The Effects of Adding Juniper Berry in Broiler Diets on Performance, Parameters of Serum, Carcass, Histopathology and Jejunum Villi Lengths

23/September/2021 Approved: 12/November/2021 ABSTRACT This study was carried out to investigate the effects of adding fruit of juniper berry ( Juniperus communis ) on performance, some carcass characteristics, serum parameters, histopathological features and jejunum villi lengths in broiler diets. A total of 150 male chicks were used in the study. The chicks were housed for 42 days. One of the experimental groups was a control group, and the diets of the 4 treatment groups included 0.5%, 1%, 1.5% and 2% dried natural juniper berries, respectively. Addition of fruit of juniper berry to the diets had positive effects on body weight, average daily weight gain, feed conversion rate and slaughter weight, feed intake, hot carcass yield and serum calcium and phosphorus levels. Juniper berry addition in the diets also increased the length of the jejunum villi. As a


INTRODUCTION
Juniperus ssp. genus is known to have about 60 species worldwide (Kesbic, 2019). Juniper berry contains up to 2% volatile oil comprising of the main compounds α-pinenes (35.4-51.85%), limonene (1.2-25.1%) and β-caryophyllene [1.3-4.2%) with smaller amounts of cadinene, α-terpinenes, tripionela, O-cymene, isopinocarveal, cedrene, elemene, thiopsene (Pandey et al., 2018). Juniper berry has been reported to have appetizing, stress preventing, immune system boosting, anti-indigestive, analgesic, hepatoprotective, antihyperlipidemic, antimicrobial, anti-inflammatory, diuretic, antioxidant, reactive oxygen species preventing, anti hypercholesterolemic, anticalaleptic activating and neuroprotective activities. It was determined that the extract obtained from Juniperus communis of fruits decreased the glycemic level of blood (Loziene and Venskutonis, 2016;Pandey et al., 2018;Kesbiç, 2019). Although studies are limited in poultry, there are many studies on the use of essential oils derived from medicinal and aromatic plants (Cengiz et al., 2015;Okoro, 2016;Sevim and Cufadar, 2017). Inci et al. (2016) reported that the addition of juniper berry at 0.5-1% level had positive effects on fattening performance and some carcass characteristics in the quail diet. However, no studies have been found showing the use of juniper berry directly in broilers rations. Therefore, Iin this study, we investigated the effects of adding 0.5%, 1%, 1.5% and 2% concentration level of Juniper berry to the broiler diets on body weight, daily gain, feed intake, feed efficiency, and parameters of serum, carcass and histopathology and jejunum villi lengths. This study was carried out to draw attention to the use of juniper berries, which are abundant in nature, in broiler rations and to shed light on future studies.

Animal and feed material and experimental design
150 three-day-old chicks of Ross-308 ® used in the experiment were obtained from CP Pullet Hatchery ® in Erzincan, Turkey. The experiment was started after the approval of protocols by the Local Ethical Committee of Van Yüzüncü Yıl University affiliated in Van, Turkey (Date: 02.11.2009, Decision No: 07). The chicks were distributed into 5 groups with 30 animals in each group. The experiment was carried out at Van Yüzüncü Yıl University Animal Research and Application Farm Directorate in Turkey (42º40'E and 37o43'). The chicks were housed in 1x1.5 m compartments with 10 chicks and under 23 hours of light followed by 1 hour darkness of photoperiods per daily cycle. One type of diet was used during the experiment, it was arranged according to the NRC (1994) ( Table 1). In the control group (C) and 4 groups which were formed by 0.5%, 1%, 1.5% and 2% dried natural berries of Juniper communis juniper berries added to the diets. Feeds and water were fed ad libitum to chicken.
Feed, performance, serum, carcass and histopathologic of analysis Feed samples were obtained at 105 °C and 16 h in oven-drying, ash for 2 h at 600 °C with furnace, fat by diethyl ether extraction method crude protein by N×6.25 Kjeldahl method (AOAC, 1990). Body weight, daily body weight gain or average daily gain (ADG), feed intake and feed conversion rate (FCR) of all birds used in the experiment were determined weekly. At the end of the experiment, 7 chickens from each group were slaughtered, and serums were removed from their blood. The values of alanine amino transaminase (ALT), aspartate amino transaminase (AST), calcium (Ca), phosphorus (P), potassium (K), chlorine (Cl) and sodium (Na) were analyzed on the auto analyzer (Cobas Integra-800 Analyzer, Roche Diagnostics GmbH, Mannheim, DE) in Van Medical Park Hospital. The birds' heads, feet, feathers and organs of digestive system except liver and kidney were separated from those trunks as "hot carcass". Division of hot carcass weight by body weight before slaughtering was calculated as "carcass yield". Fats of covers of gizzard, duodenum and intestine were weighed as "abdominal fat" and abdominal fat: hot carcass rate as "abdominal fat rate" (Nursoy et al., 2011). Liver and jejunal tissues were taken into a buffered formalin solution 10% and then routed to paraffin blocks procedures. Sections of 4µm thickness from each block were stained with hematoxylin-eosin and immunohistochemistry and examined by light microscopy and pictures were taken (Apaydın Yıldırım et al., 2017;Ertekin et al., 2017).

Statistical analysis
Data obtained were analyzed by SAS statistical program according to one-way ANOVA. The following mathematical model was applied: Yij = µ + τi + εij in which Yij represents the j-th observation on the i-th level of juniper berry content (i=0, 0.5, 1, 1.5, and 2% of the diet), μ is the overall mean response, τi is the effect due to the i-th level of juniper berry content, and εij represents the random error present in the j-th observation on the i-th treatment. The differences between the groups were performed by Duncan multiple comparison test within SAS 9.2 (SAS, 2008).

RESULTS
In general, live weights of birds were found to be high between 0-6 weeks in the control group and in 0.5-1% groups and the highest body weight was determined in 3-6 weeks and 1-6 weeks in the 0.5% group (p<0.05). Likewise, 0.5% group had the highest ADG. The lowest daily feed intake was determined in the 0.5% group between 3-6 weeks (p<0.01) and in the 0.5% group during the trial (Table 2). The slaughter weight on day 42 was found to be highest in 0.5% group (p<0.05). There was no statistically significant difference between the groups with hot carcass weight, abdominal fat weight and abdominal fat rate (p>0.05), (Table 3). Serum parameters of control and treatment groups did not statistically affect the serum ALT, AST, Cl, K and Na values of juniper fruits included in the diet (p>0.05,  Table 4). However, serum P and Ca levels were the highest in 0.5% group (p<0.01). Histopathological features were not affected by the addition of juniper fruit to the chicken diet on hepatocytes, sinusoidal cavities, mucosal, submucosa, and serosa of the liver and jejunal tissues (Figure 1 and Figure 2). The juniper groups 1% and 2% had a villi length higher than the control group (p<0.01, Table 5). In addition, the lengths of juniper groups showed a higher tendency as the consecutive fruit increase in diets increased linearly (Graph 1).

DISCUSSION
In the present study, the effects of whole, dry and grounded portions of varying concentration levels of juniper fruits were investigated on different parameters during 6 weeks. The highest body weight was found at the control group in 1-3 weeks and the highest body weight was determined at the 0.5% group in 3-6 weeks and 1-6 weeks (p<0.05, Table 2). The highest ADG was found in the control group in 1-3 weeks, and 0.5% juniper fruit group was found to have the highest ADG in 3-6 weeks and 1-6 weeks (p<0.05).

The Effects of Adding Juniper Berry in Broiler Diets on Performance, Parameters of Serum, Carcass, Histopathology and Jejunum Villi Lengths
The positive effects of juniper berry were not observed on chickens in 1-3 weeks. However, using 0.5-1% of juniper berry in the broiler diet increases body weight and ADG of 3-6 weeks. Body weights and ADG of the groups in this study are in line with the results of Lewis et al. (2003), Lee et al. (2003) and Yeşilbağ et al. (2014). The highest feed intake was found at 2% juniper group in 1-3 weeks (p<0.05) and 1-6 weeks (p<0.01) in the experiment. Feed intakes of data from the present study confirmed those values of Inci et al. (2016). In contrast, they were not compatible with studies which showed that different aromatic plant or essential oils had no effects on BW and feed intake in broilers (Zeng et al., 2015;Bozkurt et al., 2016;Simitzis, 2017).  When values of the FCR were examined in Table  2, the best utilization was determined at 0.5% group between 3-6 weeks (p<0.01) and 1-6 weeks (p<0.05). The results were supported by the values of Inci et al. (2016) that a low level of juniper added to diet increased FCR. On the other hand, other researchers (Zeng et al., 2015;Bozkurt et al., 2016;Simitzis, 2017) reported that different aromatic plant or essential oils in broiler diets had no positive effects on FCR.
The 0.5% group was found to have the highest slaughter weight after 42 days (p<0.05, Table 3). There was no statistical difference between the groups with hot carcass weight (p>0.05). Carcass yield was the lowest at 0.5% group (p<0.01). However, few researchers (Denli et al., 2004;Ocak et al., 2008;Vazquez et al., 2017) reported that different aromatic plant or essential oils did not affect hot and cold carcass characteristics. The lowest values of both abdominal fat weight (26.57 g) and abdominal fat rate (1.46%) were determined in the 2% group, but no statistical significance was found (p>0.05) in the experiment. Similar results of decreased abdominal fat weight were also reported by Ocak et al. (2008) and Denli et al. (2004). The serum parameters of the control and treatment groups did not statistically affect the serum ALT, AST, Cl, K and Na values of the juniper fruit included in the diet (p> 0.05, Table 4). However, serum P and Ca levels were found the highest in the 0.5% group (p<0.01). Histopathological features were not affected by adding juniper berry in the broiler diet on hepatocytes, sinusoidal cavities, mucosal, submucosa and serosa of the liver and jejunum tissues (Figure 1 and Figure 2). There are statistically significant differences between the averages indicated by different letters on the same line (**: p<0.01). ALT, Alanine amino transaminase; ALT, Aspartate amino transaminase; P, Phosphorus; Ca, Calcium; Cl, Chlorine; K, Potassium; Na, Sodium.

The Effects of Adding Juniper Berry in Broiler Diets on Performance, Parameters of Serum, Carcass, Histopathology and Jejunum Villi Lengths
The villi lengths of the juniper groups of 1% and 2% were higher than those of the control group (p<0.01, Table 5). In addition, the villi lengths of the juniper groups showed a higher trend with increasing juniper berry concentrations in the diets as linear effects (Graph 1). The increase in digestion and absorption of nutrients stem from increased villi lengths leading to positive effect on body weight, ADG, feed intake and FCR. Since there were very limited studies on the use of juniper berry in broilers or poultry diets, the performance, serum parameters and histopathological features data could not be compared one-to-one.

CONCLUSION
It was determined in this study that adding juniper berry in broiler diet had positive effects on body weight, ADG, feed intake, FCR, carcass characteristics, serum parameters and histopathological features. For optimum benefits, up to 1% of juniper berry can be added in broiler diet.