THESIS PROPOSAL

Name: Dao Duc BienPosition: Msc Student

Advisor:

Graduate School, Khon Kaen UniversityID: 525 030 100-0Department of Animal Science

Major advisor:Professor Dr. Metha Wanapat

1. TITLE

Effect of Sano (Sesbanina rostrata), Leucaena (Leucaena leucocephala), and Mulbery (Morus alba) meal replacement for soybean meal in concentrate mixture on milk yield and milk composition in lactating cows.

2. INTRODUCTION

Livestock plays a very important role as integral part of farming and rural life in developing countries that is providing food also the critical cash reserve and cash income for many farmers who grow crops essentially for subsistence purposes (Preston and Leng 1987). Ruminant diets in most developing countries are based on fibrous feeds and crop residues especially cereal straws. These feeds are imbalanced and are particularly deficient in protein, minerals, vitamins, and are highly lignified, consequently inadequate feed supplies is the most limiting constraint to high milk production and milk quality (Wanapat and Devendra, 1986; Preston and Leng 1987; Wanapat 1999,). Moreover, the high cost of protein sources for livestock is a perennial problem in many developing countries. Soybean oil meal (SBOM) and fish meal are still the major sources of protein in finished feeds. The increasing importation of these protein sources contributes to the financial drain in developing countries. Also, the rising cost of these feedstuffs is fast becoming prohibitive for feed millers and growers (Lopez 1989). Animal feeds generally account for up to 70 percent of the cost of production and within these costs, protein sources are likely to have a significant impact. Under the prevailing conditions and level of livestock production in Thailand, an increase in production can be anticipated. A number of local protein sources have been used in animal rations. However, soybean meal/cake and fishmeal are the major protein sources used and are mostly imported. In order to achieve the future goal of lowering imports and costs, alternative sources of competitively priced protein from different crop origins could have potential and be exploited for a more sustainable feeding system (Wanapat 2001).

Therefore, this has lead researchers to direct increased attention to nonconventional feeds, giving more emphasis to protein substitutes to deal with those of above problems with aims to improve nutritive value of feeds and reduce the cost for farmers. Efficient supplementation of locally mixed concentrate with grains or protein foliages has been demonstrated to improve rumen ecology, dry matter intake and subsequently meat and milk quantity and quality such as cassava root/hay/silage, Leucaena, Phaseolus (Wanapat 1999). One such non-protein nitrogen (NPN) source that has been used is urea. Urea is a non protein nitrogen, its low cost compare with true protein feeds and widely use as feed supplementation for ruminants. There were many researcher have shown that urea treatment could increase nutritive value, particularly the crude protein content, digestibility and voluntary feed intake by ruminant (Wanapat 1985). In addition, urea is one of the ingredients used to make concentrate with level 2 to 4 % (Khampa et al., 2009). Urea is also used in urea-molasses blocks which have been shown to improve straw or low quality roughage utilization, feed intake, rumen fermentation, end products as well as increasing live weight gain (Leng, 1984; Wanapat et al., 1991). The used of high quality feed pellet containing 10% of urea result in enhancing milk yield and compositions particularly when dairy cows were fed low quality roughage such as urea treated rice straw (Wanapat et al., 1996).

Interestingly, trees are playing an increasingly important role in agricultural production systems in the tropics particularly legumes trees. They have beneficial effects on soil fertility (by protecting soil from erosion and supplying nutrients by nitrogen fixation and incorporation of organic matter); they provide shade which helps to reduce heat stress of cattle in hot and humid areas; and they are an important alternative as forage source, due to their high production of edible, highly-acceptable biomass and drought resistance (Otsyina and McKell 1985; Preston and Murgueitio 1987). Legume crops have been considered to be suitable crop to enrich the soil fertility and for producing high protein food and feed for both human and animals consumption (Polthanee et al., 2001).

The utilization of plant leaves as possible sources of protein is one among many possibilities. Studies on shrub and tree leaves, leaf vines, grasses, and algae and other water plants have shown that, on a 90% dry-matter basis, their crude protein (CP) contents vary from 20 to 30%, crude fibre (CF) from 12 to 18%, and xanthophyll from 500 to 650 ppm. Although plant leaves are good sources of protein for both nonruminant and ruminant (Lopez 1989). Tree leaves have a high protein content (18-26% crude protein on average), and some of them have low rates of degradability in the rumen (Espinosa 1984). There are several scientists have been doing their research works

on many kind of tropical fodder trees from far in the past until now such as Manihot esculenta, Leucaena Leucocephala (Lopez 1986; Wanapat et al., 2007), Trema orientalis Castillo et al. (1981), Sesbania rostrata Sazon (1988), Moringa oleifera and Leucaena leucocephala Cariaso (1988), Muros indica, alba Castillo et al. (1980) Pisonia alba Rigon et al. (1983).These characteristics, along with those mentioned above, make them an alternative source of by-pass protein to be evaluated as a supplement for ruminant production systems in the tropics. The extent to which tree foliage protein is degraded in, or escapes from the rumen is extremely important. If the tree foliage protein is totally degraded, it provides ammonia and minerals for microbial growth (Leng 1993).

There are many available feed resources in tropics are a potential use in ruminants feeding and particularly in the development of food-feed-systems that are not only beneficial for human and animals but also for the environment (Wanapat, 2009). One amount of that is well known as sano (Sesbania rostrata). Sesbania rostrata Bremek & Oberm, a stem-nodulating species, has been the most successful green manure in increasing the yield of rainy-season lowland rice in Asia . Its potential to supply much of the N needed for moderate to high rice yields in the infertile soils in northeast Thailand (Herrera et al., 1997). Sazon (1988) was shown that Sesbania rostrata leaf meal (SLM) on a dry-weight basis, SLM has 29.7% CP, 7.6% EE, 15.3% CF, 27.6% NFE, 7.8% ash, 0.78% Ca, 0.23% P, and 467 ppm xanthophylls. Akbar et al., (1999) suggested that the supplementation of rice straw-base rations either with Sesbania or Lthyrus fodder in fresh form has increased by 0.3-0.4kg/d live weight gain and 20-26% milk production of the indigenous dairy cattle of the rural farmers in Bangladesh.

Leucaena (Leucaena leucocephala) is a deep rooted perennial leguminous tree or shrub with foliage of very high nutritive value for ruminant production. It is palatable, nutritious, long-lived and drought-tolerant. The origin of the genus generated from the North America and through Mexico, central and South America. The leucaena was introduced in philipine and South-East Asia for use as a shade plant in tea and coffee plantations by Spanish colonists around the 1600s. After that, it was used as a multipurpose plant such as: timber, fuel wood, furniture and in agroforestry systems; as a shrub, it is also used as forage for feeding of ruminant livestock. Leucaena can be considered as important protein source for ruminant in the tropical and subtropical regions. Its protein content is at high levels of 29.2 % crude protein (CP) in leaf meal and 22.03 % CP in forage (Garcia et al., 1996). Moreover, it contains condensed tannin content of 2-6% (Tropical forage) that can protect protein from rumen microbial degradation and reduce methane production (Suchitra et al. 2008). Leucaena does not cause bloat, presumably due to its tannin content (Gupta et al. 1992). In order to take

these advantages of leucaena, many studies have been done to find out the effective of leucaena utilization.

An other potential fodder tree is Mulberry (Morus alba) is growing under varied climatic conditions, ranging from temperate to tropical. The biomass yield of fresh leaves is often in the order of 25 to 30 tones/ha/year with cutting interval of about 9 to 10 weeks, while leaves have a high protein content (18% to 25% in DM) and high (75 to 85%) in viv0 DN digestibility (Ba et etl., 2005). The mulberry leaves can also be used as the main feed for sheep (Liu et al., 2004), goats (Bakshi and Wadhwa, 2007) and (Sanchez, 2000; Meijia, 2002) have been used to replace concentrates in dairy cattle, or goats diets (Anbarasu et al., 2004). With its high digestibility and high in level of crude protein, mulberry foliage can be a comparable source to commercial concentrates for ruminal feeding and production (Sigh and Makka, 2002).

However, effect of using Sesbania, Leucaena, and Mulberry leave in form of meal to replace for soybean meal in concentrate mixture on milk yield, milk quality, and economic return of dairy cows in mid lactation feed with rice straw has not investigated yet. Therefore, the objectives of this work are following as:

2.1. Objectives

2.1.1. To study the effect of Sano, Leucaena, and Mulberry leaves meal on feed intake, feed digestibility, rumen micro-organism ecology, BUN (Blood urea nitrogen), and MUN (Milk urea nitrogen) compare with those of soybean meal diet.

2.1.2. To study effect of these of feeds on milk yield, milk component.

2.1.3. Economic returns when use these of feeds replacement for soybean in concentrate.

2.2. Scientific hypothesis

2.2.1. Sano, Leucaena, and Mulberry leave meal could be replaced for soybean meal in concentrate mixture. It could be improved feed intake, feed digestibility, rumen ecology, milk yield and milk components.

2.2.2. Reduce cost of feeds and high economic return when use Sano, Leucaena, and Mulberry leave meal replacement for soybean meal in concentrate.

2.3. Expected outcomes

2.3.1. Nutritive value of those feeds will be determined and obtain in report

2.3.2. Feed intake, feed digestibility, rumen micro-organism ecology, BUN, MUN, milk yield, and milk components will be measured and obtain in report.

2.3.3. The reduced cost and calculation of economic return will be estimated in scope of the cost of concentrate feeds and price of milk.

2.3.4. The results of this work further are able to apply for replacement of soybean meal in small holder farm.

3. LITERATURES REVIEW

3.1. Dairy cattle situation in developing countries

Livestock farming is crucially important for provision of animal-based food products for the population, and as a source of income for many resource-poor farmers in developing countries. With the increase in human population and economic growth of many Asian countries, the demand for livestock products is likely to double in the coming 20 years. However, the main constraint to livestock development in these countries is the scarcity and fluctuation in the quality and quantity of the year-around animal feed supply. Increased populations and industrialization are making arable land scarce and in addition a large area of the available arable land is being degraded due to human activities (Makkar, 2006). Recent FAO statistics show that, while milking the same number of cows (about 110 million head) the developing countries (mainly located in the tropical zone) produce only 22 % of the whole fresh milk equivalent produced by the developed countries and 18 % of the total world production (461.5 million t). In addition, milk production in Asia and to a lesser extent in Africa was reduced from 1986 to 1987 due to drought and the policy measures taken by some countries. In spite of successful achievements such as “Operation Flood” in India, many failures have also been observed in the past. The problems encountered in stimulating milk production in developing countries are very complex. As in other agricultural development operations many difficulties such as pricing, marketing, etc are beyond the control of the producer. However, technical constraints including nutrition, health and breeding, have still to be, and can be overcome. (Wanapat, 1999) has mentioned that the poor quality of feed is the main factor effect on low quantity and quality of milk. Moreover, dairy cow in tropical condition are normally fed in a system base on pasture and crop residues that often lack of protein and energy source. Actually, land source is low for plant pasture for ruminant because of using for grain production of human consumption. Therefore cattle fed with dry residues or by-product crop such as rice straw, corncob, sugarcane from factories and small levels of fresh grass which are poor in protein source, mineral and vitamin as well

as. In addition, in the dry season, fresh grass strongly reduce is big problem with smallholder farm. In term of low quality of roughage source are poor digestibility and low feed intake. Improvement roughage sources and supplementation of concentrate are choices to achieve high dry matter intake, but this use or not depends on the economic return and the margin between feed cost and the price of milk. Thus, substitution of traditional feeds in the diets of dairy cows is common as economic conditions changes. Soybean meal (SBM), corn or cereal gains have long been used as a prominent source of crude protein (CP) for dairy cows, however, with its increasing price, the use results in higher cost of production (Wanapat et al., 2007). Using feed sources are use extensively for both human consumption, non-ruminants and ruminants to replace for expensive source as soybean are imported by tropical countries.

Nowadays, milk production and consumption have so far been concentrated in the developed regions - mainly Europe, North America and Australasia - though more recently Japan has also become an important milk consuming and producing country. In contrast, the developing countries, with about three quarters of world population, account for just one quarter of world milk output and, as net imports have risen, for slightly over a quarter of global consumption of milk and milk products. Milk consumption highly increase due to the increasing demand of human especially children or some countries as Thailand, the government provided for the establishment of a Milk Authority to promote milk production and consumption through a mass media- directed campaign, and initiatives such as milk-in-school. But, recent FAO statistics has shown that, while milking the same number of cows (about 110 million head) the developing countries (mainly located in the tropical zone) produce only 22 % of the whole fresh milk equivalent produced by the developed countries and 18 % of the total world production (461.5 million t). In addition, milk production in Asia and to a lesser extent in Africa was reduced from 1986 to 1987 due to drought and the policy measures taken by some countries. In spite of successful achievements such as “Operation Flood” in India, many failures have also been observed in the past.

Although, total heads of dairy cow in developing countries much higher than developed countries (Table 1), but the milk yield and milk quality is lower. Therefore, every year, developing countries still have to import milk for developed countries. The lack and low quantity and quality of feed; landless are reasons for this.

Table 1. Dairy cow production in the world, developed countries and developing countries.

Stocks (dairy cow heads) 2005 2006 2007

World 242,546.072 244,937.607 246,861.764

Developed country 46,089.606 49,590.466 49,845.120

Developing country 71,893.596 75,170.225 75,402.669

Source: FAO statistical analysis, 2008.

The light future for milk in developing countries is the satisfaction good quality of nutrients for dairy cows.

Nutrient requirement of dairy cows

Dairy cows have an enormous potential to produce animal carbohydrate, protein and fat, but they also have high nutrients requirements to achieve this potential. For example, over 12 months the quality of protein produced by Friesian cows in milk can vary from 0 to 1 kg/d. This is equivalent to beef steer just maintaining weight through the gaining weight at 8kg/d, or more than four times faster than in commercial herds. To achieve such performance levels, dairy cows must be able to consume up to 4% of their live weight as dry matter each and every day (NRC, 2001).

Protein requirement

The amount of protein a cow needs depends on her size, growth, milk production and stage of pregnancy. However, milk production is the major influence on protein needs. Crude protein needs at different levels of milk production are shown in (table 2 below). Protein is measured as crude protein, which is the sum of rumen degradable protein plus undergradable dietary protein. The digestible crude protein (DCP) is widely used to evaluate protein requirements, and it corresponds to the crude protein that remains after losses in the feces. However, a new system has been introduced which takes into account the degradability of the protein in the ration during digestion. It is a better system to calculate requirement levels, especially for high-yielding cows which have been shown to benefit from protein that escape microbial degradation in the rumen and is absorbed as amino-acids in the small intestine. Following this approach crude protein can be split into rumen degradable protein (RDP) and rumen undegraded protein (RUP). Fish

meal is for example considered as a good source of RDP and soybean is good source of RUP for dairy cattle.

Inorganic nitrogen sources from plants as well as other non-protein nitrogen, such as urea, are completely degraded by microbes in the rumen. Hence, the Rumen degradable Nitrogen (RDN) is broken down by rumen microbes and used for their protein synthesis by the microbes. Later in the digestion process the microbes are themselves digested and the microbial protein becomes available to the animal Nevertheless this microbial synthesis is only optimal when the animal receives sufficient energy supplements. Therefore, if sufficient RDP is not available, the rate of digestion of fibrous as well as concentrate rich diets will be reduced. This leads to a reduction in intake, lower energy supply and reduced milk production. On the other hand, some protein nitrogen can resist microbial breakdown in the rumen and can pass directly to the cow’s intestine. This feed protein fraction is called by-pass protein which is especially profitable for high-yielding cows. At a low level of productivity a cow can meet her protein requirements entirely from microbial protein and the diet only needs to contain degradable protein. This explains why such a cow can be fed with urea or chicken manure instead of high quality protein can meet the protein requirements. It is therefore important to have the optimum balance of RUP and RDP in the diet.

Overall, various protein sources were found for using for dairy cow such as, fish meal, soybean meal, corn meal, etc. however, the cost of these feed is very high if comparing with return source for dairy cows. Finding the feed source to replace for high price feed is importance for developing countries.

Table 2. Crude protein need of a cow at different stages of lactation.

Stage of lactation Crude protein requirements (%)

Early lactation 16-18

Mid- lactation 14-16

Late lactation 12-14

Dry 10-12

Source: Target 10, 1999 (Modified by John, 2005)

3.2. Feed resources for ruminants as well as dairy cows in tropical areas

3.3. Using sano (Sesbania rostrata) for ruminant

Legume crops have been considered as suitable crops for the intercropping systems. Legume crops are used with the aim of enhancing soil fertility through root nitrogen fixation and mulching of crop residues (Ashokan et al., 1985). In addition, under tropical condition, legumes are used for ruminant consumption because of containing high crude protein, vitamin content and low fiber content. Hence legumes normally are use as protein sources for every ruminant, including dairy cows. Moreover, condense tannin found in legume can protect high quality protein escape from rumen bacterial degradable in stead increase bypass protein.

Like many other legumes, Sesbania rostrata is a leguminous plant that can fix atmospheric nitrogen through both root and stem nodules. Its use as fodder was assessed on 4-month-old stem-inoculated plants grown in the field. The studies on Sesbania rostrata reported that the shoots had high crude protein and degradable nitrogen, but low neutral-detergent and acid-detergent fiber, lignin, total extractable phenolics and tannins, and extractable condensed tannins. Dry matter and protein degradabilities were high. S. rostrata is a potential protein supplement for ruminants (Valarini, 2003). Sesbania rostrata have been done as effect of Sesbania rostrata leaf or silage for ruminants but the study on Sesbania rostrata pellet have not been done or very scare. Therefore, the objectives of this study are to evaluation the nutritive value of Sesbania rostrata pellet on rumen fermentation, digestibility of nutrients and milk yield, milk composition of dairy cattle.

In tropic condition, legumes are used as protein source for replacement soybean meal or corn meal. Legumes provide high-quality protein and energy, often critical during the dry season when animals, feeding solely on grass, lose much of the weight they gained during the wet season. Pasture scientists in the tropical region had shown the potential and value of grass-legume pasture and an increasing number of cattle raisers are appreciating it. The nutritive value and digestibility of tropical legumes is higher than that of tropical grasses and the quality of herbage from grasses rapidly declines with increasing maturity. In contrast, herbage from the legumes remains good throughout the growing period, except for the fodder trees which become woody as they mature, although such a situation is easily overcome by regular lopping of the plants. In addition most of the tropical legumes are more productive than the grasses during the dry season, making them more valuable as sources of additional high quality feed during this period, thus increasing the year-round carrying capacity of pasture.

Sesbania is a genus of flowering plants in the pea family, Sesbania includes over 50 species (Schreb, 1770; Jacq, 1792). Among them, some species of Sesbania have been studied as protein source for ruminant, such as Sesbania grandiflora appears to be the most promising of the four foliages that were evaluated on the basis of high voluntary intake of dry matter, high digestibility and growth rate (Nhan, 1998). The study of Sesbania sesban on the milk yield of ewes and growth rate of their lambs (Mekoya et al., 2009) indicated that supplementation of S. sesban at 30% of the ration (0.98% of their body weight) during lactation improved milk yield of ewes and growth rate of lambs. In addition, the studies in legume shown that legume contain high level of condensed tannin which find in protected protein escape degradation of rumen microbes and reduce methan production.

Sesbania rostrata is one specie of Sesbania contain high crude protein levels which are usually the order of 30%, stems 7 - 12%, and whole plant 24%. P levels have been measured at 0.2 - 0.3% in both leaf and stem, and Ca levels at 1 -2% in leaves and about 0.7% in stems. NDF, ADF, hemicellulose, cellulose and lignin levels of 54.6, 39.2, 15.4, 30.5 and 8.7%, respectively, and IVDMD of 55.6% have been reported. With the nutrient value like this, sesbania rostrata can be use for ruminants as the rich protein source. Moreover, the study in goats (5 months, 9.0 kg live weight) fed Sesbania rostrata leaves ate 259 g/d DM shown that the goat could gain 38 g/day. (Akbar and Ahmed. 2006) have been conducted on indigenous lactating cows, the results indicated that increasing levels at (0; 1; 1.5 kg/head/day) of a supplement of fresh green Sesbania rostrata forage increased feed intake, possibly for two reasons: firstly, the palatability of the diet was increased; and secondly, its high CP content improved the rumen environment and hence the digestibility of the diet (33% at the higher level of supplementation). It is likely that the Sesbania increased the supply of soluble nitrogen to the rumen microbes, thereby increasing microbial activity. The contribution of Sesbania forage to increased milk yield may through its high protein content and high digestibility.

Growing of Sesbania for feeding to livestock, especially the newly introduced variety, S. rostrata, was popular among the farmers, where it was judged an improvement on the local variety, in that it contains 8% more protein and gave a 10% improvement in yield. It can be propagated through stem cuttings (not possible with the local variety), and, most importantly, its cultivation improves rice yield by 13% due to increased soil fertility. The local variety also tends to be unpalatable because of its bitter taste and smell, and is, therefore have been grown for green manure, fencing and fuel. Farmers were unaware that Sesbania was a potential animal feed. The legume forages, Sesbania, Lathyrus and Leucaena were selected for testing on-farm after laboratory analysis and an

in vitro study. When given as supplements to straw-based diets to lactating and growing cattle, increases in output were recorded. Among the forages, responses were greatest with Sesbania. The economic analysis confirmed Sesbania as the best of the legumes. The transfer of the technology of forage production and feeding has been well received by the rural farmers, and is expected to be sustainable, especially with smallholder dairy farmers (Akbar and Ahmed. 2006)

3.4. Using mulberry (Morus alba) for ruminant

Mulberry (Morus alba) is growing under varied climatic conditions, ranging from temperate to tropical. The biomass yield of fresh leaves is often in the order of 25 to 30 tones/ha/year with cutting interval of about 9 to 10 weeks, while leaves have a high protein content (18% to 25% in DM) and high (75 to 85%) un vivo DN digestibility (Ba et al 2005). Therefore the mulberry leaves have a high potential as a protein rich forage supplement for animal production (Benavides, 2000). The production of fresh mulberry leaves and total dry matter (DM) per hectare depends on climatic conditions, soil characteristics, variety, plant density, fertilizer application and harvesting techniques, but in term of digestible nutrients, mulberry produces more than most traditional forages (Sanchez, 2000). The chemical composition of the leaves varies according to variety, degree of maturity, leaf position within the branch and fertilization level (Shayo, 1997; Singh and Makkar, 2002). Thus leaves content of protein, soluble sugars and organic acids decrease with maturity, whereas fibre, fat and ash constituents increase. Moreover, the content of moisture, protein and carbohydrate of mulberry leaves is higher in temperate regions compared to the tropics (singh and Makkar, 2002).

Mulberry is cultivated on a semi-extenive scale in various parts of Greece, particularly in the northern part, mainly for the leaves to support the sericulture industry. The total acreage of mulberry plantations cultivated in Greece has been reported at around 400 ha, mulberry grows rapidly in the early stages and reaches maturity at an early age, the growth rate falls off rapidly after approximately ten years. The mulberry leavescan also be used as the main feed for sheep (Liu et al., 2004), goats (Bakshi and Wadhwa, 2007) and (Sanchez, 2000; Meijia, 2002) have been used to replace concentrates in dairy cattle, or goats diets (Anbarasu et al., 2004). With its high digestibility and high in level of crude protein, mulberry foliage can be a comparable source to commercial concentrates for ruminal feeding and production. The content of total phenols is very low (1.8% as tannic acid equivalent) and tannins by the protein precipitation capacity method were not detectable (Sigh and Makka, 2002). One of the main features of mulberry leaves is their high palatability, small ruminants avidly consume the fresh leaves and the young stems first. Even if the have never been exposed

to them before, then, if the branches are offered unchopped, they may tear them off and eat the bark, cattle consume the whole biomass if it is finely chopped, however, for ruminants, the preferred method of feeding has been branch cutting by hand, although mechanical harvesting could be employed in the future for direct feeding of fresh material on a large scale, for processing or drying.

3.5. Using Leucaena (Leucaena leucocephala) for ruminant.

Leucaena leucocephala was planted in many regions worldwide. In asia regions, its early use was as a food for humans; its first known use as forage for cattle has been more recent—Asian smallholders were feeding leucaena to cattle in Eastern Indonesia in the 1930s and in the Philippines in the 1970s. Leucaena is Shrub or tree up to 18 m tall, forked when shrubby and branching strongly after coppicing, with greyish bark and prominent lenticels. Leaves bipinnate with 4-9 pairs of pinnae, variable in length up to 35 cm, with a large gland (up to 5 mm) at the base of the petiole. It can grow on shallow limestone soils, coastal sands and seasonally dry, self-muching vertisol soils of pH 7.0-8.5. In exotic locations requires well-drained soils with pH above 5.5, or above 5.0 where aluminium saturation is very low. Leucaena thrives in around 1,000 mm rainfall environments in the humid tropics, but it is sensitive the frost and psyllid insects. It has been considered for biomass production, as its reported yield of foliage corresponds to a dried mass of 2000-20000 kg/ha/year. It is also efficient in nitrogen fixation, at more than 500 kg/ha/year (Wikipedia). Leucaena is rich in the nitritional values. The chemical composition for leucaena leaf meal (DM basis)] was 29.2 % crude protein (CP), 4.3 % mimosine, 19.2 % crude fibre (CF), 10.5 ash, 1.9 calcium (Ca), 0.23% phosphorous (P), 0.34% magnesium (Mn), 1.7% potassium (K), 237.5 ppm carotene and for Leucaena forage was 22.03% CP, 2.14% mimosine, 3.5% CF, 39.5% neutral detergent fibre (NDF), 35.1% acid detergent fibre (ADF), 4.71% hemicellulose, 18.3% ash, 0.22 sulphur, 1.80 % Ca, 0.26% P, 0.33% Mg, 1.45% K, 169.5 mg/kg zinc (Zn), and 26 mg/kg copper (Cu) (Garcia et al. 1996). Supplementation of leucaena improved the rumen fermentation and ruminant production was reported by many reseachers worldwide. The sun-dried and pelleted Leucaena foliage can be fed for goats in entire year suggesting the potential use of Leucaena as a sole feed for the maintenance of stall-fed goats (Sharma and Srivastava 1998). Preston and Hulman (1981) revealed that supplementation of fresh leucaena (1, 2 or 3% liveweight) can improve total dry matter intake, growth rates in Simmental x Friesian bulls. However, leucaena is similar to other tropical forages, it consists of antinutrional element. The mimosine content of leucaeana can cause toxicity for ruminants in some certain conditions but Leucaena toxicity does

not occur in many other tropical and subtropical area throughout the world. Australian workers were first to attribute this geographical variation in leucaena toxicosis to the presence or absence of ruminal bacteria capable of degrading and detoxifying 3,4-DHP (Hammond 1995), namely synergistes jonesii. These bacteria can be transferred successfully from a Hawaiian goat into goats and cattle in Australia .

In Thailand, Leucaena leucocephala names as krathin (กระถิ�น) and has long been grown throughout the country. About 60, 000 tons of Leucaena leaf meal in Thailand are produced annually for swine ration and poultry feed to give deep colour to egg yolks and broiler skin. However, Leucaena is not widely used for grazing and other forage purposes. In recent years, interest in Leucaena in Thailand has focused on its use for wood production, forage, soil erosion control and soil fertility improvement (Poathong and Phaikaew 2010). Poathong and Phaikaew also reported that supplementation of 2-2.5 kg/head/day of leucaena leaf meal for female American Brahman bringed the weight gain of supplemented group 63% higher than that of control group.

4. MATERIALS AND METHODS

4.1. Feed preparation.

Sano will plant at TROFREC farm then will harvest at two months after sowing that consists of leaves and young stem and then dry for 2 to 3 days under sunshine. Lecaena and mulberry leave will collect around KKU campus. After that will dry under sunshine similar to sano processing, all these dried feeds will be ground into meal form by using a grinding machine with 1-2 mm of sieve.

4.2. Animals, experimental design and feeding management.

Four, crossbred Holstein-Friesian cows (75%) will be used in the experiment. Milk yield pre-experiment are 10±3 kg/day and the body weights are 390±10 kg. Cows will be randomly assigned to a replicated 4x4 Latin square design to be allocated four treatments. The treatments are:

T1: Soybean meal 20% in concentrate mixture (T1 SBM)

T2: Sano meal replacement for soybean meal (T2 SNM)

T3: Leucaena leaf meal replacement for soybean (T3LM)

T4: Mulberry leaf meal replacement for soybean (T4 MLM) respectively

The concentrate mixture will be formulated to be iso-nitrogenous at 16 % adjusted by adding urea and feed to the cows base on their milk yields (concentrate to milk is 1:2). Urea treated rice straws will be used as main roughage source during experimental time. Concentrate mixture and roughage will be mixed together then offer to the cows twice daily at 6:00 and 16:00 after milking time with water provides ad libitum, mineral-licked-block will be available. Cows will be housed in individual pens and individually for each treatment. The experiment will be run in four periods; each experimental period lasted for 21 days, the first 14 days as a period for treatment adaptation and for feed intake measurements with the last 7 days for sample collections of rumen fluid and feces. Body weights will be measured daily during the sampling period prior to feeding. Milk yield will be recorded during the 21 day-period and samples will be collected during the last 7 day of each period. Urea treated rice straw will be prepared by using 5% (w/w) urea mixed with 100 kg of water in 100 kg of rice straw (RS) batches (50:50, water to straw) and poured over a stack of straw and then covered with a plastic sheet for a minimum of 10 days before feeding to animals (Wanapat, 1990).

Table 3. Ingredient of treatments

Item Proportion (%)T1

SBMT2

SNMT3

LLMT4

MLMcassava chip 60 59 59 59

Rice bran 4.5 3.5 3.5 4soybean meal (SBM) 20 0 0 0

Replacer 0 20 20 20coconut meal 5 5.5 5.5 5

Urea 1.5 3 3 3palm meal 5 5 5 5molasses 1 1 1 1sulphur 1 1 1 1

mineral mix 1 0.8 0.8 0.8Tallow 0 1 1 1

salt 1 0.2 0.2 0.2Total 100 100 100 100

CP (%) 16.6 16.1 16.0 16.4TDN (%) 76.3 74.0 75.0 74.9

Layout of experimental design

Period Cow No1 Cow No2 Cow No3 Cow No41 T1 T3 T2 T42 T2 T1 T4 T33 T3 T4 T1 T24 T4 T2 T3 T1

4.3. Data collection, analysis and sampling procedures

Feed intakes will be recorded daily. Feed, fecal and urine samples will be collected during the last seven days of each period. Fecal samples will be collected by rectal sampling while urine samples will be collected by spot sampling. Composites samples will be dried at 60°C and ground (1 mm screen using Cyclotech Mill, Tecator, Sweden) and then analysed for DM, Ether extract, ash and CP content (AOAC, 1985), NDF, ADF and ADL (Goering and Van Soest, 1970) and acid insoluble ash (AIA). AIA will be used to estimate digestibility of nutrients (Van Keulen and Young, 1977).

Cows will be milked twice daily, and milk weights will be recorded at each milking of each period. Milk samples will be composted daily, according to yield, for

both the a.m. and p.m. milking, preserved with 2-bromo-2 nitropropane-1, 3-dial, and stored at 4 °C until analysis for fat, protein, lactose, totals solids and solid not fat content by infrared methods (using Milko-Scan 33 Foss Electric, Hillerod, Denmark). Sub-sample of composite will be analyzed for milk urea-N (MUN) by using Sigma kits #640 (Sigma Diagnostics, St. Louis, MO) (Valladares et al., 1999).

Rumen fluid and jugular blood samples will be collected at 0, 4, 6 h post-feeding. Approximately 200 ml of rumen fluid will be taken by using vacuum pump suction at each time at the end of each period. Rumen fluid will be immediately measured for pH and temperature using a portable pH and temperature meter (HANNA instrument HI 8424 microcomputer, Singapore). Rumen fluid samples will be filtered through four layers of cheesecloth. The samples will be divided into three portions. The first portion will be used for ammonia-nitrogen (NH3-N) analysis where 5 ml of H2SO4 solution (1M) will be added to 50 ml of rumen fluid. The mixture will be centrifuged at 16,000 x g for 15 minutes (Table Top Centrifuge PLC-02, U.S.A.) and supernatant will be stored at –20 °C prior to volatile fatty acid (VFAs) analyses using a HPLC (Instruments by controller water model 600E; water model 484 UV detector; column novapak C18; column size 4 mm x 150 mm; mobile phase 10 mM H2PO4 (pH2.5)) according to Zinn and Owens (1986). Second portion will be fixed with 10% formalin solution in sterilized 0.9% saline solution. The total direct count of bacteria, protozoa and fungal zoospores will be made using the methods of Galyean (1989) based on the use of a haemacytometer (Boeco). Third portion will be cultured groups of bacteria using roll-tube technique (Hungate, 1969) for identifying bacteria groups (cellulolytic, proteolytic, amylolytic and total viable count bacteria).

A blood sample (about 10 ml) will be drawn from the jugular vein at the same time as rumen fluid sampling, separated by centrifugation at 3000 x g for 15 minutes (Table Top Centrifuge PLC-02, U.S.A.) and stored at –20 °C until analysis of fatty acid profile and blood urea nitrogen (BUN) according to the method of Crocker (1967).

4.4. Statistical analysis.

Statistical analyses will be performed using the GLM procedure of SAS (1998). Treatment means will be statistically compared using the Duncan’s New Multiple Range Test (DMRT) (Steel and Torrie, 1980).

Statistical model: Yijk = m + Ti + Cj + Rk + eijk ,

where: Yijk = The criteria under study, in treatment i; column j; row k,

m = Over all sample mean,

Ti = Effect of treatment i, Cj = Effect of column j,

Rk = Effect row k and

eijk = Error

5. EXPERIMENTAL LOCATION

• The Tropical Feed Resources Research and Development Center (TROFREC), Khon Kaen University.

• The Ruminant Nutrition and Metabolism Center, Khon Kaen University.

• Dairy Section, Department of Animal Science, Faculty of Agriculture, Khon Kaen University.

•Laboratory, TROFREC, Khon Kaen University.

Experimental timetable

Unit 2010 201110 11 12 1 2 3 4 5 6 7 8 9

Material preparation x x x

Experiment period x x x

Sample analysis x x x

Report writing x x x x

Experimental budget

No Item Budget (Baht)1.

2.

3.

4.

5.6.7.8.9.

For land preparation: Plough the land 10.000 m2, fertilizer, herbicide, and seed, etc. Labor for planting, weeding management, harvesting, and grinding for making pellet Feedstuff such as Cassava chip, rice bran, palm meal, urea, sulphur, salt, etc. Lab. Chemical, equipments, etc. Chemical analysis of DM, CP, Ash, ADF, NDFRumen fluid analyzing (VFA, NH3-N)Blood Urea Nitrogen, Urinary purine derivativeTotal microbe countsMolecular techniques for microbesMilk composition analyzing, Milk urea Nitrogen

60.000

40.000

60.000

30.000

40.00040.00030.00030.00030.000

Total 360.000

Signature………………………… Mr. Bien Dao Duc October, 2010

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