II. REVIEW OF LITERATURE. The physiological effects of probiotics as growth promoters in broiler chickens are established by various workers.

Transcription

1 II. REVIEW OF LITERATURE The physiological effects of probiotics as growth promoters in broiler chickens are established by various workers. 2.1 History and development of Probiotics Metchinkoff (1907) suggested that the health status and longevity could be improved by consumption of milk fermented by lactobacilli, as he observed it in some of the rural inhabitants of the Caucasus who consumed fermented milk throughout their life. Historically, the first use of yeasts dates back to some six thousand years, wherein ancient Egyptian aristocrats were consuming sixteen different types of beer containing yeasts (Mackenzie, 1925). Lilley and Stillwell (1965) first introduced the term "Probiotic" to describe, "growth promoting factors" produced by microorganisms. The word "probiotic" is derived from the Greek word 'probios' meaning 'for life' and has had several different meanings over the years. Parker (1974) used the term probiotics for microorganisms or substances that contribute to intestinal microbial balance. Fuller (1989) redefined the probiotic as "A live microbial feed supplement, which beneficially affects the host animal by improving the intestinal microbial balance". Miles and Bootwala (1991) noticed that the United States Food and Drug Admisistration (USFDA) directed the manufacturers to use the term

2 "Direct Fed Microbial (DFM)" rather than probiotic and they also widened the meaning of DFM as 'a source of live or viable and naturally occurring microorganisms, and this includes bacteria, yeast and fungi'. As listed by Fuller (1992) and Anonymous (2002), several microorganisms have been used as probiotics, viz., i) Bacteria belonging to genus Bifidobacterium (Bif) : Bif. adolescentis, Bif. animalis, Bif. bifidum, Bif. infantis, Bif. longum and Bif. thermophilum. ii) Bacteria belonging to genus Lactobacillus (L) : L. acidophilus, L. brevis, L. casei, L. cellobiosus, L. fermentum, L. lactis, L. plantarum, L. reuteri and L. sporogenes. iii) Bacteria belonging to genus Streptococcus (S) : S. faecium, S. intermedius, S. lactis and S. salivarius. iv) Yeast belonging to genus Saccharomyces [Sac.] : Sac. cerevisiae. v) Yeast belonging to genus Candida : Candida pintolopesii. vi) Moulds (Aspergillus niger and Aspergillus oryzae) vii) Bacillus subtilis and so on.

3 Havenaar et al. (1992) pointed out that the definition given by Fuller (1989) was restricted to feed supplements, animals and the intestinal tract. Hence, they redefined the probiotic considering both man and animals as "a mono or mixed culture of living microorganisms which beneficially affect the host by improving the properties of the indigenous microflora". 2.2 Intestinal flora of the poultry Barrow (1992) opined that the flora and its activities must be studied in relation to the host anatomy and physiology and to the conditions existing in individual sections of the alimentary tract of the poultry. The yeast was observed throughout the gut from time to time but never present in high numbers. Age, diet, immune response, oral administration of antibiotics and disease can affect the composition of the gut flora. It was also pointed out that the fermentative crop flora produced a number of organic acids, nucleotides, B vitamins and vitamin A, which were available to the host. 2.3 Physical Characters and morphology of Yeast Yeast is a fungus and lives as a saprophyte or parasite. It is 5 10 µ size with thick polysaccharide cell wall. The shape is variable like oval, circular, cylindrical, lemon shaped and triangular. Reproduction is by means of vegetative process by budding or ascospores. The number of ascospores is 1-4 per cell. Chemically yeast contains 40 per cent protein, 15 per cent fat, 5 per cent vitamins and is a rich source of minerals, enzymes and other co-factors.

4 As pointed out by Walker and Ayres (1970) and Hill (1975), the Sac. cerevisiae was one of the most commonly used yeast strain and has a number of industrial uses such as in the production of beverages, paper, food, petroleum products etc Properties of yeasts Wedberg (1966) opined that the enzymes liberated by the yeasts attacked the fermentable constituents of dough and the carbon dioxide gas that was released in the process of fermentation made the dough to rise. Yeast at higher ph value released chemical substances like nucleotides, amino acids, vitamins and enzymes such as nucleases, proteases, glucanases, mannases, lipases and amylases at higher rate and these secreted substances contributed positively to the nutrition of the animals (Rose, 1980). The improved performance of animals upon addition of yeast culture could be due to improved palatability and better nutrient utilization (Peppler, 1982 and Lyons, 1986). According to Rose (1987), chemically yeast comprised of 40 per cent protein, 25 per cent polysaccharide, 15 per cent nucleic acid and 15 per cent vitamins and minerals. He also reported that the hog farmers added dried yeast to cooked garbage to improve the pig performance. Meyen ex Hansen (1883) was the first to report the description of Sac. cerevisiae. Barnett et al. (1990) mentioned that the yeasts have been used for fermenting lactose to ethanol, producing protein from alkanes and paper pulp waste, producing various alditols, such as glycerol or D

5 glucitol and as sources of enzymes such as - D - fructofuranosidase, - D galactosidase and lipase. They also reported that Sac. cerevisiae was used as baker s yeast and it caused fermentation of D- glucose, D- galactose, maltose, sucrose, - - trehalose, melibiose, melezitose, raffinose and starch. It was also documented that the use of yeast in sausage industry by the Germans during the first and second World war was extensive. From ages, man has been consuming yeast present in beer and wine (Fuller, 1992). The living microbes such as yeasts and lactic acid bacteria produce the enzymes required for desired conversion (Buhler et al., 1998). 2.4 Properties of Lactobacillus species Lactobacilli were the first genus of bacteria proved to have beneficial health effects. They have been shown to be present in the gastrointestinal tract of most animals and birds. It is one of many friendly species of intestinal microflora considered as beneficial bacteria in its ability to aid in breakdown of proteins, carbohydrates and fats in food and help absorption of necessary elements and nutrients such as minerals, amino acids and vitamins by the host. They quickly colonized in the gut epithelium to deprive the sites for attachment of pathogens. They were also referred to as "live enzyme factory" as they produce wide range of enzymes, which can breakdown even complex carbohydrates, hence beneficial to the host (Anonymous, 2002). 2.5 Probiotics as Growth Promoters

6 Goodenough and Kleyn (1976) studied the digestion of lactose in rats deficient with lactase enzyme in the intestinal secretions after ingestion of lactic acid bacteria and attributed this character to the secretion of lactase enzyme by the supplemented bacterial probiotic. Dilworth and Day (1978) recorded improvement in growth rate and feed efficiency in broilers that were fed with diets containing probiotic cultures at the levels of , , , and per cent. The probiotics are beneficial microorganisms, which contribute to the maintenance of ideal microbial balance in the digestive tract by a mechanism known as competitive exclusion. Jernigan et al. (1985) said that probiotics were also used in animal feedstuffs as growth promoters and opined that the information available was fragmentary. Vanbelle et al. (1991) evaluated the positive effects of probiotics on development and functions of the microflora in the digestive tract of the animals. As opined by Barrow (1992) the probiotics were designed to enrich beneficial organisms or to provide the chicken with the effects of beneficial bacteria. Raibaud (1992) concluded that the bacterial interactions in the gut involve multiple mechanisms that were poorly understood and need

7 considerable work to be carried out on supplementation of bacterial probiotics. As reviewed by Rowland (1992) one of the most important ways in which a probiotic organism might exert beneficial effect on its host was to modify metabolic processes, particularly those occurring in the gut. The probiotics stimulate host enzymes involved in the digestion of complex nutrients, or provide a probiotic source of these enzymes. Also, probiotics can synthesize vitamins and other essential nutrients not provided in sufficient quantities in the diet. Vranesic (1992) reviewed the use of probiotics, live bacterial and or fungal cultures, as feed supplements and concluded that the probiotics stimulated numerous metabolic processes relating to feed digestion and absorption. It was also opined that few authors also include enzymes, yeasts and even organic acids in the group of probiotics. Hennig et al. (1993) evaluated the use of probiotics as growth promoters and opined that the experiments when supplemented with probiotics must end at a given weight. Teller and Vanbelle (1993) reviewed the available literature and reckoned that the ideal probiotic had improved growth performance atleast equal to those of antibiotics without injury to man, animal or environment.

8 Jin et al. (1997) reported improved growth performance and feed conversion in broilers, egg mass, egg weight and egg size in layers, upon addition of probiotics to the diet. According to them the mode of action of probiotics in poultry included (i) maintaining normal intestinal microflora by competitive exclusion and antagonism, (ii) altering metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production, (iii) improving feed intake and digestion, and (iv) neutralizing enterotoxins and stimulating the immune system. They also opined that if the right strain of bacteria, optimal concentration of viable cells was given under non-stress conditions, the beneficial impact of culture supplementation was significant. Panda et al. (2000) observed significant body weight gain from zero to four weeks of probiotic supplementation but no difference subsequently after four weeks of age in broiler chicks. Modirsanei et al. (2002) reported improvement in broiler performance when probiotics were added to the diet and recommended the inclusion of probiotics as growth promoter in rations of broiler chickens. Baghel and Singh (2004) opined that the probiotics have been found to improve the production performance of poultry, establish an environment to increase the digestibility of feeds and were a potential alternative to antibiotics in poultry diet.

9 Palod and Singh (2004) indicated that the 'Probiotics' in broiler feeding was becoming a new area in biotechnology and offer a possible replacement for the use of sub-therapeutic level of antibiotics in broiler feeds. The probiotics include more than 200 species of bacteria and yeast. The various probiotics available in the market are either single or combination of bacteria, yeast and fungi. The use of probiotics in broiler feed causes better growth, higher feed conversion, better digestibility and improved product quality. 2.6 Yeast (Saccharomyces cerevisiae) as a Growth Promoter Bonomi et al. (1977a) recorded improved weight gain to the extent of 7.97 per cent and 6.5 per cent increase in feed efficiency with the feeding of Sac. cerevisiae at 0.15 per cent in ration to turkeys. They also reported higher dressing percentage of 4.8 per cent in broilers fed with Sac. cerevisiae. Bonomi et al. (1977b) reported improved protein digestibility, weight gain by 9 per cent, feed efficiency by 7 per cent and plumage development by 13 per cent by feeding 0.1 per cent Sac. cerevisiae in meat type turkeys. Krueger et al. (1990) observed improved feed efficiency by 3.16 per cent upon feeding of 454 and 908 g Sac. cerevisiae per ton of feed in broiler chickens from 28 to 49 days of age. They found

10 no significant improvement in weight gain, liveability, feathering and carcass yield upon yeast supplementation. Sac. cerevisiae was a yeast employed extensively in poultry industry and encouraging results have been achieved with respect to weight gain, feed efficiency, dressing percentage, egg production, egg weight, inhibition and containment of proliferation of Escherichia coli in the intestines, phosphorus utilisation and counteracting the effect of aflatoxins in the diet. Sac. cerevisiae was also used for brewing and baking purposes (McDaniel and Sefton 1991). Wenk (1991) reported increased metabolism in growing chickens fed with the yeast and enzymes. Stanely et al. (1993) opined that the use of Sac. cerevisiae served as a source of vitamins, unidentified growth factors, enzymes and proteins which increased overall biological value of nitrogenous compounds absorbed along the digestive tract. They observed that the increase in body weight gain was per cent in chicks receiving aflatoxin contaminated feed with incorporation of 0.1 per cent Sac. cerevisiae. It was concluded, as there was a significant antagonistic interaction between aflatoxin and 0.1 per cent Sac. cerevisiae. Bradley et al. (1994) observed increased weight gain at 7, 14 and 21 days of age in poults fed with diet containing Sac. cerevisiae var boulardii (SCB) at 0.01, 0.02 and 0.06 per cent of the diet. Increased body weights were maintained from 21 to 35 days of age in poults fed 0.02 per cent SCB. They also observed decreased number of goblet cells

11 per millimeter of villus height on histological examination of ileal sections from poults at 35 days of age with a decrease in crypt depth in poults receiving 0.02 per cent SCB but no dietary differences were observed for either villus height or width. Kotrbacek et al. (1994) recorded increased weight gains in the broilers receiving yeast. The feeding of live baker's yeast at 0.2, 0.6 and 1 percent levels did not improve either the body weight or feed efficiency in broiler chicks (Yadav et al., 1994). Lyons (1995) opined that the Sac. cerevisiae was effectively used as a probiotic in monogastric animals and poultry. Sarkar et al. (1997) studied the combined effect of feeding yeast and antibiotic on the performance of broiler chicken. They did not observe significant increase in body weight gain or better feed efficiency ratio in the experimental groups, although numerical improvement in body weight was noted in few groups. Upendra (1999) recorded the significantly improved weight gain in Sac. cerevisiae supplemented group on day 14, 28 and 42 and improved FCR by per cent by the end of 42 nd day experimental trial. Silva et al. (2000) reported better feed conversion in broilers during the period 1-21 days when the diets supplemented with antibiotics and probiotics. Maiorka et al. (2001) conducted an experiment with the treatments such as T1-no additives, T2-antibiotics (Olaquindoxa and Nitrovina), T3-

12 prebiotic (0.2 per cent Sac. cerevisiae cell wall), T4-probiotic (300 ppm Bacillus subtilis) and T5-symbiotic (T3+T4) and observed better live weight gain in broilers up to 45 days of age, fed with symbiotics followed by antibiotics, prebiotics and probiotics. The total absence of additives in the diets worsened broiler chicken performance. Santin et al. (2001) reported the efficacy of Sac. cerevisiae cell walls, obtained from the brewery industry, added at 0.1 and 0.2 per cent to broiler chicken diets, on performance and intestinal mucosa development (tropic effect). The results revealed higher body weight gain for the total experimental period and higher villus height of the intestinal mucosal layer at 7 days of age in the birds fed at 0.2 per cent. Kompiang (2002) conducted an experiment to evaluate the effect of marine yeast and Sac. cerevisiae as probiotic supplements on poultry performance for 5 weeks. Their effects on bird performance were better than treatment of negative control and concluded that marine yeast or Sac. cerevisiae could replace the function of antibiotic as a growth promoter Lactobacillus species as growth promoters Tortuero (1973) observed increased weight gain, better feed conversion and although not statistically significant difference in fat digestibility and nitrogen retention in chicks fed with the probiotics.

13 In commercial broiler production, Couch (1978) recorded an increase in average body weight of 47 gram by using lactobacillus supplements at a rate of 454 g per ton of feed. Adler and DaMassa (1980) reported that feeding a lactobacillus culture to chicks resulted in an improvement in body weights and reduced the occurrence of pasted vent. Contrary to the reports of various beneficial responses of probiotic supplementation in broiler diet, Bruenrostra and Kratzer (1983) found that the lactobacillus supplemented group of broiler chicken did not show as much as good result compared to antibiotic supplemented and control groups. Watkins and Kratzer (1983) recommended that there was possibly an optimum level of lactobacilli required by the chicken that provided most of the benefits to the host. Inclusion of lactobacillus above the recommended levels resulted in undesired effect such as bacterial competition for biotin and low levels were not enough to show the desired benefits. As reported by Jernigan et al. (1985) the research related to the use of lactic acid producing bacteria as growth stimulants in broilers was not sufficient. According to them lactobacilli had an advantage that the Lactobacillus species were capable of producing large amounts of lactate from simple carbohydrates, can withstand a high degree of acidity which was usually fatal to other bacteria and have long been considered as desirable inhabitants of digestive tract.

14 Franceschi and Stocker (1991) reviewed the mechanism of action with respect to the beneficial effects of lactic acid producing bacteria on digestive system in broilers and layers. Supplementation of lactobacillus cultures at the level of 0.5 gram per kg of broiler starter increased daily weight gain and feed conversion efficiency and decreased the incidence of diarrhoea and mortality in broiler chicken (Hussein and Ashrey, 1991). As observed by Guillot and Yvore (1991) the probiotic bacillus promoted the growth of chickens. Contrary to the observations of other scientists, Maiolino et al. (1992) reported statistically non-significant mean final live weights in chickens supplemented with Lactobacillus species based probiotics. As studied by Takalikar et al. (1992) the broiler chicks supplemented with 0.02 per cent Lactobacillus sporogenes in their diet grew faster than control upto 6 weeks of age and also the experimental birds consumed less feed compared to control. Mudalgi et al. (1993) studied the effect of two probiotic cultures, such as L. acidophilus and L. bulgaricus administered through drinking water at the rate of 10 gram per liter, on performance of broiler chicken. The results revealed no significant treatment effects on growth, feed intake and feed conversion efficiency, although the birds offered probiotic

15 cultures appeared to gain numerically higher weights than those fed the control diets. Nahashon et al. (1993) reported better weight gain, feed conversion ratio, shank length, nitrogen retention and calcium retention in Lactobacillus supplemented single comb white leghorn pullets. Manickam et al. (1994) observed significantly better performance such as weight gain and feed conversion efficiency in broiler chicks given L. sporogenes compared to untreated controls. Nahashon et al. (1994a) reported improved egg weight, egg mass, egg size and body weight gains in lactobacillus supplemented cornsoybean meal when fed to single comb white leghorn layers. Only improved weight gain observed in Lactobacillus supplemented barleycorn soybean meal fed birds. Mohan et al. (1996) reported maximum weight gain in probiotic supplemented broilers. Yeo and Kim (1997) reported significantly increased average daily gain in body weight during the first three week period in broiler chicks supplemented with probiotics, L. casei. They also observed decreased urease activity in intestinal contents, which was beneficial for improving animal health and growth especially during early life. Singh et al. (1999) studied the microbial flora of the caecum in chicks fed with 0, 0.02, 0.03 or 0.04 per cent of L. sporogenes and

16 found that the microbial counts tended to decrease with increasing levels of probiotics. Ramesh et al. (2000) reported that lactobacillus-fed non-infected birds showed better FCR than control groups and also opined that feeding lactobacillus helped in preventing the development of systemic infections. Lactobacillus sporogenes was earlier called as Bacillus coagulans (Anon. 2002). Khalid et al. (2002) studied the effect of lactic acid bacteria at 1, 2 and 3 per cent levels in the diet on broiler performance and reported better FCR at all the levels of supplementations. 2.8 Combination of Saccharomyces cerevisiae and Lactobacillus species as growth promoters Burkett et al. (1977) reported better feed efficiency after four weeks in broilers with dietary supplementation of lactobacillus and / or yeast or a combination of lactobacillus and yeast. Katoch et al. (1996) observed the improved live weight and feed conversion efficiency during finishing phase of broilers when L. acidophilus, S. faecalis and Sac. carlsbergensis [Sac. uvarum] isolated from the faeces of the leopard, Panthera leo, cultured and fed to week-old chickens for 5 weeks.

17 The inclusion of Sac. cerevisiae or L. sporogenes at low levels such as per cent in the diet of broiler chicks either individually or in combination did not show any significant effect on weight gain, feed consumption, feed efficiency and liveability in a biological trial of six weeks duration (Megharaja et al., 1996). Kahraman et al. (1997) concluded that the addition of the commercial probiotic product having L. acidophilus and Sac. cerevisiae to the broiler diets had a beneficial effect on performance. Reddy (2001) opined that the combined use of lactobacillus and yeast culture in the feed and water has been shown to be effective in reducing morbidity and mortality and improving growth performance and production characteristics in poultry. Upendra and Yathiraj (2003) in a study consisting of 320 broiler chicks, reported the increase in body weight on supplementation of Sac. cerevisiae and L. acidophilus and attributed this beneficial effect to the release of vitamins, unidentified growth factor, enzymes and protein by the probiotics. 2.9 Effects of Mixture of Probiotics Baidya et al. (1994) reported a slight increase in body weight in broiler chicken fed with a commercial preparation of probiotic, G- Probiotic and it was non-significant. The weekly body weight gain in gray Partridges from one week age to 22 weeks of age was recorded when supplemented with Biovet. The overall mean body weight of the birds in experimental group was

18 significantly higher than that of control indicating beneficial effect of biovet on increase in feed consumption and the metabolic rate (Mishra and Khan, 1994). Schneitz et al. (1998) evaluated the commercial probiotic preparation, Broilact in broiler chicken on salmonella colonization, nutrient digestibility, metabolizable energy and production of volatile fatty acids. They reported improved degradation of ß- glucans and arabinoxylans which was due to increased enzymatic activity in Broilact treated group. Georgieva et al. (2000) observed a significant weight gain by less feed consumption at 49 days of age in broiler chicken when supplemented with a commercial probiotics, Lacto-Sacc compared to controls and antibiotic treated groups. Bhat et al. (2003) reported that the probiotic mixture containing L. sporogenes 30,000 million cfu., L. acidophilus 30,000 million cfu., Sac. cerevisiae SC 47 1,25,000 million cfu., Alpha amylase 5 gm and sea weed extract 50 gm per kg when fed to broiler chicken at the rate of 0.1 per cent in feed improved the body weight gain, feed consumption and feed conversion ratio.

19 2.10 Development of digestive tract Dror et al. (1977) opined that the ability of alimentary tract to digest large amounts of food was presumably related to relative size of the digestive tract as studied in light and heavy breeds of chicks. Nitsan et al. (1991a) reported the allometric growth of pancreas and small intestine by 4-fold and 2-fold, respectively by 11 days of age and also studied the levels of different digestive enzymes in relation to age in broiler chicken. Sell et al. (1991) opined that the development of the gastrointestinal tract and secretion of digestive enzymes was an important aspect of growth, especially during the early post-hatching period. Uni et al. (1995) reported that the development of the gastro intestinal tract during the post hatch period had a major role in influencing the rate of growth in the chicken. Shapiro et al. (1997) reported evident changes in the relative weights of the gastrointestinal tract segments by one week of age in broiler chicken compared to stunting syndrome affected chickens. Sklan and Noy (2000) observed increased small intestinal weight in the 48 h post hatch chicks with free access to feed compared to chicks with restricted access to feed.

20 At the immediate post-hatch period the increase in weight of small intestine was rapid and maximal at 6-10 days in the chick (Sklan, 2001) Organ weights Forner et al. (1991) reported highest liver weights in chicks fed with protein rich diet by day 21 of age, due to increased cell proliferation. Organ body weight ratio was taken as one of the criteria to assess immune status of the birds (Arun, 1992). Dietary supplementation of probiotic in broiler chicken ration did not show any influence on the carcass yield and weight of internal organs (Mandal, 1994; Kaistha et al., 1996; Mohan et al., 1996). Palo et al. (1995) studied the development of the gastrointestinal tract of broiler chicks. They recorded the weights of proventriculus, gizzard, small intestine, pancreas and liver at 7, 14, 21, and 41 days of age and found that the weights of gastrointestinal organs were increased significantly in ad libitum fed birds compared to feed restricted birds from 7 to 14 days of age. Uni et al. (1995) observed non striking increase of the organs of digestive tract such as the gizzard and pancreas in their relative size. Panda et al. (2000) reported probiotic had no influence on dressing percentage or weight of internal organs such as liver, heart and gizzard Digestive enzyme in crop contents

21 Bolton (1965) and Pritchard (1972) reported that certain carbohydrates are degraded in the crop and the breakdown was attributed to enzymes of dietary origin or bacterial fermentation. Sturkie (1976) reported that the amylase, ptyalin, was present in the saliva and its scrapings from the mouth and esophagus of the fowl. The presence of enzymes in the crop and their role in digestion have been subjects of controversy. A number of workers have reported the presence of proteolytic and amylolytic enzymes in the crop or its contents. It was also opined that the enzymes of crop may not play a significant role in digestion and absorption from the crop was minimal. Amylase has been found in the saliva of the poultry, which helps in a little digestion of carbohydrates in the mouth. Amylase found in the crop may originate from the salivary glands, ingested food, and bacteria in the crop or regurgitated duodenal contents. It was believed that a significant amount of starch digestion occurs in the crop of the chicken as the result of bacterial action (Duke, 1996) Assay of Digestive enzymes in intestinal contents Borgstrom et al. (1959) reported that feces of conventional rats contained appreciable amounts of trypsin and invertase, whereas feces of germ-free rats showed no activity of these enzymes.

22 Lepkovsky et al. (1964) reported slightly higher but negligible activities of amylase and lipase in conventional chickens compared to germ-free chickens. Tortuero (1973) reported apparent fat digestibility and nitrogen retention in chicks up to 15 days of age when implanted with L. acidophilus. Nitsan et al. (1974) opined that the overall secretion of digestive enzymes increased parallel to the increase in food consumption, in respective of force fed and ad libitum fed groups. Pisharody and Nair (1974) conducted a comparative study of the activity of some of the digestive and metabolic enzymes in the mucosa of the gastrointestinal tract of chicks and ducklings to establish the ability to digest and absorb nutrients that reflected on the enzyme activity of their digestive system. They reported higher level of lipase in the intestinal mucosa of ducklings that was correlated with rapid growth rate and greater feed requirements compared to chicks. Lipase activity was lowered in high protein diet fed ducklings. Similarly, high fat ration decreased protease activity in chicks and ducklings. They reported ± 75.11, ± and ± protease units per gram of tissue in proventriculus for control, high protein and high fat treated groups, respectively, in chicks. Lipase activities were ± 6.28, ± and ± units per gram of tissue at first part of small instestine in control, high protein and high fat consumed groups, respectively. Amylase levels from first part of small intestine were

23 48.75 ± 6.06, ± 5.78 and ± Somogyi units per gm of tissue in control, high protein and fat treated chicks respectively. Nir et al. (1978) observed that overfeeding had no effect on the specific activities of the pancreatic digestive enzymes in young chicks of light and heavy breeds. Philips and Fuller (1983) assayed the activities of amylase and trypsin like protease in the gut contents of germ free and conventional chickens at 3 days and 14 days of age. They observed increased proteolytic activity and decreased amylolytic activity in conventional chicks. The protease activity was 600 ± 58.8 µg per g of intestinal contents in conventional chicks against 134 ± 25.8 µg per g in germ free chicks on 3 rd day and they were 1124 ± and 255 ± 35 µg per g, respectively on 14 th day. The amylase activity was 240 ± 23.8 units per g of contents from lower small intestine in conventional chicks against 313 ± 32.9 in germ free chicks on 3 rd day of age. On 14 th day of age the amylase activity was 414 ± 15.9 and 541 ± 60.3 units per g, respectively, in conventional and germ free chicks. Escribano et al. (1988) opined that the efficient hydrolysis of dietary triglycerides required the coordinated action of lipase, colipase, bile salts and phospholipids, together with an adequate degree of intestinal motility. They reported linearly increased lipase activity from 4 days after hatching to 16 days in young turkeys.

24 Krogdahl and Sell (1989) opined that the development of intestinal lipase activity seemed to depend on dietary fat level. They also reported increased activities from day 1 until day 21. Angel et al. (1990a) reported unaltered effect on pancreatic lipase, amylase and trypsin activities in stunting syndrome induced turkeys. Nitsan et al. (1991b) studied the digestive enzyme levels in different lines of chickens differing in body weight. They found that the levels of trypsin, chymotrypsin and amylase in the intestinal contents differed from those in the pancreas and appeared to be line dependent. Shao and Han (1991) reported significantly increased activity of amylase in the duodenal contents from one-day-old to four weeks old in Siji geese. Nir et al. (1993) opined that the secretion of digestive enzymes in newly hatched meat type chicken could be a limiting factor in digestion and, consequently, in food intake and growth. Shapiro and Nir (1995) recorded the lowered amylase, trypsin and chymotrypsin activities in the intestinal chyme of broiler birds infected with stunting syndrome compared to controls. Noy and Sklan (1995) reported enhanced secretions of lipase, trypsin and amylase enzymes in the young chicks at 10 days of age which was correlated with increased feed intake.

25 The small intestine was the primary site of chemical digestion where it occurs as a result of pancreatic enzymes and microbial activity as well as by intestinal secretions (Duke, 1996). Shapiro et al. (1997) reported stunting syndrome-infected birds had a significant increase in pancreatic trypsin activity in intestinal contents during weeks 1 and 2 in broiler chicks. They also observed unaffected amylase activity in broiler chicks infected with stunting syndrome up to 2 weeks of age. Sklan and Noy (2000) measured the levels of amylase, lipase and trypsin in the intestinal contents and found that the activities of these enzymes increased in chicks ingesting feed compared to chicks without ingesting feed up to 48 hours post hatch. Jakab et al. (2001) reported significantly higher levels of alphaamylase, lipase, trypsin and total-protease activities in the small intestinal contents of broiler chicken at 49 days of age supplemented with microelement preparation. This increase was responsible for the improvement of slaughter weight, carcass weight and killing-out per cent age that was not significant. Sklan (2001) reported that the increased activities of intestinal enzymes correlated with both intestinal and body weights in chicks.

26 Palod and Singh (2004) mentioned various ways by which the yeast culture added in broiler feed produced the beneficial effects. Live yeast culture was capable of releasing phytase for phosphorus digestion, lipase for fat digestion and endotryptase for protein digestion. It triggers the growth of cellulolytic bacteria resulting into digestion of fiber portion of feed. The yeast also facilitates the growth of lactobacillus and streptococcus. Yeast ferments the carbohydrate. The fermentation products inactivate the intentional toxins, which reduce the appetite of birds. Yeasts were also a source of Vitamin B complex Assay of digestive enzymes in intestinal brush border cells Laws and Moore (1963) determined the maltase activity of intestinal contents of chicks. March (1979) opined that the bacterial enzymes enriched the digestive capacity in the lower intestine. Henning (1985) reviewed the genetic, dietary and hormonal influences on the activities of intestinal disaccharidases in rats and humans. Allen (1987) opined that the activity of brush border enzymes was the index of maturity and absorptive capacity of mucosal cells in chicken gut. Sell et al. (1989) opined that the digestion of oligo and disaccharides was vital for the nutrition of animals, but a very little information was available about intestinal disaccharidases of avians. The

27 complete digestion of these carbohydrates depends, in large measure, on the activity of disaccharidases located on the microvilli of enterocytes. Appreciable maltase and isomaltase activities were observed in high carbohydrate diet fed young turkeys. Angel et al. (1990 b) assayed disaccharidases in turkeys and found that the maltase specific activities were lower in stunting syndrome birds than control. Collington et al. (1990) reported a significant effect on the development of sucrase, lactase and tripeptidase activities before weaning in pigs upon the addition of an antibiotic or a probiotic in the diet. Al Batshan et al. (1992) observed the increased specific activities of maltase and sucrase in the jejunum of con poults treated with virginiamycin. Mahagna and Nir (1996) reported highest activities of intestinal disaccharidases such as maltase and saccharase in jejunum compared to duodenum in broiler and layer type chicks. Shapiro et al. (1998) recorded slightly decreased activities of maltase and saccharase in the jejunal contents of broiler chicks at 2 nd and 3 rd week of inoculation of stunting syndrome causative agent compared to controls. Ingledew (1999) reviewed that the yeast was used commercially to produce sucrase, maltase, lactase and trehalase. King et al. (2000) measured sucrase and alkaline phosphatase activities of homogenates from the small intestinal mucosa and found

28 that the sucrase activity per unit body weight did not differ in white pekin ducks Serum glucose content Hazelwood (1976) reported the range of normal blood glucose levels in control chickens as mg per 100 ml. Singh et al. (2001) reported significantly increased levels of blood glucose in broiler chicken than the pure strains and crosses of guinea fowl Serum protein levels Griminger (1976) reported the serum protein levels of 2.68 g per 100 ml at first week and 4.63 g per 100 ml at 12 weeks of age in chickens. The supplementation of Sac. cerevisiae at 0.05 and 0.1 per cent levels in broiler chicken showed serum total protein value of 2.42 and 3.45 grams per 100 ml, respectively, compared to control level of 3.49 grams (Victor et al., 1993). Shapiro et al. (1997) reported significantly increased blood plasma protein levels in stunting syndrome chickens from 1 to 3 weeks compared to naïve chickens.

29 As reported by Gohain and Sapkota (1998), the serum protein levels at 7th week of age in broiler chickens were 3.26 ± 0.19 and 3.23 ± 0.09 g per 100 ml, for control and probiotic supplemented groups, respectively, and it was non-significant. Swain et al. (2001) studied the effect of different litters on blood biochemical parameters of broilers. They observed that the serum total protein, calcium, and phosphorus concentrations did not differ among the chicks belonging to different litter groups Serum calcium and phosphorus levels According to Mandal et al. (1994) there was no significant increase in body weight gain in Bioboost, a commericial probiotic containing Sac. cerevisiae and Bacillus coagulans (L. spororgenes), supplemented group. They also reported serum biochemical components such as serum protein (5.70 ± 0.50 g per 100 ml), serum calcium (9.00 ± 0.42 mg per 100 ml) and serum phosphorus (7.20 ± 0.42 mg per 100 ml) which did not differ significantly between control and probiotics supplemented groups. Nahashon et al. (1994b) studied the effect of direct fed microbials along with two levels of available phosphorus in single comb white leghorn layers. They reported the higher phosphorus retention with the Lactobacillus supplementation when 0.25 per cent available phosphorus was included in the diet. Nahashon et al. (1994c) observed

30 positive correlations for nitrogen and calcium retentions, daily feed consumption and egg size in lactobacillus-supplemented diets in layer chicken. Shapiro et al. (1997) reported that the stunting syndrome infection caused a significant increase in plasma calcium during 2 and 3 weeks, accompanied by a significant reduction in plasma phosphorus at week 2 only. Singh et al. (2001) reported significantly different levels of serum calcium and phosphorus in pure strains, crosses of guinea fowl and broiler chicken Serum Lipid profile in Broiler chickens According to Fontana et al. (1993), the plasma VLDL and LDL levels were significantly lower in broiler chicken with restricted feed for 7 days than the ad libitum fed birds at 49 days of age. However, they observed higher levels of plasma triglycerides and lipoproteins in feed restricted chicks compared to controls. This was attributed to increased lipogenesis in the body due to induction of metabolic shift in feed restricted birds. The effect of supplementing Sac. cerevisiae at 0.05 and 0.1 per cent in broiler chicken on serum cholesterol levels was significantly lower compared to control. The reported values were and mg per 100 ml at 0.05 and 0.1 per cent inclusion, respectively, against the control cholesterol level of mg per 100 ml of serum (Victor et al., 1993).

31 Probiotics received broiler chickens showed significant decrease in serum cholesterol levels at the age of six weeks. It was significantly reduced from mg per dl in control group to a lower value of 94 mg per dl in probiotic supplemented group (Mohan et al., 1996). Shapiro et al. (1997) reported significantly reduced blood plasma cholesterol in broiler chickens with stunting syndrome during second week of age. As studied by Gohain and Sapkota (1998) the serum cholesterol level was reduced numerically, but not significantly reduced statistically from 174 ± 8.31 mg per 100 ml in the control group of broilers to a mean value of 149 ± 2.88 mg per 100 ml in the probiotic fed group. Aziz and Shukla (2001) reported the increased total plasma cholesterol levels at 20 weeks of age in white leghorn layers and it was attributed to increase in egg mass production. Panda et al. (2001) reported a significant reduction in the total serum cholesterol concentration from ± 8.87 to ± 6.45 mg per 100 ml in probiotics supplemented broiler chicken, but no probiotic effect on high density lipoproteins (HDL), very low density lipoproteins (VLDL), low density lipoproteins (LDL) and triglycerides. They also explained that the cholesterol decreasing effect could be due to conjugating effect of bile salts with Lactobacillus acidophilus in the intestine thereby preventing them from acting as precursor in cholesterol synthesis and reducing the serum cholesterol.

32 Singh et al. (2001) reported significantly decreased levels of blood cholesterol and serum protein in pure strains and crosses of guinea fowl than broiler chicken. Bhatti et al. (2003) observed lowest cholesterol contents in desi F2 chickens that were attributed to reduced lipogenic activity in desi F2 generation. Whereas in other strains such as Fayoumi, Rhode Island Red, parent desi and desi F1 strains of chickens there was least significant difference Liver glycogen levels Griminger (1976) reported the normal liver glycogen values as 248 mg per 100 g of liver in domestic fowl. As reported by Warriss et al. (1999) the glycogen levels in the liver of broiler chicken were depleted from 16.5 mg per gram of tissue at the poultry farm to the levels 4.6 mg per gram of tissue after transportation for 3 hrs to lairage and holding them up to four hours in lairage Crude Protein in meat sample Forner et al. (1991) reported highest levels of protein contents in breast muscle and serum in chicks fed with most protein by day 21 of age, due to increased cell proliferation. Buche et al. (1992) found maximum retention of protein in broilers supplemented with 0.05 per cent probiotics.

33 Emmans (1995) reviewed the problems in modeling the growth of poultry. It was also opined that there was direct relationship between growth and feed intake. Further, chemical composition of the body needs to be fully elucidated to know the relationship. Bhatti et al. (2003) conducted an experiment to analyze the crude protein, crude fat, total ash and moisture contents in the breast muscle of Fayoumi, Rhode Island Red, Parent desi, desi F1 and desi F2 strains of chickens and observed that there were no significant differences in the parameters studied except the lowest dry matter content in parent desi stocks DNA content in muscle Scanes et al. (1975) reported the Deoxyribonucleic acid (DNA) levels in muscle tissue of bovine growth hormone treated broiler chicks as 138 to 156 µg per gram of tissue. Goll et al. (1977) reported the normal levels of DNA in porcine muscle as 25 to 30 mg per 100 g of tissue. Forner et al. (1991) reported highest levels of DNA in breast muscle in chicks fed with most protein by day 21 of age, due to increased cell proliferation. Fontana et al. (1993) determined the levels of DNA in pectoral muscle tissue in broilers fed ad libitum and severely feed restricted groups. They found and mg DNA per gram of muscle in ad libitum and feed restricted birds, at 28 days of age, respectively, and they

34 were not statistically significant. They concluded that protein metabolism in broilers was minimally affected by feeding regimens imposed in their studies Bone density Leterrier and Nys (1992) calculated the bone density as the ratio of hydrated weight to volume, where volume was measured by water displacement in a measuring cylinder. The density was ranging from 0.97 ± 0.07 to 1.19 ± 0.07 g per cubic centimeter in chicken from hatching until the chicks weighed 500 grams Effect of probiotics on Bone ash content Guevara et al. (1978) reported that at 1.5 per cent yeast inclusion, the phosphorus utilisation was better as observed in tibial ash. This effect was more prominent in male birds. Maximum mineral retention was noticed in broilers supplemented with 0.05 per cent probiotics (Buche et al., 1992). Leterrier and Nys (1992) reported similar bone volume and ash per cent with equal body weights in chicks from hatching until the chicks weighed 500 grams. The bone ash per cent on day 8 in slow growing and rapidly growing chicks were 36.3 ± 1.7 and 35.6 ± 0.8, respectively. On day 15 the respective ash per cent were 38.9 ± 1.8 and 40.4 ± 1.4. They finally concluded that an increased rate of growth was associated with a lower mineral density and higher porosity of the tibial cortices.

35 Research into the skeletal biology of farm species was relatively neglected and hence there was a need for better understanding of the possible manipulation of skeletal growth and development (Loveridge, 1999) 2.24 Effect of Probiotics on competitive exclusion and immune response Lloyd et al. (1977) coined the term 'competitive exclusion' for the mechanism of protecting the host against the proliferation of pathogenic bacteria in the alimentary tract with the administration of probiotics. The administration of live cultures of Chinese, Japanese and Russian strains of Lactobacillus acidophilus, Streptococcus faecalis, and Bacillus subtilis by mouth, mixed with feed, or added to drinking water caused disappearance of symptoms of diarrhoea in livestock and fowls and it was attributed to competitive exclusion of pathogens by probiotics (Guo et al., 1991). Certain strains of Lactobacillus and Pediococcus species are probiotic candidates for use in the microbiological control of enteropathogens (Juven et al., 1991). The probiotics competed with the pathogenic bacteria in utilizing the nutrients available in the gastrointestinal tract and hence deprived the pathogenic bacteria (Seema and Johri, 1992). Barrow (1992) reported that the use of probiotic preparations were necessary to maintain or restore a stable microflora of the gut of

36 chickens which can resist the colonization of pathogens and growth depressing bacteria. Vandevoorde et al. (1993) opined that the lactobacilli play a distinctive role in the microbial balance of the chicken gut. The use of yeast can be extended to counteract the effect of aflatoxins in poultry through adsorption of aflatoxins by the mannan-oligosaccharides present in the yeast cell wall (Devegowda et al., 1994). Perdigon et al. (1995) reported that the L. casei prevented the enteric infections on dose dependent basis and stimulated immune response in mice. Ingledew (1999) reviewed that the proliferation of pathogens in the gut was prevented by addition of live saccharomyces yeast in poultry. Yi et al. (2001) reported the chemical composition of mannanoligosaccharides (MOS) from Sac. cerevisiae as its protein had relatively high proportions of serine, threonine, aspartic acid, glutamic acid and methionine and found to decrease the number of Escherichia coli, significantly in the intestines.