period for chemicalanalysis. The occasional refused feed was individuallysampled and frozen immediately at −18 °C. At the end ofeach experimental period these individual samples of refusalswere thawed and sent to a laboratory for chemical analysis.From each concentrate in the experiment 1 kg from everydelivered batch was collected for analysis, giving a total ofthree samples per concentrate corresponding to the threeperiods in the experiment.

The cows were hand-milked and at each milking the yieldwas weighed and recorded. From each cow and milking asample of 100 ml was collected and immediately refrigeratedat 4 °C. Milk samples from each cow were pooled into onesample per week during the data collection period and thenanalyzed for fat and protein content, the same milk sampleprocedure was used for the organoleptic test.

2.3. Feed preparation

The branches with leaves and soft twigs used for theproduction of MLM were collected from M. oleifera trees inthe experimental area by cutting every 45 days. They werethen sun-dried for 24 h before the partially dried leaves wereremoved by threshing and then sun-dried again approxi-mately 48 h on black plastic sheets. The dried leaves werefinely ground in a hammer mill, packed in sacks and stored ina well-ventilated storeroom. During the production of MLMfor the experiment, the amount of fresh material harvestedand the amount of MLM produced was weighed. Further-more, the work used for harvesting Moringa and the amountof work used in the drying process was registered.

The other concentrate ingredients were purchased in thelocal market and the experimental concentrate mixtures(Table 1) were produced at the Feed Concentrate Plant of theNational Agrarian University, Nicaragua. Before the start ofthe experiment, the field with Elephant grass (P. purpureum)was divided into several plots that were harvested at regularintervals before experimental start. This was done to enableregrowth cuts at similar intervals with the objective toharvest at approximately 45 days regrowth throughout theexperiment. During the experiment, the elephant grass washarvested daily at 46±3 days regrowth and offered fresh tothe cows, chopped into pieces of approximately 2 cm using atractor mounted forage chopper.

2.4. Digestibility study

Every day during the last week of each period, all thefaeces from each cow were collected manually. During thecollection period cows were supervised 24 h daily and anytime when a cow adopted the defecation position a shovelwas put under her tail to collect the faeces and avoidcontamination from urine and dirt from the stall floor.

Table 1Proportions of feedstuffs in % wet weight in the concentrate mixtures.

Ingredients MLMconcentrate

Isoconcentrate

SBMconcentrate

Sorghum 30.1 55.3 30.1Rice polishing 41.0 25.1 41.0Sugar canemolasses

5.9 5.9 5.9

Peanut meal 1.0 9.4 1.0Calcium carbonate 1.5 1.5 1.5Salt 0.5 0.5 0.5Moringa leaf meal 20.0 0.0 0.0Soybean meal 0.0 2.3 20.0

MLM: moringa leaf meal, SBM: soybean meal.

12 B. Mendieta-Araica et al. / Livestock Science 137 (2011) 10–17

Thereafter, the faecal material was put into a large,individually marked container for the cow and coveredwith a lid to avoid evaporation. Once daily, the faecalcontents in the containers of each cow were weighed andthoroughly mixed. Five percent of the daily faecal contentsfrom each cow was then taken as a subsample and frozen.When the collection was complete, the subsamples werethawed and mixed together into one homogeneous sampleper cow and period. Approximately 300 g of the mixturefrom each animal was then taken as a faecal sample. Theresults of the chemical analysis of faeces and feed wereused together with intake data to estimate apparentdigestibility.

2.5. Chemical analysis

The samples of offered feed, refused feed and faeces weredried at 60 °C and ground through a 1 mm sieve beforeanalysis. Ash and DM were analyzed using AOAC (1990)procedures. The CP content was determined using theKjeldahl method (AOAC, 1984). Neutral detergent fibre andADF were analyzed as described by Van Soest et al. (1991).The apparent digestibility coefficient for DM was calculatedby comparing the dietary intake of constituents and theamounts recovered in faeces. Nitrogen content in the milkwas determined using the Kjeldahl method and milk proteincontent was calculated as N×6.38. Milk fat was determinedusing the Babcock method (Pereira, 1988), while total solidsand casein were analyzed according to AOAC (1984)procedures. An organoleptic evaluation of the milk wasperformed by an experienced panel of 15 persons. A triangledifference test (Witting de Penna, 1981) was applied using amilk sample with normal sensory characteristics (colour,smell and taste) as standard.

Organic matter digestibility and the metabolisable energy(ME) of the elephant grass were determined by in vitroincubation in rumen liquid: 0.5 g dried sample mixed with49 ml buffer and 1 ml rumen fluid was incubated 96 h at38 °C. The residues were combusted to get the digestibilitycoefficient of the organic matter and ME was then estimatedusing equation presented by Lindgren (1979). For concen-trates, ME was calculated based on the Weende analysis, asdescribed by McDonald et al. (1988).

2.6. Statistical analysis

The data was analyzed using the GLM procedure in theSAS Version 9.1.2 (SAS, 2004). Tukey's pairwise comparisonprocedure was used whenever the overall F-test of treatmentmeans showed a significant result. The mathematical modelused was

Yjk=μ+Pk+Cj+Tl+εjk, with j=1,…,6 and k=1,2,3,where μ was the overall mean, Pk the fixed effect ofperiod, Cj the random effect of cow, Tl the fixed effect oftreatment and εjk the random residual error.

Carry-over effects from previous periods, the interactionbetween periods and treatments were tested initially, asdescribed by Patterson and Lucas (1962), but were excludedfrom the final model because of lack of significance (PN0.10).

3. Results

3.1. Feed intake and apparent digestibility

The MLM used in this experiment contained 292 g CP,161 g neutral detergent fibre (NDF), 151 g acid detergentfibre (ADF), 68 g lignin and 94 g ash per kg DM. The chemicalcomposition of the feeds presented in Table 2 shows that thebasal Elephant grass diet had a low CP content (34 g kg−1

DM) and a high fibre content. The CP content in the SBMconcentrate was typical for commercial concentrates inCentral America.

The feed intake and apparent digestibility coefficients areshown in Table 3. There were no significant differencesamong treatments with regard to feed intake or the intakeand digestibility of DM, organic matter, NDF and ADF(Table 3). The CP and ME intake was higher for cows fedthe SBM concentrate when compared with the other treat-ments. Compared to the NRC requirements (2001) the CPintake covered 104%, 76% and 73% of requirements for theSBM, MLM and Iso diets, respectively. The CP content of thediet was 102 g kg−1 DM for cows fed SBM concentrate and79 g kg−1 DM for the other treatments. The starch intake wasalso higher for cows fed the Iso concentrate. Furthermore, thedigestibility of CP was higher when cows were fed SBMconcentrate compared with Iso concentrate.

3.2. Milk yield and composition

The average daily milk yield during the experiment was11.8 kg energy correctedmilk (ECM). Bothmeandailymilk andECM yield were significantly (Pb0.05) higher when cows werefed SBM concentrate compared with the other treatments(Table 4). However, there was no significant difference in milkcomposition between treatments and, in average, the milkcontained 34.9 g kg−1 fat, 34.5 g kg−1 protein, 126.1 g kg−1

total solids and 27.4 g kg−1 DM casein. The hypothesis thatMLM would influence the organoleptic characteristics of themilk proved to be wrong: the colour, smell and taste of milkfrom all treatments were classified as normal.

Table 2Chemical composition of the concentrates and basal diet used in theexperiment, means and standard deviation (in parenthesis) n=3.

Nutrients Feeds

Elephantgrass

MLMconcentrate

Isoconcentrate

SBMconcentrate

DM, g kg−1 173 (33) 837 (12) 836 (15) 858 (14)g kg−1 DMCrude protein 34 (8) 154 (5) 153 (13) 221 (5)Neutral detergentfibre

673 (52) 129 (11) 119 (8) 106 (7)

Acid detergentfibre

558 (23) 86 (2) 84 (8) 72 (0)

Lignin 105 (18) 28 (1) 25 (2) 21 (1)Ash 119 (10) 91 (4) 76 (8) 82 (2)Starch nd 337 (21) 462 (18) 354 (16)G+F nd 25 (2) 16 (4) 14 (2)ME⁎ MJkg−1DM 7.1 (0.4) 12.9 (0.3) 12.9 (0.2) 13.4 (0.2)

DM: drymatter,MLM:moringa leafmeal, SBM: soybeanmeal, G+F: glucose+fructose, ME: metabolizable energy, * calculated, nd: not determined.

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3.3. Labour requirements for Moringa leaf meal production

Weighing of the freshly harvested Moringa and the driedleaves showed that 1 ton of fresh Moringa gave 123.5 kg ofdried Moringa leaves. At a planting density of 100,000–170,000 Moringa plants per ha and a harvesting interval of45 days, the work required for 1 person to harvest 1 ton ofMoringa was 2.4 days. The work involved in spreading outthis amount of harvested Moringa, for threshing and the finaldrying on plastic sheets was 0.8 days for 1 man. The leaveswere, as mentioned earlier, ground at a feed plant and norecord of the work needed for manual grinding or crushing ofleaves could therefore be registered. The total amount ofwork to produce dry Moringa leaves from 1 ton of freshMoringa was therefore 3.4 man days giving approximately120 kg of Moringa leaves.

4. Discussion

The method used in the present experiment for theproduction of MLM was a simple and cheap method of sun-drying on plastic sheets and this method can easily beadopted by small farmers. There are several other methods of

producing MLM such as freeze-drying (Richter et al., 2003),drying in the shadow (Sarwatt et al., 2004 and Kakengi et al.,2007) and combined air- and floor drying (Murro et al., 2003)but the sun-dryingmethod has been studied and proved to besimple and adequate (Olsson and Wilgert, 2007).

It is important to realize that the chemical composition ofMLM can vary considerably mainly depending on the amountof smaller branches and twigs included along with the leavesin the leaf meal. This was shown by Fujihara et al. (2005),who analyzed different fractions of Moringa (leaves, seedcake, soft twigs, and bucks). The leaves and seed cake had a CPcontent of approximately 250–300 g kg−1 DM while leaveswith soft twigs had a CP content of 195 g kg−1 DM. The CPcontent of soft twigs alone was yet somewhat lower but thisfraction can be used for animals with lower nutrientrequirements such as dry cows, who readily consume thisfraction. The drying method used in the present experimentallows dried leaves to be easily removed from the coarserfraction, giving a highly digestible product with a highnutrient content. The MLM used here had an NDF contentof 161 g kg−1 DM. This value is similar to the NDF content159 g kg−1 DM in the experiment of Richter et al. (2003)where only leaves were used and much lower than the NDF

Table 3Least square means of intake and apparent digestibility of dairy cows fed different concentrates.

Items Treatments SE Significancelevel

MLM concentrate Iso concentrate SBM concentrate

Feed intake (kg DM day−1)Elephant grass 9.45 9.22 10.38 0.44 nsConcentrate 5.82 5.54 5.92 0.08 nsNutrient intake kg day−1

Dry matter 15.27 14.76 16.30 0.42 nsOrganic matter 13.62 13.41 14.58 0.37 nsCrude protein 1.21b 1.16b 1.66a 0.02 ***Neutral detergent fibre 7.07 6.92 7.52 0.30 nsAcid detergent fibre 5.70 5.73 6.20 0.05 nsLignin 1.15 1.13 1.22 0.04 nsStarch 1.96b 2.56a 2.10b 0.04 *Metabolizable energy, MJ day−1 142b 138b 153a 2.97 *

Apparent digestibility coefficientDry matter 0.74 0.74 0.73 0.01 nsOrganic matter 0.77 0.77 0.76 0.01 nsCrude protein 0.67ab 0.62b 0.70a 0.02 *Neutral detergent fibre 0.69 0.70 0.67 0.01 nsAcid detergent fibre 0.68 0.68 0.66 0.02 ns

abc within a row means without common superscript differs. MLM: moringa leaf meal, SBM: soybean meal, SE: standard error, ns: not significant.

Table 4Least square means of milk yield and milk composition for cows fed different concentrates.

Items Treatments S.E. Significancelevel

MLM concentrate Iso concentrate SBM concentrate

Milk (kg day−1) 12.3b 12.1b 13.2a 0.20 *Energy corrected milk (kg day−1) 11.6b 11.3b 12.4a 0.19 *Milk fat (g kg−1 milk) 35.3 34.5 34.9 0.51 nsTotal solids (g kg−1 milk) 126.6 125.5 126.2 0.08 nsNon fat solids (g kg−1 milk) 91.3 91.0 91.2 0.05 nsMilk crude protein (g kg−1 milk) 34.7 34.3 34.6 0.23 nsCasein (g kg−1 milk) 27.6 27.2 27.4 0.22 ns

MLM: moringa leaf meal, SBM: soybean meal, S.E.: standard error.

14 B. Mendieta-Araica et al. / Livestock Science 137 (2011) 10–17

content 306 g kg−1 DM reported by Murro et al. (2003)where twigs were included in the meal. Furthermore, thesimple sun-drying method used here does not seem todecrease the CP concentration and the MLM used in thepresent experiment was 292 g kg−1 DMwhich is in the rangeof 250–297 g kg−1 DM reported in other studies where theleaf meal is produced almost entirely from the leaf fraction(Richter et al., 2003; Kakengi et al., 2007). Moringa seems tobe a promising feed resource with a high nutrient content.However, the in vitro studies of Fujihara et al. (2005) showedthat although the CP content in MLM was high with a highrumen degradability, the proportion of protein potentiallydegrade in the lower tract was lower compared to L.leucocephala, another interesting protein source used intropical regions. It would therefore be valuable if in vivostudies could be initiated in the future to study proteinavailability on Moringa diets in more detail.

The CP content in the Elephant grass used in thisexperiment, 34 g kg−1 DM, is below the range of 49 to82 g kg−1 DM reported for tropical countries (Mendieta-Araica et al., 2009; Sarwatt et al., 2004). However, in tropicalregions, animals are often fed roughages with very lowprotein contents, similar to that of the elephant grass used inthe present experiment (Cândido et al., 2007 and Cavali et al.,2010).

The CP content in the MLM concentrate and Iso concen-trate diets were especially low; indeed, far below 143 g kg−1

DM as is recommended by the NRC (2001) for small breeds inearly to mid lactation producing 12.5 kg milk day−1. Underthe production conditions for farmers who cannot afford tobuy concentrate supplements, these protein levels are,however, common. Although diets with low protein contenthave been reported to depress intake (M'hamed et al., 2001)the DM intake in the present experiment was comparativelyhigh. In fact the low CP content in the diets in this experimentwas to some extent compensated by high DM intakes.Estimated intake by NRC (2001) for small breeds in early tomid lactation with 12.5 kg ECM is 11.2 kg DM per day andcombined with the recommended CP content it renders a CPintake per day of 1.6 kg for 12.5 kg ECM milk production. Inthis experiment the CP allotment was 104% compared to theNRC recommendation for the SBM diet, 76% for the MLM dietand 73% for Iso diet. Although no significant differences inintake were observed in the present experiment, it is notablethat total intake was approximately 1–1.5 kg DM lower in thetreatments where animals were fed diets with a lower CPcontent (i.e. the MLM concentrate and Iso concentratetreatments).

Forages with less than 80 gCP kg−1 DM are defined byLeng (1990) as low quality forages. Although the CP contentpresented in Table 2 shows that the Elephant grass used inthis experiment was a low quality forage, the NDF contentwas within the expected range of 521 to 784 g kg−1 DMreported by Mendieta-Araica et al. (2009) and Sarwatt et al.(2004) and together with a high fibre digestibility this couldbe the reason that intake of elephant grass was as expected.

The chemical composition of the MLM and Iso concen-trates was similar with the exception of a higher starchcontent in the Iso concentrate, which can be attributed to itshigh proportion of sorghum. The difference between thenutrient content of the SBM concentrate and the other

concentrate mixtures was, as expected, substantial, mainlywith regard to CP content but also to a certain extent withregard to energy content.

The DM and organic matter digestibilities averaged 0.74and 0.77, respectively, without significant differences amongtreatments, which is within the range of 0.70 to 0.75 for DMdigestibility reported by Murro et al. (2003) when MLMreplaces cottonseed cake as the protein source in concen-trates for growing sheep and Nouala et al. (2006) when MLMreplaces 25% of commercial concentrates in an in vitro gasproduction study. The lack of significant differences in DM,OM, NDF and ADF digestibility among treatments might bedue to the similar DM digestibility values of MLM and SBM,which have been reported as 0.82 and 0.81, respectively(Sarwatt et al., 2004; Loerch et al., 1983). Although there wasa difference in CP digestibility between the SBM and Isoconcentrate treatments, no difference in CP digestibility wasfound between SBM and MLM concentrates, thus showingthat the CP in the Moringa diet was as digestible as in thesoybean diet.

The MLM and Iso diets in the present experiment had alow CP content. This was largely due to the forage used. Theforage to concentrate ratio was also chosen to representproportions commonly used by farmers in Central America.Therefore, the results of this experiment are directly relevantfor the farming situation of the region.

Milk yield was higher (Pb0.05) from cows fed the SBMconcentrate compared with the other concentrates (Table 4).However, the difference was moderate: approximately 7%lower when cows were fed the MLM concentrate (Table 4). Itis the local prices of SBM compared with milk that willdetermine whether or not MLM is an economical alternativeto SBM in concentrates. The difference in milk yield can beexplained by the higher ME and CP intake when cows werefed the SBM concentrate. There were no significant differ-ences in either milk yield or ECM between MLM and Isoconcentrates, indicating that the protein sources used in thesetwo concentrate mixtures were of similar quality with regardto milk production. Milk composition was similar in all of thetreatments, which is consistent with previous studiesshowing that low protein diets for dairy cows have little orno effect on the fat and protein content in milk (Frank andSwensson, 2002; Nielsen et al., 2003).

There is a common opinion among farmers that dairycows fed fresh Moringa will produce milk with a bad taste orsmell. Makkar and Becker (1997) attribute this bitter taste infresh Moringa to alkaloids, saponins and glucosinolates. Theresults of the organoleptic analysis of milk in this experimentshowed no evidence of quality problems for any of thetreatments. The values for taste, smell and colour were alltypical, with no significant differences among treatments. It ispossible that the earlier mentioned substances disappeared inthe drying and storage process used to produce the leaf meal.

Moringa has been reported to give a fresh matter yield of71.4 Mg ha−1 year−1 corresponding to a dry matter yield of13.5 Mg ha−1 year−1 at 45 days of cutting frequency (Reyes-Sánchez et al., 2006). Even though this was at plantingdensities of 250,000–750,000 plants per ha, which is higherthan the 100,000–170,000 plants per ha used in the presentexperiment, it shows that the production potential ofMoringa is substantial. The amount of work to produce

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approximately 120 kg dried Moringa leaves was 3–4 mandays in the present experiment and the method is thereforeinteresting in situations where labour is available but cashmoney is scarce. It is therefore possible for small farmers inmarginal areas to cultivate Moringa and produce leaf mealthemselves to use as a supplement to their animals during thedry season when there is a crucial shortage of protein in theavailable feed. Still, more studies with MLM in other commontropical diets are needed to obtain more detailed results withregard to how feedingMoringa affects dairy production underconditions where protein quantity and quality are limitingfactors.

5. Conclusion

Moringa leaf meal is a potential source of protein tosupplement poor-quality forage such as Elephant grass. It cansuccessfully replace commercial concentrate constituents fordairy cows as long as the substitution is isocaloric andisoproteinic.

Acknowledgments

The funding for this research provided by the SwedishInternational Development Agency (SIDA) is gratefullyacknowledged. Ing. Pablo Valdivia, the owner of Santa AnaFarm, is also acknowledged for his help and supportthroughout the experimental period.

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17B. Mendieta-Araica et al. / Livestock Science 137 (2011) 10–17

ΙII

Silage quality when Moringa oleifera is ensiledin mixtures with Elephant grass, sugar caneand molasses

B. Mendieta-Araica*, E. Sporndly*, N. Reyes-Sanchez†, L. Norell‡ and R. Sporndly*

*Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala,

Sweden, †Faculty of Animal Science, Universidad Nacional Agraria, Managua, Nicaragua, and ‡Unit of Applied

Statistics and Mathematics, Department of Economics, Swedish University of Agricultural Sciences, Uppsala,

Sweden

Abstract

Fourteen different silages were prepared using mixtures

of Moringa (Moringa oleifera), Elephant grass (Pennise-

tum purpureum cv Taiwan) or sugar cane (Saccharum

officinarum). Molasses from sugar cane was used in the

amounts of either 10 or 50 g kg)1 fresh matter (FM) in

treatments without sugar cane. A completely random-

ized design with three replicates of each treatment was

used. The silages were prepared in 1800 mL micro silos

and opened after 120 d. The presence of Moringa and

Elephant grass in the silage changed the pH by )0Æ8 and

+0Æ7, respectively (P < 0Æ001), indicating a favourable

effect of Moringa on silage pH. Overall differences were

found among treatments for dry matter content, crude

protein and acetic acid concentrations, weight loss, CO2

production and silage pH after spoilage (P < 0Æ001).Weight loss was proportionately 0Æ034 and 0Æ014 in

silages with and without sugar cane respectively

(P < 0Æ001). Overall, differences (P < 0Æ05) were also

found for neutral-detergent fibre and lactic acid con-

centrations, lactic acid bacteria counts, clostridial counts

and time to spoilage of the silages. Treatments contain-

ing Moringa had higher lactic acid concentrations

(+16 g kg)1 DM; P < 0Æ01) compared to treatments

without but the presence of Moringa decreased time

to spoilage by 67 h (P < 0Æ05). No differences were

found in propionic acid concentration or fungal growth

of the silages. It is concluded that Moringa can be used

as a component of high quality silages which also

contain high concentrations of crude protein.

Keywords: Moringa oleifera, Elephant grass, sugar cane,

tropical feedstuffs, ensiling, spoilage

Introduction

In tropical countries where livestock production is

mainly based on grass-dominated pastures, herbage

mass during the dry season is generally not sufficient

to satisfy the nutritional requirements of livestock. To

mitigate this, silage is one of the alternative feeds as it

is relatively simple to produce and utilizes the surplus

in herbage production from the rainy season. Elephant

grass (Pennisetum spp) is commonly used for silage but

authors report that the herbage has a low content of

dry matter (DM) and low concentrations of water

soluble carbohydrates (WSC) and crude protein (CP)

(Andrade and Melotti, 2004; Mtengeti et al., 2006;

Zanine et al., 2006). The composition of the herbage

makes it difficult to produce high quality silage

without the use of additives. Adding molasses, or other

sources of WSC, is widely used to promote a low pH

and high proportions of lactate in such silages (Umana

et al., 1991; Andrade and Melotti, 2004; Mtengeti

et al., 2006). Silage made from sugar cane (Saccharum

officinarum), either pure or mixed with grasses, is also

used in tropical areas. Undesired ethanol production,

however, leads to substantial DM losses and low

quality silage when pure sugar cane is ensiled (Pedroso

et al., 2005).

The low quality of tropical silages is often caused by

the low nutritive value of the herbage ensiled. McDo-

well (1972) studied 312 tropical and 760 temperate

species and found that in more than half of the tropical

grasses the concentration of total digestible nutrients

(TDN) was up to 15 units less than that of temperate

grasses. The need for higher quality silage in tropical

areas calls for new solutions using unconventional

Correspondence to: R. Sporndly, Department of Animal

Nutrition and Management, Swedish University of Agri-

cultural Sciences, P.O. Box 7024, 75007 Uppsala, Sweden.

E-mail: [email protected]

Received 5 February 2009; revised 18 June 2009

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373 doi: 10.1111/j.1365-2494.2009.00701.x364

forage species and, according to Cardenas et al. (2003),

the use of tree foliage as a feed for livestock has

increased. Tree foliage can be used to obtain silage with

higher CP concentrations and offers the possibility to

replace conventional concentrates (Cardenas et al.,

2003).

While there are many agro-forestry species of inter-

est, one of the most interesting trees is Moringa oleifera,

commonly referred to as ‘Moringa’. It is one of the most

widely utilized species (Makkar and Becker, 1996,

1997). Moringa is a fast-growing tree which can reach

12 m in height at maturity and yield up to 88 t ha)1

fresh matter (FM) annually when planted very densely

for use as a forage. The CP concentration in leaves is

about 200–250 g kg)1 DM with a negligible amount of

tannins in all fractions of the Moringa plant and

high levels of sulphur-containing amino acids (Reyes-

Sanchez et al., 2006).

The principal aim of this experiment was to evaluate

the effect on fermentation characteristics whenMoringa

is introduced for ensiling in various mixtures with at

least one of the components of Elephant grass, sugar

cane or sugar-cane molasses. Particular attention was

paid to losses and the aerobic stability of silages. The

hypothesis tested was that the introduction of Moringa

oleifera, as a silage component to improve the nutri-

tional value of silages used for supplementary feeding in

the tropics, will produce silages with fermentation

characteristics and aerobic stability that are comparable

to traditional silages based on pure sugar cane or

Elephant grass with molasses as an additive.

Materials and methods

This experiment was carried out at the National

University of Agriculture (UNA) in Managua, Nicaragua

between March and June 2007. In the laboratory, the

average relative humidity was 55Æ1% and the average

temperature was 31Æ5�C, with the highest temperatures

occurring towards the end of April and a large variation

in both humidity and temperature.

Micro-silo preparation

Non-irrigated, unfertilized Moringa (Moringa oleifera)

foliage (leaves and branches of <5 mm diameter),

Elephant grass (Pennisetum purpureum cv Taiwan) and

sugar cane (Saccharum officinarum) were harvested from

the experimental fields of UNA. Sugar-cane molasses

(molasses) was purchased from an agricultural feed

supplier. The chemical composition of the materials

used is presented in Table 1.

All forages were hand-cut with a machete. Moringa

and Elephant grass were cut 45 d after pruning. Sugar

cane was cut 9 months after the previous cut. All leaves

and roots were removed from the stems and only the

stems were ensiled. Due to the low nutrient content of

the sugar-cane leaves, it is common practice to remove

the leaves along with the roots before ensiling. After

cutting, forages were chopped into pieces of approxi-

mately 2 cm in length using a mechanical chopper.

Once the forages were chopped, fourteen treatments

were prepared from different proportions of Moringa,

Elephant grass and sugar cane for ensiling (see Table 2).

In treatments without sugar cane, molasses without any

Table 1 Dry matter (DM) content and concentrations of

crude protein (CP), water-soluble carbohydrates (WSC),

neutral-detergent fibre (NDF), acid-detergent fibre (ADF) and

lignin of ensiled feeds.

Feeds

Moringa

Elephant

grass

Sugar

cane

Sugar-cane

molasses

DM content

(g kg)1)

193 197 223 721

Concentration

(g kg)1 DM) of:

CP 268 49 27 22

Ash 15 18 15 28

WSC <50 <50 81 472

NDF 521 737 669 ND

ADF 361 374 380 ND

Lignin 119 102 63 ND

ND, not determined.

Table 2 Proportions of Moringa (M), Elephant grass (E), sugar

cane (Sc) and molasses of sugar cane (Sm) in the treatments.

Feeds

Treatment

designation

Moringa

(g kg)1)

Elephant

grass

(g kg)1)

Sugar

cane

(g kg)1)

Molasses

(g kg)1)

M99E0Sm1 990 – – 10

M95E0Sm5 950 – – 50

M67E0Sc33 667 – 333 –

M66E33Sm1 660 330 0 10

M63E32Sm5 633 317 0 50

M33E33Sc33 333 333 333 –

M33E66Sm1 330 660 – 10

M32E63Sm5 317 633 – 50

M33E0Sc67 333 – 667 –

M0E99Sm1 – 990 – 10

M0E95Sm5 – 950 – 50

M0E67Sc33 – 667 333 –

M0E33Sc67 – 333 667 –

M0E0Sc100 – – 1000 –

Silages made from tropical grasses and Moringa 365

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

water dilution was added at rates of 10 or 50 g kg)1 FM.

Mixtures for each treatment were prepared and from

these batches fresh material was taken to fill on average

1564 g FM in glass jars with a nominal volume of

1800 mL. The fresh material was pressed into the jar to

remove as much air as possible. The weight of the fresh

material in each jar was calculated from the difference

between empty and filled jars. The glass jars, subse-

quently referred to as micro-silos, were fitted with

water-locks on their lids to let fermentation gases

escape. Each of the fourteen treatments had three

replicates, giving a total of forty-two micro-silos. The

temperature and relative humidity of the store room

was monitored three times daily. The water-locks were

refilled when needed. Micro-silo weight was recorded

every 2 d at 08:00 h.

All micro-silos were opened and analysed 120 d after

being closed. The contents from each micro-silo were

transferred into a plastic bag, thoroughly mixed and

then samples taken for analysis. The DM content of

each sample was determined by oven-drying at 105�Cfor 12 h. Concentrations of ash, CP (as 6Æ25 · N

concentration) and WSC were determined as described

by AOAC (2000). A Thermo Scientific Orion 2-Star

Benchtop pH meter (Thermo FisherScientific Inc.,

Waltham, MA, USA) was used to determine pH. The

concentrations of neutral-detergent fibre (NDF) and

acid-detergent fibre (ADF) were determined according

to Van Soest et al. (1991). Concentrations of lactic acid,

propionic acid and acetic acid were determined by High

Performance Liquid Chromatography (HP 1100, Agilent

Technologies, Santa Clara, CA, USA) with 40 mL silage

fluid. Before being injected into the chromatograph, the

samples were centrifuged for 10 min at a temperature

of approximately 4�C to prevent loss of volatiles.

Clostridium perfringens, lactic acid bacteria (LAB), aerobic

enterobacteria and fungal growth were determined

according to APHA (2001).

Aerobic stability phase

From each micro-silo, a 250-g sample was taken to

evaluate aerobic stability using the method of Ashbell

et al. (1990) to measure CO2 production as an estimate

of microbial activity. These samples were aerobically

stored and protected by nets against insects. Both the

sample and ambient temperature were measured

during the deterioration process every 2 h.

Once a sample temperature of 5�C above room

temperature was recorded three times in a row, the

sample was considered spoiled and sent, together with

its potassium hydroxide solution, for chemical analysis.

The concentration of CO2 was measured according to

Ashbell et al. (1990). From 10 g, blended for 5 min in a

laboratory blender with 90 mL distilled water, filtrate

pH was determined using a Thermo Scientific Orion

2-Star Benchtop pH meter.

Experimental design and statistical analysis

A completely randomized experimental design was

used, with fourteen treatments and three replicates

for each treatment. The treatments are presented in

Table 2 and, as can be seen in the table, the design was

not symmetric with regard to proportions of compo-

nents. Small amounts of sugar-cane molasses (10 or

50 g kg)1) were included in silages with Moringa and

Elephant grass but not in mixtures with sugar cane. The

reason was that ensiling of sugar cane using sugar-cane

molasses as an additive rarely occurs in practice. Each of

these two components generally contains readily

fermentable WSC, which are favourable for the ensiling

process, and it is regarded uneconomical and unneces-

sary to add molasses when the mixture already contains

sugar cane. A symmetric design would have included a

number of treatments with both sugar cane and sugar-

cane molasses.

A preliminary analysis including the DM content as a

covariate was undertaken. The variable did not improve

the analysis and was omitted. Thus, the variables were

analysed by the one-way model:

yij ¼ lþ si þ eij; i ¼ 1; . . . ;14; j ¼ 1;2;3

where l = overall mean, si = treatment effect and

eij � N(0,r2) is a random error following the normal

distribution. In addition to the overall F-test of treat-

ment effects, pair-wise differences were studied. A

difference was considered as significant if P £ 0Æ05,where the P-values were adjusted for multiple compar-

isons according to Tukey’s method.

The averages of the groups of treatments including a

feed component were compared with averages of the

treatments without the same component to summarize

the effect of including that component in the silage

mixture. It should be remembered that the results of

these comparisons are somewhat uncertain as the out-

come is also influenced by the proportions and combi-

nations of the other components used in the two groups.

To enable an analysis of effects of the proportion of

silage components, mixture experiment models were

investigated, see e.g. Atkinson and Donev (1992). The

first-order model was

yij ¼ b1xi1 þ b2xi2 þ b3xi3 þ b4xi4 þ eij;

i ¼ 1; . . . ;14; j ¼ 1;2;3

where xi1,…,xi4 denote the proportions of Moringa,

Elephant grass, sugar cane and molasses respectively. In

addition to the usual restrictions xi1 + � � � + xi4 = 1 and

xik ‡ 0, the special restrictions xi3 + xi4 ‡ 0Æ01, xi4 £ 0Æ05and xi3xi4 = 0 were added, cf. first paragraph in this

366 B. Mendieta-Araica et al.

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

section. The second-order model for the mixture

experiment was

yij ¼b1xi1 þ � � � þ b4xi4 þ b12xi1xi2 þ b13xi1xi3þ b14xi1xi4 þ b23xi2xi3 þ b24xi2xi4 þ eij

The product xi3xi4 is excluded from the model since

xi3xi4 = 0 implying that b34 is not possible to estimate.

Goodness-of-fit tests were performed by using the

difference of the sums of squares of the one-way model

and of the mixture experiment model as the numerator

in an F-test. In cases where the second-order model was

applied, the product effects were successively tested and

removed until only significant products remained. In

parallel with significance testing of mixture experiment

models, descriptive correlation coefficients of the pre-

dicted values and the treatment means were calculated.

Variables not showing sufficient goodness-of-fit with

the mixture experiment models were analysed by the

one-way model only.

The procedure of GLM in SAS (2004), including the

options MEANS, PDIFF and ESTIMATE, was used for

the numerical calculations.

Results

Chemical composition of silages

The DM content and concentrations of CP and NDF of

each silage treatment 120 d after ensiling are presented

in Table 3. In general, there was a narrow range in DM

content among treatments: 210–269 g kg)1. Elephant

grass treatments had the highest DM contents regard-

less of their content of molasses.

Crude protein concentrations increased markedly

with increasing proportion of Moringa, with the highest

CP concentration being found at the highest proportion

of Moringa (treatment M99E0Sm1, 150 g kg)1 DM)

and the lowest CP concentration with pure sugar cane

(treatment M0E0Sc100, 25 g kg)1 DM). Furthermore,

the ANOVAANOVA model estimated that treatment groups with

Moringa contained significantly higher (+56 g kg)1

DM; P < 0Æ001) concentrations of CP compared to

treatment groups without Moringa, while the presence

of Elephant grass or sugar cane gave significantly lower

CP concentrations, )35 and )30 g kg)1 DM (P < 0Æ001)respectively. Treatment M66E33Sm1 had an unexpect-

edly low CP concentration (Table 3).

The concentration of NDF in silage decreased when

the proportion of Moringa increased. An inexplicably

low NDF concentration was recorded for treatment

M95E0Sm5, 397 g kg)1 DM. It was not possible to

re-analyse the sample for NDF concentration to deter-

mine whether this was an error. All silage treatments

had WSC concentrations lower than the detection limit

of 50 g kg)1 DM with the only exception being the

treatment composed entirely of sugar cane (treatment

M0E0Sc100) which had a WSC concentration of

71 g kg)1 DM.

Fermentation profile

Silage pH, fermentation products and weight loss of

silage from the ANOVAANOVA model are presented in Table 4.

The effect of treatment on silage pH (P < 0Æ01), lacticand acetic acid concentrations (P < 0Æ01 and P < 0Æ001respectively), and weight loss (P < 0Æ001), were signif-

icant in the model. DM content had no significant effect

either on silage pH, fermentation products or weight

loss in the model and was, therefore, as mentioned in

the section on Experimental deign and statistical anal-

yses, excluded from the model.

When tested in the ANOVAANOVA model, the presence of

Moringa in treatments decreased pH values by 0Æ8 (P <

0Æ0001). Correspondingly, the presence of Elephant

grass increased pH values by 0Æ7 (P < 0Æ001). No such

effect was seen with the presence of sugar cane.

The presence of both Moringa and Elephant grass, as

well as the proportion of molasses, affected the lactic

acid concentration by 16 g kg)1 DM (P < 0Æ001),)21 g kg)1 DM (P < 0Æ001) and )12 g kg)1 DM

(P < 0Æ05) respectively. The presence of sugar cane

decreased acetic acid concentration (P < 0Æ05). When

sugar cane was the only silage component, the acetic

acid concentration was below the detection limit.

According to the ANOVAANOVA analysis, there were signif-

icant differences (P < 0Æ001) among treatments with

Table 3 Dry matter (DM) content and concentrations of

crude protein (CP) and neutral-detergent fibre (NDF) of silage

treatments after ensiling for 120 days.

Treatments

DM

content

(g kg)1)

CP

concentration

(g kg)1 DM)

NDF

concentration

(g kg)1 DM)

M99E0Sm1 212 150 583

M95E0Sm5 217 144 397

M67E0Sc33 216 101 517

M66E33Sm1 240 67 518

M63E32Sm5 233 107 628

M33E33Sc33 235 71 622

M33E66Sm1 223 67 691

M32E63Sm5 253 68 562

M33E0Sc67 210 64 614

M0E99Sm1 264 41 689

M0E95Sm5 269 42 710

M0E67Sc33 258 45 776

M0E33Sc67 247 37 651

M0E0Sc100 237 25 740

Silages made from tropical grasses and Moringa 367

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

regard to weight loss of silage. The presence of sugar

cane caused a significantly (P < 0Æ001) greater propor-

tional weight loss from 0Æ014 to 0Æ034 (see Table 4).

Microbiological composition of silages

The microbiological composition of the silage treat-

ments is shown in Table 5. The highest proportion of

Elephant grass resulted in the lowest number of

clostridia (2Æ0 log cfu g)1). A tendency for higher

Clostridia numbers was also observed in treatments

with sugar cane rather than molasses. Intermediate

LAB numbers were found in treatments with Moringa

while the highest number (6Æ5 log cfu g)1, P < 0Æ01)was observed with a combination of Elephant grass and

sugar cane (treatment M0E67Sc33) and the lowest with

a combination of Elephant grass and molasses (treat-

ment M0E95Sm5). There were no significant differ-

ences in fungal growth among treatments. All silages

had Enterobacteria numbers below the detection limit

of log 0Æ5 MPN g)1.

Aerobic stability of silages

Results from the aerobic stability phase are presented in

Table 6. The production of CO2 (P < 0Æ001), time to

spoilage (P < 0Æ01) and pH after spoilage (P < 0Æ001)were all significantly affected by treatment in the

model.

When the estimates were tested in the ANOVAANOVA model,

the presence of Moringa decreased time to spoilage by

67 h (P < 0Æ05). The presence of Moringa caused a

significant (P < 0Æ001) increase in CO2 production

while the presence of Elephant grass caused a decrease

(P < 0Æ001). Increasing the proportion of molasses from

0Æ01 to 0Æ05 decreased time to spoilage by 103 h

(P < 0Æ05); however, treatment M66E33Sm1 had an

unexpectedly long time to spoilage and treatment

Table 4 The concentrations of lactic,

acetic and propionic acids, pH and weight

losses (WL) in silage treatments after

120 days of ensiling. Means from ANOVAANOVA

model with standard error of mean and

level of significance of treatments are also

given.

Treatments

Lactic acid

(g kg)1 DM)

Acetic acid

(g kg)1 DM)

Propionic acid

(g kg)1 DM) pH

WL

(g kg)1)

M99E0Sm1 93abc 31ab 0Æ3 3Æ70b 9g

M95E0Sm5 106a 26ab 0Æ2 3Æ53b 13efg

M67E0Sc33 98ab 17bcd 7Æ0 3Æ49b 24cd

M66E33Sm1 79abc 26ab 2Æ8 4Æ06ab 14efg

M63E32Sm5 93abc 24ab 4Æ5 3Æ49b 11fg

M33E33Sc33 90abc 3d 5Æ1 3Æ71b 24c

M33E66Sm1 73abc 17bcd 1Æ8 5Æ09ab 14efg

M32E63Sm5 75abc 15bcd 5Æ5 3Æ99ab 14efg

M33E0Sc67 99ab 2d 4Æ5 3Æ54b 38b

M0E99Sm1 55c 36a 6Æ0 4Æ70b 20cde

M0E95Sm5 74abc 21abc 6Æ5 4Æ20ab 19def

M0E67Sc33 64bc 4cd 3Æ2 5Æ84a 28c

M0E33Sc67 85abc 2d 2Æ7 4Æ22ab 41ab

M0E0Sc100 92abc >1 2Æ7 3Æ56b 47a

s.e. of mean 5Æ7 3Æ1 1Æ6 0Æ08 0Æ17

P-value <0Æ001 <0Æ001 NS <0Æ01 <0Æ001

Columns without common superscripts differ significantly (P < 0Æ05); NS, not

significant.

Table 5 Concentrations of clostridia, lactic acid bacteria

(LAB) and fungi of each silage treatment after 120 days of

ensiling with standard error of mean and levels of significance of

treatments.

Treatments

Clostridia

(log cfu g)1)

LAB

(log cfu g)1)

Fungi

(log MPN g)1)

M99E0Sm1 3Æ8ab 3Æ6abc 1Æ3

M95E0Sm5 3Æ6ab 2Æ5abc 1Æ6

M67E0Sc33 3Æ2ab 2Æ9abc 1Æ1

M66E33Sm1 3Æ1ab 2Æ4abc 0Æ5

M63E32Sm5 3Æ5ab 3Æ2abc 0Æ8

M33E33Sc33 3Æ5ab 3Æ5abc 0Æ5

M33E66Sm1 3Æ4ab 2Æ9abc 0Æ5

M32E63Sm5 3Æ2ab 3Æ0abc 0Æ5

M33E0Sc67 3Æ3ab 2Æ7abc 0Æ5

M0E99Sm1 2Æ0b 1Æ9bc 0Æ5

M0E95Sm5 2Æ5ab 0Æ8c 0Æ5

M0E67Sc33 4Æ2ab 6Æ5a 1Æ3

M0E33Sc67 4Æ4a 4Æ9ab 0Æ5

M0E0Sc100 3Æ5ab 1Æ9bc 0Æ5

s.e. of mean 0Æ42 0Æ77 0Æ31

P-value <0Æ05 <0Æ01 NS

Columns without common superscripts differ significantly

(P < 0Æ05); NS, not significant.

368 B. Mendieta-Araica et al.

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

M63E32Sm5 had an unexpectedly short time. The

presence of Moringa caused a significant (P < 0Æ001)increase in pH after spoilage; when the estimates were

tested in the ANOVAANOVA model, the presence of Moringa

increased pH after spoilage by 1Æ55 (P < 0Æ001).

Mixture experiment models

With the exception of fungi, all variables in Tables 4–6

were analysed in mixture experiment models and the

variables that showed goodness-of-fit for first or second

order models are presented in Table 7 along with

P-values and the descriptive correlation coefficients

with sample means. The results of the model estima-

tions must be considered while keeping in mind the

restriction of not having the components sugar cane

and molasses in the same mixture.

A mixture experiment model was considered relevant

if the P-value of the goodness-of-fit test was greater

than 0Æ05 and the descriptive correlation coefficient

versus the sample means was at least 0Æ90. A second-

order model was used only if the first- order model was

insufficient. The variables that showed a relevant fit to

mixture experiment models are given in Table 7.

The predicted values based on the first-order model

are given in Table 8. Under the restrictions for the

x values, described in the section on Experimental

design and statistical analyses, the estimated first-order

model for lactic acid concentration has its maximum

at x1 = 0Æ95, x4 = 0Æ05, corresponding to treatment

M95E0Sm5, which also had the highest sample mean

(see Table 4). The minimum value according to the

first-order model is attained at x2 = 0Æ99, x3 = 0Æ01, acomposition not included in the experiment. Among

the studied treatments (see Table 4), the lowest mean

lactic acid concentration of 55 g kg)1 DM was obtained

for treatment M0E99Sm1 with x2 = 0Æ99, x4 = 0Æ01,

Table 6 Carbon dioxide production during the aerobic

stability phase, time to spoilage (TTS) and pH after spoilage of

the treatments with standard error of mean and level of

significance.

Treatments

CO2 production

(g kg)1 DM)

TTS

(h) pH

M99E0Sm1 42ab 81abc 6Æ25bcd

M95E0Sm5 43a 115abc 7Æ50ab

M67E0Sc33 39abc 106abc 4Æ56de

M66E33Sm1 34bcd 303abc 9Æ32a

M63E32Sm5 39abc 39bc 5Æ51cde

M33E33Sc33 34bcd 194abc 7Æ77ab

M33E66Sm1 34bcd 180abc 7Æ73ab

M32E63Sm5 31cde 151abc 6Æ75bc

M33E0Sc67 31cde 237abc 4Æ13e

M0E99Sm1 25e 354a 5Æ18cde

M0E95Sm5 26de 246abc 4Æ62de

M0E67Sc33 30cde 33c 5Æ00cde

M0E33Sc67 28de 191abc 5Æ99cde

M0E0Sc100 31cde 314ab 4Æ54de

s.e. of mean 1Æ7 53Æ0 0Æ38

P-value <0Æ001 <0Æ01 <0Æ001

Columns without common superscripts differ significantly

(P < 0Æ05).

Table 7 Variables [concentrations of clostridia, Lactic acid

bacteria (LAB), lactic acid concentration, weight loss (WL)

and CO2 production] with goodness-of-fit in first or second

order mixture experiment models; p1, p2 and r1, r2 are the

P-values of goodness-of-fit tests and descriptive correlation

coefficients of first and second order models, respectively,

vs. sample means.

Variable p1 r1 p2 r2

Model order

chosen

Clostridia 0Æ081 0Æ54 0Æ84 0Æ96 2nd

LAB 0Æ005 0Æ38 0Æ28 0Æ92 2nd

Lactic acid 0Æ805 0Æ95 0Æ71 0Æ97 1st

WL 0Æ308 0Æ99 0Æ54 1Æ00 1st

CO2 production 0Æ227 0Æ95 0Æ57 0Æ98 1st

Table 8 Variables modelled by the first order mixture

experiment model [ concentration of lactic acid, weight loss

(WL) and CO2 production]. Estimated coefficients with

standard errors (s.e. of coefficient) for predicting equation y and

values of components x1 (Moringa), x2 (Elephant grass), x3(sugar cane) and x4 (molasses) giving proportions of these

components at maximal (prop. at max.) and minimal (prop. at

min.) predicted values. Corresponding sample mean �y is givenprovided the composition exists in Table 2.

Variable x1 x2 x3 x4 y �y

Lactic acid

Coefficient 93Æ8 55Æ0 98Æ5 309Æ9

s.e. of coefficient 4Æ0 4Æ3 4Æ7 91Æ5

Prop. at max. 0Æ95 0 0 0Æ05 104Æ6 105Æ5

Prop. at min. 0 0Æ99 0Æ01 0 55Æ4 Non-

existent

WL

Coefficient 1Æ03 1Æ88 4Æ91 0Æ72

SE of coefficient 0Æ12 0Æ13 0Æ13 2Æ76

Prop.at max. 0 0 1 0 4Æ91 4Æ70

Prop. at min. 0Æ95 0 0 0Æ05 1Æ01 0Æ89

CO2 production

Coefficient 41Æ7 25Æ8 30Æ2 53Æ1

SE of coefficient 1Æ2 1Æ3 1Æ3 27Æ6

Prop. at max. 0Æ95 0 0 0Æ05 42Æ3 43Æ1

Prop. at min. 0 0Æ99 0Æ01 0 25Æ8 Non-

existent

Silages made from tropical grasses and Moringa 369

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

which in the interpretation of the first-order model is

very close to x2 = 0Æ99, x3 = 0Æ01.The final second-order mixture models after removal

of non-significant product effects are presented in

Table 9. The maximum value of the variable clostrid-

ium, according to the second-order model, is obtained

at x2 = 0Æ42, x3 = 0Æ58 with a predicted value of 4Æ49 log

cfu g)1. The two treatments with the highest sample

means of 4Æ41 and 4Æ22 log cfu g)1 (see Table 5)

correspond to x2 = 1 ⁄ 3, x3 = 2 ⁄ 3 and x2 = 2 ⁄ 3,x3 = 1 ⁄ 3. These treatments with predicted values of

4Æ44 and 4Æ07 log cfu g)1 are, in a mathematical sense,

close neighbours to x2 = 0Æ42, x3 = 0Æ58 and the max-

imum value of 4Æ49 log cfu g)1 is a consequence of the

curvature due to the product effect of x2x3. For LAB, the

second-order model attains its maximum value of 5Æ98log cfu g)1at x2 = 0Æ50, x3 = 0Æ50, a treatment not

included in the experiment. The closest treatment

neighbours, i.e. those with x2 = 2 ⁄ 3, x3 = 1 ⁄ 3 and

x2 = 1 ⁄ 3, x3 = 2 ⁄3, yielded the sample means �y ¼ 6:50

log cfu g)1and �y ¼ 4:92 log cfu g)1 respectively. This is

contradictory. However, comparisons of the difference

between the second-order model predicted values, i.e.

y ¼ 5:55 log cfu g)1and y ¼ 5:51 log cfu g)1and the

corresponding sample means using t tests do not give

significant results, t = )1Æ73 and t = 1Æ10 respectively.

For the minimum value, the difference of y ¼ 1:69 log

cfu g)1and �y ¼ 0:78 log cfu g)1 neither leads to a

significance, since t = 1Æ61. The fit of the mixture model

for LAB is not as good as for clostridia, cf. Table 7.

An important reason for the lack of significances is that

the sample means are based on only three observations.

Discussion

Increasing the proportion of Moringa elevated the CP

concentration of silage. The results (range of CP

concentrations of 84–148 g kg)1 DM) are in agreement

with other studies in that foliage from fodder trees

increases the CP concentration of silages (Cardenas

et al., 2003; Phiri et al., 2007). Very little WSC remained

after fermentation, with the exception of silage made

entirely with sugar cane. All treatments without sugar

cane had WSC concentrations below the detection limit

of 50 g kg)1 DM, which is consistent with results from

McDonald et al. (2002). The NDF concentration of

the silages ranged from 396 to 775 g kg)1 DM and

decreased when the proportion of Moringa was

increased. These results are similar to those reported

by Phiri et al. (2007) and could be attributed to the low

concentration of NDF in Moringa.

In silages made from tropical grasses, a pH value of

4Æ2 has been reported as the maximum to consider

silage to be well-preserved (McDonald et al., 2002;

Cardenas et al., 2003). Weissbach (1996) presented a

critical limit for good quality silage depending on DM

content in which pH should be no higher than 0Æ0257DM content (expressed as %) + 3Æ71. With one

exception, pH of the silages where the proportion of

Elephant grass exceeded 0Æ33 was above the limit

suggested by Weissbach (1996), suggesting that Ele-

phant grass had a negative impact on silage quality.

High-quality silage is likely to be achieved when

lactic acid is the predominant acid produced, as it is

the most efficient fermentation acid and reduces silage

pH more efficiently than other fermentation products.

According to the mixture experiment model, the

highest concentration of lactic acid is obtained with a

mixture of 0Æ95 Moringa and 0Æ05 molasses giving

a predicted lactic acid concentration of 105 g kg)1 DM,

a value very close to the value actually obtained for

this silage, 106 g kg)1 DM. This provides evidence that

Moringa silages have lactic acid concentrations supe-

rior to silage made from Elephant grass or sugar cane

and in contrast to findings reported by Cardenas et al.

(2003) and Phiri et al. (2007). Higher concentrations of

fermentation acids are associated with lower DM

contents (McDonald et al., 2002; Pinho et al., 2004)

but, since the DM content was not significantly lower

for the treatments high in Moringa when tested as a

covariate in the ANOVAANOVA model, it is not a valid

explanation. The higher CP concentration in Moringa

should also lead to a higher buffering capacity in

mixtures with high proportions of Moringa although

Table 9 Variables modelled by the second order mixture

experiment model [concentrations of clostridia and lactic acid

bacteria (LAB)]. Estimated coefficients with standard errors (s.e.

of coefficient) for final predicting equation y and values of

components x1 (Moringa), x2 (Elephant grass), x3 (sugar cane)

and x4 (molasses) giving proportions of these components at

maximal (prop. at max.) and minimal (prop. at min.) predicted

values. Corresponding sample mean �y is given provided the

composition exists in Table 2.

Variable x1 x2 x3 x4 x2 x3 y �y

Clostridia

Coefficient 3Æ45 2Æ21 3Æ31 10Æ04 6Æ74

s.e. of coefficient 0Æ27 0Æ31 0Æ31 6Æ26 1Æ54

Prop.at max. 0 0Æ42 0Æ58 0 4Æ49 Non-

existent

Prop. at min. 0 0Æ99 0Æ01 0 2Æ28 Non-

existent

LAB

Coefficient 3Æ45 1Æ99 1Æ87 )3Æ97 16Æ22

s.e. of coefficient 0Æ53 0Æ61 0Æ61 12Æ24 3Æ02

Prop.at max. 0 0Æ50 0Æ50 0 5Æ98 Non-

existent

Prop. at min. 0 0Æ95 0 0Æ05 1Æ69 0Æ78

370 B. Mendieta-Araica et al.

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

this was not determined in this experiment. A high

buffering capacity also results in an elevated lactic acid

production in order to reach a satisfactory low pH

(McDonald et al., 2002).

There was a great variation in acetic acid concentra-

tion among treatments which has already been reported

for tropical silages (Ferrari and Lavezzo, 2001; Pinho

et al., 2004). Even so, the acetic acid concentration did

not reach 60 g kg)1 DM in any of the treatments, which

is considered as the highest recommended level (Card-

enas et al., 2003). Silages usually contain traces of

propionic acid and indeed the concentrations found in

the treatments studied here are within or slightly below

the range 0Æ6–39 g kg)1 DM reported by others (Pand-

itharatne et al., 1986; Lavezzo et al., 1990; Chiou et al.,

2000; Ferrari and Lavezzo, 2001; Pinho et al., 2004).

The model for estimating weight loss during the

ensiling process gave a comparatively high coefficient

for the component, sugar cane, showing clearly that

silage with sugar cane gave high weight losses. Similar

results were reported by Pedroso et al. (2005, 2008)

who pointed out that increased ethanol formation was

associated with increased weight loss.

Many microorganisms are found in fresh forages and,

under the anaerobic conditions that characterize the

ensiling process, some of the most important microor-

ganisms are LAB, clostridia, Enterobacteria and fungi.

Clostridia is not uncommon in raw material, thus, its

mere presence in feedstuffs may be unavoidable. When

clostridia are found in silage it is most likely the result of

faecal or soil contamination. Clostridia were taken into

account for their primary role in organoleptic and

nutritional decay associated with foul smell and

increased DM losses. Among the spore-forming clostri-

dia commonly found in silages, Clostridium perfringens

was chosen since it also can be a potential health hazard

for livestock (Berghaus et al., 2005). Clostridia grow

best at pH values of 7Æ0–7Æ4. They cannot tolerate acid

conditions and a pH of 4Æ2 is usually considered as being

low enough to inhibit growth. In the present experi-

ment, treatments with higher pH values also had more

clostridia growth. The variables studied in the second-

order mixture experiment model were clostridia and

LAB. Concerning clostridia, a mixture of 0Æ42 of

Elephant grass and 0Æ58 of sugar cane gave the highest

counts of clostridia while 0Æ99 of Elephant grass and

0Æ01 of sugar cane gave the lowest. The means obtained

in the ANOVAANOVA analysis also showed that the mixtures

with mainly Elephant grass had lower clostridium

values, near the minimum obtained in the model,

whereas the mixtures with Elephant grass and sugar

cane had high values near the obtained maximum

value of the mixture experiment model. In treatments

containing sugar cane, cultivation and harvest methods

lead to a high probability of soil contamination which

could be the main reason for the high clostridia levels

among these treatments.

In order to preserve high-moisture forages such as

Moringa, a high LAB activity is necessary. Therefore,

the rapid establishment and consequent maintenance

of anaerobic conditions throughout ensiling is essential

for LAB proliferation. For silages made from temperate

grasses, LAB levels of at least 3Æ9 log cfu g)1 are

desirable for a good fermentation process but lower

values have been reported as normal in silages from

tropical grasses (Tjandraatmadja et al., 1994a,b; Pedroso

et al., 2005). The low concentration of lactic acid

combined with high LAB counts in treatment

M0E67Sc33 is unusual but could indicate that there

was an unintentional minor air leakage leading to the

presence of aerobic fungi that consumed the lactic acid

as it was produced. In the second-order mixture

experiment model used in the present experiment, a

mixture of 0Æ50 of Elephant grass and 0Æ50 of sugar cane

gave the highest LAB count. This is not quite in line

with the highest lactic acid concentration, produced in

the mixture of 0Æ95 of Moringa and 0Æ01 of Molasses,

demonstrating that counts of viable bacteria and lactic

acid concentrations do not always harmonize.

When exposed to oxygen in the air, all silages

deteriorate as a result of aerobic microbial activity. As

soon as a silo is opened, it is exposed to the air for the

entire feed-out period. Therefore, silage quality is not

only characterized by its chemical composition, but also

by how long it will remain stable and resist deteriora-

tion once the silo has been opened and the contents are

fed to livestock. The start of the deterioration process is

characterized by a rise in temperature in the silage

indicating increased microbial activity as the silage

spoils.

Treatment M66E33Sm1 had an unexpectedly long

time to spoilage and M63E32Sm5 unexpectedly short,

a difference which was significant in the ANOVAANOVA

analysis. When the first-order mixture experiment

model was used for CO2 production during the storage

stability phase the mixture with the highest CO2

production was 0Æ95 of Moringa and 0Æ05 of molasses.

The model suggested 0Æ99 of Elephant grass and 0Æ01of sugar cane would have the lowest CO2 production.

This indicates that silage with Moringa could have a

somewhat lower aerobic stability after opening the

silo. Nevertheless, there was a great variation in time

to spoilage values observed in this study, something

that has also been presented in other reports under

tropical conditions (Danner et al., 2003; Pedroso et al.,

2008). A relationship between acetic acid and stability

was proposed by Danner et al. (2003) who claimed

that increasing acetic acid concentrations inhibit spoil-

age organisms, thereby promoting exponential

increases in stability.

Silages made from tropical grasses and Moringa 371

� 2009 Blackwell Publishing Ltd. Grass and Forage Science, 64, 364–373

Conclusion

The CP concentration of silage based on Elephant grass

or sugar cane was substantially increased by adding

Moringa. The present study shows that the inclusion of

Moringa also has a positive influence on silage quality.

The pH is lowered and the production of lactic acid is

elevated. Furthermore, ensiling mixtures of Moringa

and sugar cane decreased DM losses compared with

silage based on only sugar cane. By using the mixture

experiment model when evaluating the result of the

fourteen treatments, modelling of other proportions

than those used in the experiment could be performed.

Such operation indicates that, even if ensiling Moringa

alone would give the best fermentation pattern, the

aerobic stability would be better when Moringa is

ensiled in mixtures with Elephant grass or sugar cane.

Equal proportions of Moringa and Elephant grass with

0Æ05 molasses added or equal proportions of Moringa

and sugar cane would provide both acceptable fermen-

tation patterns and stability after opening while still

maintaining the nutritive value of the silage. The

experiment also showed that Moringa alone with 0Æ01–0Æ05 molasses added produces a good silage quality.

Acknowledgments

The funding of this research by the Swedish Interna-

tional Development Cooperation Agency (SIDA) is

gratefully acknowledged.

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ΙV

ORIGINAL RESEARCH

Feeding Moringa oleifera fresh or ensiled to dairycows—effects on milk yield and milk flavor

Bryan Mendieta-Araica & Eva Spörndly &

Nadir Reyes-Sánchez & Rolf Spörndly

Accepted: 10 February 2011 /Published online: 23 February 2011# Springer Science+Business Media B.V. 2011

Abstract Moringa oleifera, either fresh or ensiled, wascompared with Elephant grass as a main feedstuff for dairycows. To test the effects feed had on milk yield, milkcomposition, ration digestibility, and the organoleptic charac-teristics of milk, six lactating dairy cows were used in aChangeover 3×3 Latin Square experiment, replicated twice.With equal intake of metabolizable energy the intake ofprotein and fiber differed (p<0.001) between all diets wherefresh Moringa had the highest and the Elephant grass diethad the lowest intake. Compared with the control diet,ensiled Moringa had higher digestibility (P<0.05) of bothprotein and fiber. With the exception of DM digestibility, nodigestibility differences were found between fresh Moringaand Moringa silage treatments. Milk yield did not differbetween any of the treatments and averaged 13.7 kgcow day−1. Milk composition was similar among all treat-ments. Milk from the fresh Moringa treatment, however, hada grassy flavor and aroma, significantly different from theother two treatments, even though it was normal in color andappearance. No organoleptic differences were found betweenmilk from the control treatment and the Moringa silagetreatment. The conclusion is that Moringa silage can be fed to

dairy cows in large quantities to produce the same quantityand quality of milk as traditional diets.

Keywords Fresh Moringa . Silage . Dairy cows .

Milk yield . Organoleptic characteristics

Introduction

Livestock production is a very important part of theagricultural sector in many tropical countries, representingup to 40% of the agricultural gross domestic product(Steinfeld et al. 2006). The dairy industry, in particular, isof increasing economic importance. For example, milkproduction in Latin America increased by 12% between2003 and 2007 (FAO 2010).

Although regional variation occurs throughout Centraland South America, it is fair to describe intensive dairyfarming in Latin America as consisting of medium- tosmall-scale farms with 10–17 ha and a herd of 15–21 cows(Holmann et al. 2003). These farms generally practice stall-feeding, providing mainly planted Elephant grass (Pennisetumpurpureum) as roughage and either molasses, locally avail-able by-products, or commercial feeds as concentrates. PureHolstein–Friesian cattle are the most popular and produceapproximately 16 kg milk per day (range, 5–27 kg) atconcentrate feeding rates of 1–10 kg per day (de Leeuw et al.1998). Even though such roughage has a low nutritionalcontent, few farmers can afford to sufficiently supplement thediet with conventional concentrates during the dry seasonbecause they cost too much.

Despite the enormous potential for biomass productionin tropical regions, forages are only abundant during therainy season and many authors (Seré et al. 1995; Holmannet al. 2003; Ruiz 2005) agree that the main restriction to

B. Mendieta-Araica : E. SpörndlyDepartment of Animal Nutrition and Management,Swedish University of Agricultural Sciences,P.O. Box 7024, SE-750 07 Uppsala, Sweden

N. Reyes-SánchezFacultad de Ciencia Animal, Universidad Nacional Agraria,P.O. Box 453, Managua, Nicaragua

R. Spörndly (*)Department of Animal Nutrition and Management,Swedish University of Agricultural Sciences,Kungängen Research Station, SE-753 23 Uppsala, Swedene-mail: [email protected]

Trop Anim Health Prod (2011) 43:1039–1047DOI 10.1007/s11250-011-9803-7

increased milk production is the low quality and quantity offeed resources during the dry season.

Grass silages can be used to overcome feed shortageduring the dry season; however, tropical grasses maturequickly and produce low quality silages due to the lownutritive value of the biomass ensiled (Panditharatne et al.1986; Tjandraatmadja et al. 1994). The problem remainsto find inexpensive solutions to the nutritional deficitcreated by scarcity of good quality forages during the dryseason.

The potential for agroforestry to supply animals withhigh quality feed has been recognized by many authors(Pezo 1991; Fernández-Baca 1992; Szott et al. 2000;Mauricio et al. 2008). Trees and bushes are droughttolerant and the foliage is often rich in crude protein (CP)(Benavides 1994; Cárdenas et al. 2003). Furthermore, theyoften show a tolerance to a wide range of managementpractices (Paterson et al. 1998) and may thus enhance thesustainability of the farming system (Reyes-Sánchez2006).

One of the most interesting trees is Moringa oleifera,commonly referred to as “Moringa” or “Drumstick tree”,which grows throughout the tropics. It is one of the mostwidely used species for fodder (Makkar and Becker 1996).It can grow in all types of soils (Duke 1983) and cantolerate dry seasons lasting up to 6 months. The total drymatter (DM) yield from Moringa can be up to24 Mg ha−1 year−1 (Reyes-Sánchez et al. 2006a). Further-more, Moringa contains negligible amounts of antinutri-tional factors, has a high CP content and containssignificant amounts of vitamins A, B, and C in the foliage(Makkar and Becker 1996; Ferreira et al. 2008).

As fresh forage, Moringa has been included into thediets of many different animals. Positive effects on thefeeding behavior in goats (Manh et al. 2005) and on thegrowth rate in sheep (Ben Salem and Makkar 2009) havebeen reported. Fresh Moringa in dairy cow diets has alsogiven favorable production results (Reyes-Sánchez et al.2006b) as has Moringa leaf meal as a protein source in theconcentrate (Mendieta-Araica et al. 2010). Studies havealso shown that Moringa can be ensiled alone or inmixtures with Elephant grass or sugar cane to increase thenutritive value of the silage (Mendieta-Araica et al. 2009).However, diets based almost entirely on Moringa orMoringa silage have not been previously studied in detailfor dairy cows.

Therefore, the aim of this study was to evaluate the milkyield, milk composition and digestibility of three differentdairy cow diets: (1) a conventional Elephant grass diet withcommercial concentrate, (2) fresh Moringa only, or (3)ensiled Moringa only. Special attention was even paid tothe organoleptic characteristics of milk produced by thecows on these diets.

Materials and methods

Experimental design

The experiment was designed as a Changeover 3×3 LatinSquare, replicated in two orthogonal Latin squares, asdescribed by Patterson and Lucas (1962). Each experimentalperiod consisted of 2 weeks for treatment adaptation and2 weeks of data collection with regard to milk yield and feedintake. The last week of each period was used fororganoleptic testing and to estimate digestibility.

Location

The experiment was carried out during the dry season at theNational Agrarian University (UNA) farm in Managua,Nicaragua. The farm is located 12° 08′ 15″ N and 86° 09′ 36″W. The average annual temperature is 27°C and the meanannual rainfall is 1,440 mm, with a marked dry season betweenNovember and May.

Feed preparation

The fields with Elephant grass (P. purpureum cv CT 115)and M. oleifera were already divided into regularlyharvested plots before the start of the experiment in orderto enable circa 45-day harvest intervals throughout theexperiment. Each day, Elephant grass and Moringa (leavesand soft twigs) from a new plot was harvested, choppedmechanically into 2 cm lengths and offered fresh to thecows, resulting in re-growth intervals of 46±3 days.Moringa silage was prepared 120 days before the experi-ment by cutting a field of M. oleifera 45 days after aprevious harvest. After removing the thicker twigs, thematerial was chopped and sugar-cane molasses was addedto at a rate of 5% according to fresh weight. The materialwas then put into 55×97 cm polythene bags and com-pressed by hand. The bags were then sealed to serve assilos. In total 180 bags, weighing approximately 45 kgeach, were stored indoors on a concrete floor until opening.Feed concentrate was mixed at the UNA Feed ConcentratePlant using commercially available feedstuffs: 41% ricepolishing, 30% sorghum, 20% soybean meal, 6% molasses,1.5% calcium carbonate, 1% peanut meal, and 0.5%sodium chloride.

Management

Six dairy cows of the Brown Swiss breed from the farmherd, each in their second or third lactation and weighingon average 456 kg (SD 50), were used in the trial. Thecows were in their fourth week of lactation at the start ofthe experiment. Prior to the experiment, they were injected

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with vitamin A (625,000 IU), vitamin D3 (125,000 IU), andvitamin E (125 IU), treated against external and internalparasites and vaccinated against anthrax. All of the cowswere treated according to EC Directive 86/609/EEC foranimal experiments.

The cows were kept loose in individual, well-ventilatedstalls with concrete floors. Water and minerals were suppliedad libitum. Except during the weeks when fecal samples weretaken, the cows were allowed to exercise daily in a commonarea while the individual stalls were cleaned.

Treatments

The cows were fed three different isocaloric treatmentsduring three periods. The diets in these treatments arepresented in Table 1. They were designed to fulfill DM andmetabolizable energy (ME) requirements (NRC 1988). Theexpected DM intake was calculated with the NRC (2001)equation using the average body weight and milk yield forthe entire herd at the start of the experiment. The controltreatment (60% Elephant grass roughage and 40% com-mercial concentrate) represents the current recommendationfor dairy farmers in Central America (Vélez 1997 andCastro Ramírez 2002). The Moringa treatments containedonly Moringa with 1 kg added molasses to promotepalatability.

Roughages were offered twice daily to each cow at 0700and 1700 hours, while the concentrate of the controltreatment was fed during milking, at 0500 and 1600 hours.The Moringa treatments contained the supplement molasseswhich was thoroughly mixed with the roughages beforefeeding. The DM content of roughages was determinedtwice a week using a microwave oven according to theprocedure described by Undersander et al. (1993) so thatthe DM allowance could be adjusted according to thefeeding plan.

The amounts of feed offered were weighed daily andsampled. Sampling was performed as follows: 1 kg of offeredroughage per cow per day was collected and immediatelyfrozen at −18°C. The frozen samples from each period were

then thawed and pooled into one sample for chemical analysis.The occasional refusals were individually weighed and frozensamples were sent for chemical analysis at the end of eachexperimental period. One kilogram of concentrate wascollected as a sample every week during data collection forchemical analysis (see below).

The cows were hand-milked twice daily with approxi-mately 12-h interval. At each milking, the yield from eachcow was weighed and recorded, and 100-ml samples weretaken in sterile vials. The milk samples were immediatelyrefrigerated at 4°C and then pooled into one sample percow per data collection period for fat and protein contentanalysis. The same milk sampling procedure was repeatedduring the last 3 days of each experimental period fororganoleptic testing.

Digestibility study

The cows were weighed before the start of the experimentand after each period. During the last week of each period,rubber mats were placed in the stalls while all of the fecesfrom each cow were manually collected. During thoseweeks, the cows were supervised around the clock.Whenever a cow adopted the defecation position, a shovelwas put under her tail to collect the feces, therebyminimizing urine and dirt contamination. The feces fromeach cow were put into a large container and covered with alid to avoid evaporation. Once daily, the feces from eachcontainer were weighed and thoroughly mixed. Fivepercent were taken as a subsample and frozen before thecontainers were emptied. When the collection was com-plete, the subsamples from each cow were thawed andmixed together. Approximately 300 g of this mixture wasthen taken as a fecal sample for each animal andexperimental period to be used in the chemical analysis.

Chemical analysis and organoleptic tests

The feed samples and fecal samples were analyzed so as tobe able to estimate apparent digestibility. The samples weredried and ground through a 1-mm sieve before analysis.Ash and DM content were analyzed using AOAC (1990)procedures. CP content was determined using the Kjeldahlmethod (AOAC 1984). Neutral detergent fiber (NDF) andacid detergent fiber (ADF) contents were analyzed usingsodium sulfite as described by Van Soest et al. (1991). Anapparent digestibility coefficient for DM was calculated bycomparing the dietary intake with recovery in the feces.Organic matter digestibility of the roughages was determinedusing in vitro incubation in rumen liquid. To determine ME,0.5-g dried sample was incubated with 49-ml buffer and 1-mlrumen fluid for 96 h at 38°C. Thereafter, the residues wereweighed and combusted and the organic matter digestibility

Table 1 Feeding treatments offered to dairy cows in a Changeover3×3 Latin Square experiment, replicated twice

Treatment Control Fresh Moringa Moringa silage

Feed (kg DM day−1)

Elephant grass 6.65 – –

Moringa foliage – 10.31 –

Moringa silage – – 10.40

Molasses – 1.00 1.00

Concentrate 4.44 – –

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coefficient could be determined. ME was estimated using theequation presented by Lindgren (1979), except for in theconcentrate where the analysis was provided by the feedproducer (UNA Feed Concentrate Plant, Nicaragua).

Nitrogen content in the milk was determined using theKjeldahl method and milk protein content was calculated asN×6.38. The Babcock method used to determine milk fatcontent and the methods used to analyze total solids andcasein were all according to AOAC (1984) procedures.

An experienced panel evaluated the organoleptic char-acteristics of the milk samples. A triangle difference test(Witting de Penna 1981) was applied using a milk samplewith normal sensory characteristics (flavor, aroma, color,and appearance) as standard. Twenty judges were asked todetermine the sensory characteristics in the milk samplesaccording to the score sheet presented in Table 2, where 5 isthe maximum score for flavor and aroma and 3 themaximum for color and appearance. The total score foreach trait is the sum of scores awarded by all of the judges,while classification is the most common score awarded.

Statistical analysis

The data were analyzed using the GLM procedure in SASSoftware Version 9.1.2 (SAS 2004). Tukey's pairwisecomparison was used whenever the overall F test oftreatment means showed a significant result. The mathe-matical model used for digestibility, milk production, andorganoleptic data was the following:

Yijkl=μ + Bi+Pj+Tk+Cl+εijkl, with i=1,2, j=1,…,6 andk=1,2,3, where μ was the overall mean, Bi the fixed effect

of block, Pj the fixed effect of period, Tk the fixed effect oftreatment, Cl the random effect of cow, and εijkl the randomresidual error.

Carry-over effects from previous periods and interactionbetween period and treatment were tested, and then excludedfrom the final model as they were not significant (P>0.10).

Results

Nutrient intake and apparent digestibility

The chemical compositions of the feeds used in the threetreatments are presented in Table 3. Both fresh Moringa andMoringa silage had high CP contents and low NDFconcentrations compared with the Elephant grass used inthe experiment. The CP content in the concentrate wastypical for commercial concentrates in Central America.

Intake of DM was approximately as planned (Tables 1and 4) as there were no refusals in the control treatment andfeed refusals in the Moringa treatments were minor,representing only 0.97% of the daily offered amount. Therewere no significant differences between treatments in MEintake, showing that the treatments were isocaloric asplanned. There was also no difference in the live weightchange between the treatments (data not shown) but a slightdecline in live weight over the complete experiment period(0.19 kg day−1) indicated an underfeeding equivalent to6.5 MJ ME day−1 (NRC 1988).

Nutrient intake and apparent digestibility coefficientsamong the three treatments are presented in Table 4. There

Table 2 Descriptive terms and scores for testing the organoleptic characteristics of milk (adapted and shortened from Witting de Penna 1981)

Aroma Score Flavor Score Color Score Appearance Score

Undoubted characteristic 5 Undoubted characteristic 5 Yellowish white 3 Homogeneous when shaken 3

Normal 4 Normal 4 Markedly white 2 Small lumps after shaking 2

Subtly lack of freshness 3 Subtly impure 3 Abnormal coloration 1 Big lumps after shaking 1

Subtly grassy 2 Subtly grassy 2

Markedly impure 1 Markedly grassy 1

Constituents Elephant grass Fresh Moringa Moringa silage Molasses Concentrate

DM (g kg−1) 153(17.3) 193(1.2) 267(9.8) 727(0.1) 845(10.4)

CP (g kg−1 DM) 108(2.0) 241(5.2) 226(5.7) 22(0.3) 184(1.6)

NDF (g kg−1 DM) 510(7.7) 453(5.8) 435(9.1) nd 91(3.1)

ADF (g kg−1 DM) 314(8.1) 299(5.3) 291(4.0) nd 58(1.1)

Lignin (g kg−1 DM) 30(5.8) 103(6.1) 99(3.3) nd 43(4.5)

Ash (g kg−1 DM) 181(0.3) 93(3.2) 116(3.2) 28(0.1) 77(4.0)

ME (MJ kg−1 DM) 8.5(0.08) 10.9(1.50) 10.8(1.72) 9.2(0.00) 14.6(0.12)

Number of samples 6 6 6 6 6

Table 3 The chemicalcomposition of feedstuffsfed to dairy cows presentedas means with standarddeviations in parenthesis

DM dry matter, CP crudeprotein, NDF neutral detergentfiber, ADF acid detergentfiber, ME metabolizableenergy, MJ megajoule,nd not determined

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were significant differences among treatments with regard toall nutrient intakes with the exception of ME. Significantdifferences (P<0.05) were also found for DM, CP, organicmatter, NDF and ADF digestibility. Compared with thecontrol diet, the Moringa silage diet had a significantlyhigher digestibility of all the measured variables, while thefresh Moringa diet only had a higher digestibility of CP andNDF. With the exception of DM digestibility, no differencesin digestibility were found between Moringa treatments.

Milk yield and composition

The average daily milk yield during the experiment was13.7 kg. There were no significant differences betweentreatments with regard to yield or milk composition. On

average, the milk contained 35.1 g fat kg−1, 123 g totalsolids kg−1, 34.5 g protein kg−1 protein and 27.3 gcasein kg−1 (Table 5).

Organoleptic characteristics

Results from the organoleptic characteristic tests arepresented in Table 6 and can be compared with the scoringsystem in Table 2. The color and appearance of milk fromall three treatments were classified as normal. No differ-ences in these traits were found among treatments.However, there were significant differences (P<0.001) inthe scores for flavor and aroma between treatments. Themilk from the control and Moringa silage treatments wereclassified as normal by most judges, while milk from the

Table 4 Least square means of nutrient intake and apparent digestibility of three different feeding treatments fed to dairy cows

Items Treatments Standard error Significance level

Elephant grass+concentrate Fresh Moringa Moringa silage

Nutrient intake (kg day−1)

Dry matter 11.1b 11.2ab 11.3a 0.03 *

Organic matter 9.6c 10.2a 10.1b 0.03 **

Crude protein 1.53c 2.48a 2.39b 0.01 **

Neutral detergent fiber 3.79c 4.64a 4.49b 0.02 **

Acid detergent fiber 2.35c 3.06a 3.00b 0.01 **

Lignin 0.39c 1.06a 1.01b 0.01 **

Metabolizable energy (MJ day−1) 121 120 120 0.41 ns

Apparent digestibility coefficients

Dry matter 0.64b 0.69b 0.76a 0.02 *

Crude protein 0.74b 0.81a 0.83a 0.01 *

Organic matter 0.64b 0.70ab 0.77a 0.02 *

Neutral detergent fiber 0.37b 0.54a 0.66a 0.04 *

Acid detergent fiber 0.33b 0.43ab 0.61a 0.04 *

ns not significantabcWithin a row means without common superscript differs

*P<0.05, **P<0.001

Table 5 Least square means of milk production and milk composition from cows fed three different treatments

Items Treatments Standard error Significance level

Elephant grass+concentrate Fresh Moringa Moringa silage

Milk (kg cow−1 day−1) 13.9 13.6 13.7 0.13 ns

Energy corrected milk (kg cow−1 day−1) 13.0 12.9 12.8 0.13 ns

Milk fat (g kg−1 milk) 34.9 35.3 35.1 0.17 ns

Total solids (g kg−1 milk) 122 123 123 0.37 ns

Milk crude protein (g kg−1 milk) 34.5 34.6 34.2 0.19 ns

Casein (g kg−1 milk) 27.3 27.3 27.3 0.17 ns

ns not significant

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fresh Moringa treatment was classified as subtly grassy bymost judges (Tables 2 and 6).

Discussion

Latin American farmers commonly feed Elephant grass as thebasal diet to dairy cows. However, Elephant grass maturesrapidly, losing protein content, thus resulting in the need tosupplement the basal diet with commercial concentrates. Aninteresting feeding alternative is to replace Elephant grass withMoringa in dairy cow diets. Previous studies have includedMoringa in the roughage to reduce the need for other proteinsupplements (Reyes-Sánchez et al. 2006b). In the presentexperiment, M. oleifera constitutes the entire roughage dietwithout any protein supplement and only complementedwith 1 kg of molasses. This is a level of Moringa in dairycow diets that to our knowledge, has not previously beentested under experimental conditions.

Elephant grass can be fed either fresh or ensiled. In orderto ensure high quality, the Elephant grass must be cut atregular intervals. Prolonged harvest intervals result indramatic decreases in both digestibility and CP content. Itis increasingly common that Latin American farmers ensileElephant grass at the right stage of development to ensurequality (Reyes-Sánchez et al. 2008, 2009). The nutrientcontent of Moringa is less sensitive to cutting intervals andretains better digestibility and CP content over longerperiods (Reyes-Sánchez et al. 2006a). Moringa can alsobe ensiled, either alone or in combination with other crops(Mendieta-Araica et al. 2009). Although it is not asnecessary to ensile Moringa as it is Elephant Grass, it ispractical to feed Moringa from a silo rather than harvestingand transporting the roughage daily.

CT-115, an improved variety of Elephant Grass devel-oped by the Cuban Institute of Animal Science throughtissue culture, was used in the present experiment. The

mean CP content (108 gkg−1 DM) is within the range(71–143 gkg−1 DM) reported by other researchers for thisvariety (Valenciaga et al. 2001, 2009) but higher than therange (49–82 gkg−1 DM) reported for other Elephant grassvarieties (Mendieta-Araica et al. 2009; Sarwatt et al. 2004).

The CP contents of fresh and ensiled Moringa reported inTable 3 are considerably higher than the 144 gkg−1 DMreported previously (Mendieta-Araica et al. 2009). This isexplained by the fact that only the leaves and soft twigs wereused in this experiment, whereas leaves, twigs, and brancheswere used in the previous study. A similar pattern is presentedby Fujihara et al. (2005) who report 265 g CP kg−1 DM inleaves compared with 195 g CP kg−1 DM in a mixture of softtwigs and leaves. Makkar and Becker (1997) also report agreat variability in CP content for different parts of Moringa:leaves contain 264 g CP kg−1 DM, while twigs and stemscontain 72 and 62 g CP kg−1 DM, respectively.

The main objective of this study was to see if Moringacould maintain the same level of production as thetraditional diet of Elephant grass and concentrates, thusenabling farmers to produce high-quality feed for theirdairy cows without having to purchase concentrates.Moringa has a high CP content and as the diets weredesigned to cover the energy requirements of the cows theMoringa diets contained approximately 60% more proteinthan the control diet. This excess protein was highlydigestible as can be seen from the digestibility values(Table 4). The higher protein feeding did not lead to ahigher milk production and the excess nitrogen had to beexcreted in the urine at an energy cost for the animal. Thehigher digestibility of the nutrients in the Moringa treat-ments could theoretically provide this extra energy.

The digestibility of Moringa silage has not previouslybeen studied; however, the digestibility values observedwere better than for silages from others forage trees andshrubs. Cárdenas et al. (2003) reports DM in vitrodigestibility values for four forage tree silages between

Table 6 Organoleptic evaluation scores and average sensory classification for milk from cows fed one of three treatments

Attribute Max score Elephant grass+concentrate Fresh Moringa Moringa silage Significance level

Total score Classification Total score Classification Total score Classification

Flavor 100 79a 4 45b 2 81a 4 *

Aroma 100 80a 4 47b 2 79a 4 *

Color 60 60 3 60 3 59 3 ns

Appearance 60 59 3 59 3 59 3 ns

Total scores are the sum of the scores given by 20 experienced judges

High scores correspond to high milk quality

ns not significantab Scores within a row without common superscripts differ significantly

*P<0.05

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45% and 58% and organic matter in vitro digestibility forthe same silages between 46% and 59%. In a study withmixed silages, Acacia boliviana with maize had a DMapparent digestibility of 53.5% and Leucaena leucocephalawith maize 56.5% (Phiri et al. 2007).

As mentioned earlier, there were no significant differ-ences in milk production between treatments despitedifferences in CP intake at a production level of13.7 kg cow day−1 for milk yield, or 12.9 kg cow day−1

ECM. According to Oldham (1984), lactating cows feddiets which meet their ME and CP requirements are notexpected to respond to increased protein levels but areduced ME utilization can sometimes be observed withexcessive CP intakes. A higher CP intake, however, is oftenassociated with a higher dry matter intake (Kokkonen et al.2002). Restricted feed allowance, as practiced in the presentexperiment, did not allow a higher feed intake and thisaspect could therefore not be studied.

Milk composition was not affected by the treatments,which is consistent with previous findings that show norelationship between milk protein content and CP content inthe diet when the diets have similar energy concentrations(Sutton 1989; Schingoethe 1996; Reyes-Sánchez et al.2006a).

In countries where farmers feed Moringa to dairy cows,there are reports that it gives an off-flavor to the milk(Reyes-Sánchez 2006). To avoid a grassy flavor and aromain the milk, Agrodesierto (2010) recommends that during atleast 3 h prior to milking, fresh Moringa should not be fedto cows. This recommendation is, however, difficult forintensive dairy farmers to follow. In a study by Reyes-Sánchez et al. (2006a) where 20% of the diet consisted ofMoringa, no effects in milk flavor were observed. In thepresent experiment, the cows fed fresh Moringa withmolasses produced milk with inferior organolpetic charac-teristics. It is important to note that we found that the samequantity of ensiled Moringa with molasses did notnegatively affect milk quality.

How flavor is passed on to milk has been widely studied(Dougherty et al. 1962; Shipe et al. 1962; Shipe et al.1978). Makkar and Becker (1997) attribute the off-taste inmilk from cows fed fresh Moringa to glucosinolates. Shipeet al. (1962) report that the presence of esters gives off-tasteto milk when introduced into either the rumen or the lungs.Accordingly, Bennet et al. (2003) report appreciableconcentrations of 4-(α-Lrhamnopyranosyloxy)-bensylglu-cosinolate, three monoacetyl isomers of this glucosinolateand the esters 3-caffeoylquinic and 5-caffeoylquinic inMoringa leaves.

Ensiling results in up to a 90% reduction of glucosino-lates concentrations (Nash 1985; Fales et al. 1987; Pancieraet al. 2003). That Moringa silage did not lead to the sameoff-flavor and aroma in milk as fresh Moringa did could be

due to a reduction in the concentration of glucosinolatesalready mentioned.

In conclusion, the results of this study show thatensiled Moringa can be fed to dairy cows in largequantities without any negative effect on nutrient intakeor digestibility. Cows fed large quantities of Moringaproduce milk in as much quantity and as high in qualityas cows fed conventional Elephant grass diets. Whereas afresh Moringa diet can lead to an off-flavor and aroma inmilk, a Moringa silage diet leads to good organolepticmilk characteristics.

Acknowledgments The funding for this research, provided by theSwedish International Development Agency (Sida), is gratefullyacknowledged. Börje Ericsson, is also acknowledged for his helpand support throughout the laboratory analysis.

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