PREVENTATIVE EFFECTS OF PROBIOTIC (MIACLOST) ON EXPERIMENTALLY INDUCED HYPOCALCEMIC RICKETS IN BROILER CHICKS

A trial was conducted to study the effects of probiotic (Miaclost) supplement on experimentally induced hypocalcemic rickets in broiler chicks, a total of 180 one-day-old broiler chicks (Ross 308) were randomly divided into three equal groups 60 chicks per group with 3 replicates (20 birds /replicate) the dietary treatments consisted of a normal ration for G1, calcium-deficient ration 5% for G2 and calcium-deficient ration with addition of probiotics in drinking water for G3.Initial signs of rickets have been observed at 35-day of age in G2.while, in G1 and G3 no clinical signs observed, the gross lesions appeared enlargement of parathyroid gland, costochondral junction and increase in the width of growth plate of tibial bone of G2 whereas no gross lesions recorded in G1 and G3, the histopathological examination of parathyroid gland in G2 there were a focal parathyroid hyperplasia and increasing in numbers of syncytial cells and normal in G1 and G3, no intestinal histopathological changes in G1 and G2 and increase in height and width of the intestinal villi in probiotic group G3. Marked increase in the thickness of proliferation zone within growth plate of tibia bone in G2 and normal thickness in G1 and G3. the serum biochemical analysis of calcium of G2 recorded significantly low level in G2 and high level in G3 comparatively with G1, finally the serum alkaline phosphatase values were high significantly in G2 and normal in G3, it is concluded that probiotic (MiaClost) can be used as prophylaxis to prevent hypocalcemic rickets in broiler chicks


INTRODUCTION
Producers of broiler chicks started use antibiotics as feed additives to increase the digestive system performance of broiler chickens, they are mainly used to prevent infections, treat infected animals and to stimulate growth (22). Unfortunately, the use of these medicines was wildly exaggerated and bacteria began to create resistance to them (22,36).These situations demanded solutions to growth promoters and prevent infection. A search for fresh dietary alternative, namely probiotics (pro.) has been given increased attention. Pro. (MiaClost) was as efficient as antibiotics in controlling efficiency, including the ratio of body weight gain and feed conversion, and improving gut integrity by raising Villus Height and Villus Height: Crypt Depth ratio in broiler chicks threatened by E Coli, suggesting that MiaClost can act as a substitute for antibiotics to suppress E coli infection (29). Pro. are a preparations or substance containing a sufficient amount of efficient, specified live microorganisms (e.g. Lactobacillus and Bifdobacteria) that give the host a health benefit when administered in adequate quantities (49). In the gastrointestinal (GI) tract, these microorganisms compete with ingested pathogens, generate volatile fatty acids and stimulate immune responses to foreign microorganisms (7), Display inhibition action against bacterial infections (Staphylococcus aures, Pseudomonas aeruginosa) (39), also (26) showed that the presence of active compounds in chamomile including (flavonoids, kamasolen and bisaboldaxid essential oils) that may act like pro. By enhancing the gastro intestinal normal microbial population and improving nutrient absorption. Additionally the pro. enhance the quality characteristics of Eggs in hens (23). Furthermore pro. and prebiotic can be used to lengthening the life span of fermented dairy products (25). Rickets is a metabolic condition, defined by thickening of bone growth plates and inadequate mineralization in young growing chicks. Etiology is generally associated with the deficiency of nutritional calcium (Ca), phosphorus (P) and vitamin D3, impaired Ca/ P ratio (2:1) or impaired absorption of one or both of these macroelements (11,12). False feed mixing may also cause outbreaks, the adequate calcification of the cartilage is impaired in both types (Ca-deficiency and Pdeficiency) (11). To our knowledge, there is no data available on the impact of pro. on calcium deficiency rickets in broiler chicks. Therefore, the aim of this study is to explore the possibility of using pro. for prevention of calcium deficiency rickets in broiler chicks.

MATERIAIS AND METHODS
A field experiment was conducted at the agricultural experiment unit, College of Agriculture Engineering Sciences, University of Sulaimani, to study the effects of Miaclost pro. (Bacillus subtilis and Enterococcus faecium) supplements on experimentally induced hypocalcemic rickets in broiler chicks, A total of 180, one-day-old, broiler chicks (Rose 308), obtained from Kosar Company for Agriculture and Poultry were randomly assigned into 3 groups (3 replicates each, 20 chicks / replicate) and housed in separate, temperature, ventilation & light-controlled pens. The experimental rations were formulated by manufactured company to contain 0.5% calcium and fixed levels of Phosphorus, the diets were prepared to fulfill the standard requirements of broiler chicks (with no antibiotics or growth promoter supplementations). From one day old the chicks in G2 received ration deficient in calcium without MiaClost pro. while G3 received ration deficient in calcium with MiaClost pro. as directed by manufactured companies in drinking water, whereas the control group's chicks (G1) were left without treatment free drinking water and normal ration. At the end of weeks 1, 2, 3, 4, 5, and 6, two chicks were randomly selected from each replicate (8 birds / group), euthanized by slaughtering and the procedure of blood samples collection was carried out followed by putting blood samples obliquely in sterile clean plastic test tubes without anticoagulants. This procedure is achieved for estimation of serum calcium, serum phosphorus and serum alkaline phosphatase weekly. After that the slaughtered chickens dissected for two reasons; first to observe the gross lesions in the affected organs and second to obtain tissue samples (parathyroid gland, intestine and tibia bone) for histopathological examination. the histological preparations and staining achieved according to (46), and 2 cm long tibial bones specimens were decalcified according to (5). The SAS statistical package (PROC GLM) was used to determine significance of main effects (SAS, 2013). Duncan's multiple range tests was used to detect the differences between individual treatment means. Statistical significant was declared at (p<0.05).

RESUITS AND DISCUSSION
Generally, at the end of each week (1, 2, 3 and 4) of the chickens age there were no changes or statistical significant differences recorded of all studded parameters between the groups of the experiment, while, following this period the pathological changes were start to observed and there were a significant differences at (P<0.05) between the mean values of parameters of the study Clinical signs: basically, in calcium deficient groups the initial signs of rickets have been observed at and after 35 day of age, manifested with usual signsvarious extent of leg weaknessfrom unstable gait to complete inability of chickens to stand up, lying down on their bent legs or on their side. Some of affected chickens used their wings for support when moving  The main organ affected by calcium deficient ration is parathyroid gland, normally this organ is very small and it is difficult to recognized easily by naked eye, while, when there is calcium deficiency in the ration it is recognized easily due to sever enlargement of the organ this condition is clearly appeared in affected chickens of G2

. Active (dark) chief cells, 2.Inactive (light) chief cells and 3.Multinucleated syncytial cells. H&E, X400
On the other hand, the microscopic examination of the parathyroid glands in chickens of the control positive group (G2) shows focal parathyroid hyperplasia and increasing in numbers of syncytial cells (Fig  11 and 12). The hyperplastic focus consisted mainly from hypertrophied light staining (inactive) chief cells.     Whereas, the tibial bones of chickens in G2 showed a marked increase in the thickness of the proliferation zone ( fig 18) resulting in formation of cartilaginous projections extending out from the growth plate and intersecting with the diaphyseal primary spongiosa (Fig 19). The chondrocytes in these cartilaginous projections do not show any sign of degeneration or necrosis.  Regarding to the biochemical examinations of the experiment the results were as follows, from the beginning of the experiment till the 5 th week of the chickens age there were no significant differences at (P<0.05) between the mean value of serum calcium of all the groups of the experiment, while, following this period there were a significant differences at (P<0.05) between the mean values of serum calcium at and after 35-37 day of chickens age recorded in the groups of experiment were as follow (G1 9.487±0.103), (G2 9.287±0.103), (G3 9.564±0.103), as shown in Table 1. G1 recorded the highest mean value among the groups while G2 recorded the lowest mean value between the groups of the experiment. Additionally there were no significant differences at (P<0.05) between the mean value of serum calcium of the groups G1 and G3, at and after 35-37day of chickens age.
Concerning to the mean value of serum alkaline phosphatase of the groups of experiment mainly, from the starting of the experiment until the week 5 th of the chickens age there were no significant differences between the mean value of serum alkaline phosphatase (ALP) of all the groups of the experiment, whilst, following this duration there were a significant differences at (P<0.05) between the mean values as follow (G1 676.346±11.008), (G2 715.556±11.008), (G3 705.889±11.008), as shown in Table 2 . G1 recorded the lowest mean value in comparison with other groups and G2 recorded the highest mean value comparatively with remaining groups of the experiment also there were no significant differences between the mean value of serum ALP of the groups G1and G3 at and after 37 day of chickens' age.

05).The symbol (G) represents the group and (W) represent Week
Probiotics may have a possible influence on bone accumulation, which may occur through microbial activity of metabolites or enzymes or synthesis of vitamins (18,8). The point is that the significant histological improvements in calcium deficient ration groups supported by pro. happened in the chickens intestine indicates that they may have suppressed and occupied the growth of rachitic bone lesions due to calcium deficiency in the experiment; Thus, there were no visible clinical signs in the experiment group 3 till the termination of the experiment. The clinical signs of G1 (fed balanced rations), revealed normal clinical signs, in contrast, the clinical signs in chickens of G2 (fed calcium-deficient rations) showed typical signs of hypocalcemic rickets. This finding can be attributed to the effects of calcium deficiency which is in agreement with those of (11, 12). In G3 (and fed on calciumdeficient rations supplemented with pro. in drinking water), showed approximately normal clinical sings. This finding indicates a reliable prophylaxis effect of pro. against the effects of calcium-deficiency in the diet through several normocalcemic-inducing mechanisms such as increasing the solubility of minerals & increasing the absorption surface area of the intestinal epithelium (40), enhancement of vitamin D receptor expression on the intestinal epithelial cells (38) and increasing the rate of calcium absorption by the small intestinal tract (40,48,30).

Gross Findings of the Parathyroid Glands:
the enlargement of the parathyroid gland in G2, can be attributed to the hypertrophy and hyperplasia of the chief cells of the glands as an attempt to neutralize the effects of calcium deficiency (37, 4).On the other hand, normalsized parathyroid glands in G3, indicates that such supplementation could relieve the increase for functional demand of the parathyroid glands because of the dietary calcium deficiency by increasing the rate of calcium absorption through the small intestinal tract (17,48,30), increasing the solubility of minerals & increasing the absorption surface area of the intestinal epithelium (40). Related to Gross findings of the ribs and tibial bones G2 These findings which are generally in agreement with related works (3,13) can be attributed to the abnormal configuration of the metaphysis due to accumulation of nonmineralized osteoid in the zone of provisional calcification of the growth plates (10,44). In addition, softness of the tibial bones. This finding which is in agreement with those of (3) and (42) can be ascribed to the decrease in bone mineral density (24) because of the dietary-induced hypocalcemia. While, in G3 showed normal manifestation of the costochondral joints, and normal morphology of the tibial growth plates. These findings which are in agreement with (15) and (1)  mineral content and bone density and they indicate that the supplementation by pro. could provide a reasonable prophylaxis against the injurious effects of diet calcium-deficiency on the bone development process in the chickens perhaps through increasing calcium absorption through the small intestinal tract (48,30) and through enhancing microbial synthesis of metabolites, enzymes or vitamins such as D, C, K and folate which are involved in calcium metabolism and synthesis of the bone matrix and accretion of the bone (8,40). Related to histopathological examination of the parathyroid glands in the G1 revealed normal morphological characteristics of the gland. Oxyphil cells, which are present in the parathyroid glands of mammals such as human, equines and cattle (21) were not seen in the present study. This result is compatible with the results of other avian parathyroid studies which reported the lack of oxyphil cells (9). Interestingly, multinucleated syncytial cells have been observed and reported for the first time during the present study in the avian parathyroid glands. These cells, which are believed to be formed by cytoplasmic fusion of adjacent chief cells, have been observed in human, canine and murine parathyroid glands (41). In contrast, the microscopic examination of the parathyroid glands in G2 showed focal parathyroid hyperplasia. This finding which is in agreement with those of (14,47) can be attributed to the gland response to the stress of calcium deficiency because the parathyroid glands play an important role in regulation and maintenance of a normal calcium levels in the blood (4). The hyperplastic focus consisted mainly of hypertrophied light staining (inactive) chief cells. This result is constant with those of (41) and it can be ascribed to the fact that the light staining chief cells have become inactive after passing through a period of activity during which they release their secretory products (32).In addition, increasing in numbers of syncytial cells. This interesting finding possibly indicates a proportional correlation with the parathyroid gland activity. In the G3 chickens, the microscopic examination of the parathyroid glands showed approximately normal parathyroid morphological characteristics. This finding indicates a reliable prophylaxis effect of this supplement against the effects of calciumdeficiency in the diet on the parathyroid histomorphlogy through several normocalcemic-inducing mechanisms such as increasing the solubility of minerals & increasing the absorption surface area of the intestinal epithelium (40),enhancement of vitamin D receptor expression on the intestinal epithelial cells (38) and increasing the rate of calcium absorption by the small intestinal tract (48,30). The Histopathological Findings of the Small Intestine: Compared to G1 and G2 which showed normal gross appearance, normal microscopic morphology and normal height, depth and width of the intestinal villi; the microscopic examination of the small intestine in chickens of the G3 showed marked increase in height and width of the intestinal villi and consequently marked increase in the absorption surface area. This improvement is possibly attributed to the ability of the pro. bacteria to enhance the intestinal mucosa through amelioration of the epithelial injury (43), inhibition of epithelial cells' apoptosis (50), regulation of the expression & distribution of the epithelial tight junction proteins (31,35), prevention of pathogens adherence (45) and regulation of mucus secretion (6). The improvement in the intestinal morphology seen in this study is in agreement with similar findings reported by (19) and (16). However, it disagrees with the findings of (2) who stated no significant differences in height, depth, or width of the intestinal villi; this disagreement can be attributed to differences in the dose, way of administration & species of microorganism found in the concerned pro. and to animal characteristics (species, age, stage of production and health status) & housing conditions (30). Histopathological Findings of the tibial Bones of G2 This increase in thickness of the proliferation zone within the growth plate and the excessive osteoid deposition are possibly attributed to the delay in the calcification process due to hypocalcemia (27) and they are generally in agreement with the findings of (33) who revealed that the trabecular bone of hypocalcemic growing rats' tibial bones showed that the central parts of the trabeculae were calcified, whereas the peripheral bone matrix were composed of thick, uncalcified osteoid borders and they are also in agreement with the results of (28) who observed deficient mineralization and irregular, disordered columns of a continued cartilaginous proliferation in the growth plate resulting in penetration of the other zones in the growth plate in the fore limbs' bones of a 6-month-old monkey with rickets. In chickens of G3 showed approximately normal growth plate zones. This finding, which is in agreement with (34) who observed that the pro. increased the calcium absorptionlevel, bone mineral content and bone density in the growing rats. This prophylactic effect of the pro. is referring to a conceivable prophylaxis effect of these supplements against the deleterious effects of calcium-deficiency in the diet on the process of bone development in the chickens via increasing the solubility of minerals (40) and facilitating mineral absorption through the small intestinal tract (30) and via microbial synthesis of metabolites, enzymes or vitamins (8) because certain vitamins such as D, C, K and folate are involved in calcium metabolism and are also required for synthesis of the bone matrix and accretion of the bone (40). Biochemical analysis: This finding indicates that such supplementation(probiotic) could relieve the increase for functional demand of the parathyroid glands because of the dietary calcium deficiency by increasing the rate of calcium absorption through the small intestinal tract (30), increasing the solubility of minerals & increasing the absorption surface area of the intestinal epithelium (40). Regarding to the serum alkaline phosphatase , this finding of G2 result of hyperactivity under physiological conditions (hypocalcemia) and is an attempt to compensate the stress result from calcium deficiency in the ration (37,4). While, the normal levels of serum ALP of the G3, indicates that such supplementation could relieve the increase for functional demand of Serum ALP because of the dietary calcium deficiency by increasing the rate of calcium absorption through the small intestinal tract (30), increasing the solubility of minerals & increasing the absorption surface area of the intestinal epithelium (40). Totally and finally, pro. can be used as prophylaxis to prevent hypocalcemic rickets in broiler chickens, also the Broiler chickens in this study appeared to be highly efficient at utilizing Ca at low levels of dietary Ca (0.5%). This improved efficiency might have been due to increased intestinal absorption of Ca as a result of up-regulation of plasma and intestinal 1,25dihydroxycholecalciferol concentrations, with a consequent increase in the concentration of duodenal calbindin, a protein which lines the gut and binds Ca from the lumen., this result is in accordance to the result of (47) whom showed increase in calcium retention, when levels of reduction of Ca are higher than 30%.