EFFECT OF PEPPERMINT SUPPLEMENTATION AS POWDER OR EXTRACT ON BROILER PERFORMANCE, SERUM BIOCHEMICAL CONTENT AND GUT HEALTH UNDER E COLI CHALLENGE

The aim of this study was to evaluate the efficacy of different levels of peppermint (Mentha piperita) on broiler performance, jejunum morphology, organs percentage and serum biochemical content. A total of 576-day old ross 308 chickens was divided randomly into 12 treatments with 4 replicates pen per treatment and 12 birds each pen. Treatments were arranged in a 2 × 6 factorial arrangement of treatments was employed. Factors were challenge, or +; control, antibiotic, 0.5 % peppermint in feed, 1% peppermint in feed, 0.5% peppermint in water and 1% peppermint in water. The result of study was showed that the peppermint supplementation was significant effect on feed intake, feed conversion ratio and it has effect on body weight at day 24 and 35. The peppermint supplementation either in feed and water at 24 day had a significant effect on jejunum muscle thickness and villus/ crypt ratio while not effect on villus height and crypt depth. In conclusion, supplementation of peppermint has positive effect on broiler performance and gut morphology.


INTRODUCTION
Commercial poultry egg and meat production dramatically increased all over the world between 1970 and 2005, poultry production was increased faster than other animal production like beef (26). This due to efficiency of the feed conversion and lower production costs related with intensive poultry production. There are many challenges related to food safety, animal well-being, production efficiency of the animal, housing environment, management (19). They face many challenges of bacterial and viral infection. Colibacillosis and salmonella are considered major bacterial diseases in the poultry industry worldwide (11). Escherichia coli belongs to the enterobacteriacea family, gram-negative bacteria, growing aerobic and anaerobic with varying size and shape (20). They mainly affect intestine of birds which cause many diverse diseases, in which Escherichia coli (APEC) is the main reason of colibacillosis in broiler chickens (15). It has a negative economic impact on poultry production worldwide. Antibiotics have been used in the poultry industry to fight against infection disease. However, the frequent use of antibiotics may increase the incidence of antibiotic resistance. Which leads to economic losses and treatment failure, and there are also concerns about human health about the presence of antimicrobial residues in meat (7,9). It is important to find an effective way to replace antibiotics to control infectious diseases and reduce the spread of resistant bacteria (14). Medicinal herbs have been used as alternatives to antibiotics. Medicinal plants and their products including plant extracts or essential oils are introduced as candidates for use in broiler diets and they have beneficial effects on broiler health (6). The mechanism of action of those medical plants may be through stabilization of normal gut microflora, prevention of pathogens colonization and play important role of digestive enzymes production and activities improvement (13). Peppermint is considered therapeutic herbs in the world is member from the Labiate family. It is broadly utilized in herbal medicine (21). The peppermint consists of many chemical compounds which play important role as antibacterial activities, for instance, menthol in which main phenolic compound in oil peppermint. Some studies indicated that essential oils of peppermint inhibit the growth of Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, and Candida albicans (22). Thus this study aimed to determine the effect of different levels of peppermint (Mentha piperita) in feed and water on broiler performance (weight gain, feed intake, FCR) at 10,24,35 day and histology of jejunum (villus height, crypt depth, villi/crypt ratio, muscle thickness) and organs percentage and serum biochemical at day 24.

MATERIALS AND METHODS
The study was conducted at the animal house of animal production department, College of Agricultural Engineering Sciences, University of Duhok, Kurdistan region of Iraq.

Animal husbandry
A total of 576-day old Ross 308 chickens were divided randomly into 12 treatments with 4 replicate pen per treatment and 12 birds each pen (wire mesh partitioned at (120*75cm) will assign in two room to prevent birds from infect E. coli. The rooms temperature at the first day was set on 33-34 °C and then decreased by 3 °C until the end of week. Temperature and light program were adjusted according to the Ross 308 guide (Aviagen 2012). The water and feed were provided ad libitum the feeding program consist three periods the first is starter from 0 to 10 day and second is grower from 10-24 day, third finisher from 24 to 35. The ingredient and composition of basal diet was showed at Table 1. The primary determinants of performance, i.e., cumulative pen weight, feed intake (FI), and feed conversion ratio (FCR) were measured at d 10, 24 and 35.

Dietary treatment
In this study the negative control (no additive and non-challenge) and negative antibiotic (non-challenge and the diet 0.33 g/kg zinc bacitracin). 0.5% peppermint in feed (5 g/kg peppermint in feed and non-challenge), 1%peppermint in feed (10g/kg peppermint in feed and non-challenge) and (0.5% peppermint in water and non-challenge) (1%peppermint in water and non-challenge). However, the positive control (there is no additive and challenge and positive antibiotic (challenge and addition 0.33 g/kg zinc bacitracin in diet). 0.5% peppermint in feed (5 g/kg peppermint in feed and challenge), 1%peppermint in feed (10g/kg peppermint in feed and challenge) (0.5% peppermint in water and challenge), (1%peppermint in water and challenge).

Preparation of powder peppermint
Fresh peppermint plant was obtained from Duhok during July and August month in 2018. At the beginning the thick stem was removed and spread on a clean cloth under the sun for 2 days to dried up.

Challenge of E. coli
These bacteria were isolated in a laboratory from local farms. Incubated E. coli was occurring at 37 °C overnight in the MacConkey broth sterile (100 ml), Then 0.1 ml of the previous broth Subsequent incubations in Eosin methylene blue agar for accounting colony. 1000 mL of MacConkey broth was used to inoculate a colony from Eosin methylene blue agar to get the vaccine challenge. Birds were inoculated with a 1.5 ml of the suspension E. coli (3.8 × 108 CFU per mL) at 8 and 9 day of the bird's age.

Measurements studied traits Jejunum morphology
The fixed samples were dried, disinfected and embedded in paraffin wax for histological analysis subsequently. Successive longitudinal Superfrost® slices (Thermo Scientific, Rockville, MD, USA) and were staining with eosin and hematoxylin. The Crypt depth and Villus height was measured by Dino-eye software and images was taken by using a color video camera (Dino-eye 20). The height of villi and depth of crypts and thickness of muscles from each replication was measured ten times. The means of crypt depth, villus height was obtaining and also villus height per crypt depth ratio was determined. Relative organ percentage From each pen 1 birds was picked for eviscerating to calculate the heart, spleen bursa, liver and gizzard weight at day 24.

Blood biochemical
Blood samples were collected from bird at day 24 old. Two birds from each cage were randomly selected and slaughtered. The blood was collected from jugular vein, centrifuged at 3000 rpm for 15 min and serum were taken, frozen at -20E°C. Determination of plasma total protein (TP), albumin, cholesterol, glucose, creatinine (CR), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), triglyceride (TG) and globulin were determined by automatic analyses Biolis (Tokyo Boeki Medical system) 24i by using respective biochemical kits of Cormay company the Cat. No 4-204 and 4-404 and the application of the procedure are available on (prestige 24i Biolis ).

Statistical analysis
The comparison between means were carried out according to Duncan's multiple range test (P < 0.05) using a computerized program of SAS (SAS, 2013). The results of this study showed a significant effect of additives on broiler performance ( Table 2). From 0-10 day the inclusion of antibiotic significantly increased weight gain and feed intake over all treatments. However, the birds fed 1%peppermint had lower feed intake compared to the control and antibiotic (P<0.002). Moreover, birds fed peppermint either in water or feed decreased feed intake when compared to birds fed antibiotic. A significant challenge ×additive interaction was observed in feed intake (p<0.05). The nonchallenge 1% peppermint in feed treatment significantly had decrease feed intake when compared with challenged control and antibiotic. From 0-24 day the effect of additives was visible (Table 3). Birds fed Antibiotic had higher (P< 0.01) body weight gain over other additives. All additives had better (P<0.001) feed conversion ratio when compared to control. Significant challenge × additive interactions (P < 0.01) were observed in body weight gain and feed conversion. the non-challenged birds fed antibiotic significantly increase body weight gain over challenged control and non-challenged 0.5% peppermint in water. On the other hand, the challenged or no challenged birds fed peppermint either in water or feed (except non-challenged 0.5% peppermint in water) were not different in body weight gain in compare to non-challenged birds fed antibiotic. In term of feed conversion ratio, the challenged control had poorer(P<0.001) feed conversion ratio over all treatment. The nonchallenged antibiotic, non-challenged 1% Peppermint in feed, challenged 0.5% and 1% Peppermint in water significantly improved feed conversion ratio in compare to nonchallenged control. The performance results from day 0-35 are present in table 4. There was no significant effect of challenge on body weight gain, feed intake and feed conversion ratio. The bird's performance was markedly different upon the feed additive treatments. The highest (P<0.001) body weight gain and feed intake was recorded for birds fed antibiotics. No significant different were found between birds fed control and peppermint either in water or feed for body weight gain. Birds fed control diet had significantly higher feed intake than birds fed 0.5 and 0.1 peppermint in water. All additives, except 1% peppermint in water, significantly improved feed conversion ratio over control. Challenge × additive interactions (P<0.001) was observed for body weight gain, feed intake and feed conversion ratio. Body weight gain of challenged antibiotic birds were significantly higher overall treatments except non challenged antibiotic. Challenged birds fed control diet had lower body weight gain when compared to non-challenge 1% peppermint in feed, no challenged control and challenged control. Challenged birds fed antibiotic had significantly higher feed intake in compare to all treatments, whereas challenged birds fed 1% peppermint in water had lower feed intake, except 0.5% peppermint in water, compared to other treatments. Poorer feed conversion ratio was recorded for challenged birds fed control diet. All treatments (challenged and nonchallenged) significantly improved feed conversion ratio over challenged control.

Jejunum morphology
The results of jejunal villus height, crypt depth, villi/crypt ratio and muscle thickness at day 24 are present in table 5. Additives had a significant effect on jejunum muscle thickness and villus/ crypt ratio. The birds fed 0.5% and 0.1% peppermint either in water or feed significantly reduced the muscle thickness compared to birds fed control diet. All additives had higher (P<0.01) villus/ crypt ratio over control. There were significant challenge*additive observed in crypt depth and villus/ crypt ratio. The inclusion of additive is non-significant effect on villus height, crypt depth, muscle thickness) at the 24 day only the significant effect appears on villi/crypt ratio p<0.05 in which the different level of peppermint had significant effect on villi crypt ratio that is increase villi crypt ratio compared with control. Also, there is no significant effect between challenge and nonchallenge birds p>0.05 and there is nonsignificant challenge ×additive interactions observed p<0.05 in villus height, crypt depth, villi/crypt ratio, muscle thickness.

Organs percentage
Organ percentage from live body weight results are presented in table 6. The inclusion of all peppermint additives was not affect organ percentage but only the antibiotic has decrease intestine percentage. Also there is no significant effect found between challenge and non-challenge bird and also there is no significant × additive interaction was observed in percentage of internal organ.

Serum biochemical
Serum biochemical results are present in Table  7 refer the effect of antibiotic, different levels of peppermint (Mentha piperita) in feed or water on serum biochemical (glucose, creatinine, total protein, albumin, cholesterol, triglyceride, GOT(ASAT), ALAT, globulin). At day 24 the effect of challenge was clearly visibly. Challenged birds have higher (P<0.001) glucose, creatinine and albumin than non-challenged birds. However, nonchallenged birds had significantly higher total protein and globulin than challenged birds. No significant effect of additives was observed in glucose, creatinine, total protein, albumin, cholesterol, triglyceride, (ASAT), ALAT, globulin. There were challenge*additive interaction observed for glucose, total protein, albumin and globulin. Challenged control birds had highest serum glucose concentration compare to non-challenged treatments. While the birds fed challenge 1% peppermint in water had significantly lower serum glucose compared to challenged treatments. The serum total protein concentration of challenged birds fed 1% peppermint in feed or water and 0.5 % peppermint in water were significantly lower than birds fed challenged antibiotic and nonchallenged 0.5 peppermint in water. The serum albumin concentration of challenged birds fed antibiotic and control was higher (P<0.001) than all non-challenged treatments and challenged 1% peppermint in feed or water. The peppermint treatment in water and feed and antibiotic under non-challenge condition and negative control had significantly increased serum globulin compared with this treatment under E. Coli challenge.    The peppermint supplementation in feed decreased feed intake at 10 days. The results were in agreement with the finding of Aroche (5) who reported that the supplementation of mixed leaves powder of medicinal plant reduce feed intake. Also Amasaib (4) who showed that the addition of different levels of spearmint (Mentha spicata) improved feed intake. While, the results were in contrast with finding of Ocak (18) who reported that the dietary supplementation of dried peppermint (Mentha piperita L.) and thyme (Thymus vulgaris L.) leaves don't have any considerable effect on feed intake. At the 24 day the peppermint supplementation either in feed and water had improve feed conversion ratio. Also, the all peppermint additive except 1% in water had significantly improved feed conversion ratio at day 35. This result is similar to Gurbuz (10) who found that the peppermint supplementation had a significantly affect on FCR compared with control. Also Ahmed (2) they reported the addition 250 mg peppermint extract and supplement 1 g flavomycin at the grower and Finisher periods had decrease the feed conversion ratio compared with other treatment. Also the results were agreed with Mustafa (17) study who reported that (1% betony+ 1% peppermint) had significant effect on feed conversion ratio in which have better when compared with control at age 25-42 day. In contrast, Witkowska (27) who reported the essential oil mist in peppermint no effect on feed conversion ratio from (1 to 42 days). Most of medical plants consist of different essential oil in which stimulate digestive enzyme secretion and the effect on microbial that present in gut through the balance of microbial (6). The peppermint supplementation either in water or feed have effect on weight gain on day 24 and 35. The result was agreement with finding Witkowska (27) study who reported the addition peppermint oil mist had increase the mean BW and WG than control and birds exposed to thyme oil mist. While the result was in disagreement with the finding of Ocak (18) who recorded the addition peppermint had not any effect on body weight at 42 of age. The reason the peppermint improve bird performance may be due to that the herbs like peppermint contain different his was variety of active components which affect process of digestion in which stimulate saliva secretion and enhance bile acid synthesis in liver and bile excretion which affect process digestion and lipid absorption (8). The peppermint supplementation either in feed or water at 24 day had a significant effect on jejunum muscle thickness and villus/ crypt ratio while not affect on villus height and crypt depth. The result were agreement with Yang (28) whom reported that the addition of essential oils and organic acids in bird diet during finisher period were increased villus height to crypt depth ratio when compared to the bird fed diet with 0.15 g per kg enramycin at grower period. While, the result disagree with finding of Ahmed (2) whom recorded that the extract of peppermint had desirable effect on crypt depth and villus height in ileal in small intestine. The increase of villus height: crypt depth ratio can indicate an increase in the turnover of epithelial cells in the intestinal part due to the increase of beneficial bacteria in mint groups (16). According to this study, the all peppermint additive in water or feed and antibiotic don't observed any considerable effect on internal organ percentage (intestine, liver, gizzard, pancreas, spleen) at day 24. The results were similar to the finding of Toghyani (24) who reported that the internal organ weight are not affect by dietary addition of peppermint . Also Ocak (18) who reported the dried peppermint (Mentha piperita L.) or thyme (thymus vulgaris L.) in diet supplements was not have any significant effect on the weights of edible organs , pancreas and gizzard. Abdel-Wareth (1) found the addition of menthol and leaves peppermint had no effect on relative weight of pancreas ,gizzard, heart ,liver, spleen .While the result was in contrast with the finding Mustafa (17) who showed the supplementation of dried peppermint 2% in broiler diet had significant increase liver and heart and gizzard percentage. According to this study, peppermint supplementation either in water or feed and antibiotic had not considerable effect on serum biochemical at day 24. The results were in agreement with finding of Khursheed (12) who reported that the supplementation of mint leaves with or without enzyme in both 1 or 2% levels were not observed any significant effect on serum glucose, total protein, cholesterol, SGPT and SGOT when compared with control. Also the results in this study were similar to the finding of Toghyani (24) who showed that the addition of different levels of black seed (Nigellasativa) and peppermint (Mentha piperita) had no significant effect on triglyceride , protein, albumin, total cholesterol. In contrast, Ahmed (2) indicated that peppermint had significant effect in which they increase the cholesterol and total protein ,globulin and liver enzyme. According the study of Akbari (3) supplementation of essential oil of peppermint and chromium picolinate under heat stress decrease the concentration of triglycerides and glucose. Also Tayeb (23) in their study found the medical plants had significant affect on albumin, globulin, cholesterol and glucose concentration.
The result of serum biochemical, in this study were varied there are more factors affecting on serum biochemical. It can be nutritional situation and