Abstract: Objective: This experiment was conducted to assess the use of Chinese Royal Jelly (RJ) over a 6 weeks period on performance, behaviour and blood parameters of broilers. Methodology: Approximalty,180 one-day-old Ross broiler chicks were randomly assigned to six treatment groups as follows; Group 1: Control (LSD; 8 birds, per meter square and no supplementation), Group 2: High stocking density (HSD; 16 birds per meter square and no supplementation), Group 3: LSD-RJ 250 (8 birds per meter square and 250 mg kg–1), Group 4: LSD-RJ 500 (8 birds per meter square and 500 mg kg–1 b.wt.), Group 5: HSD-RJ 250 (16 birds per meter square and 250 mg kg–1 b.wt.) and Group 6: HSD-RJ 500 (16 birds per meter square and 500 mg kg–1) RJ given orally. Scan sampling for comparative behavior in all groups was carried out at 2 min intervals during 2-20 min periods for 5 days each week. Results: The highest Feed Intake (FI), Feed Conversion Rate (FCR) and Body Weight Gain (BWG) were recorded in the LSD group (p<0.05) fed on diet supplemented with Chinese Royal Jelly (RJ) compared to other treatments during the experimental period. Also, FI, FCR and BWG were significant (p<0.05) improved in HSD-RJ 250 and HSD-RJ 500 compared to other groups. It was found that a significant higher proportion of chicks in RJ supplemented groups were engaged in feeding, resting, walking, standing, foraging and preening behaviour however, drinking behaviour was not significant influenced (p>0.05) by RJ supplementation. Chicks fed 500 mg of RJ were found to have the highest values of the heterophils and lymphocytes. Serum total protein, albumen and globulin concentrations were significantly (p<0.05) boosted in the RJ groups compared to the controls. Conclusion: The study was completed concluding that Chinese royal jelly supplementation can alleviate the deleterious effect of high stocking density in Ross broiler chicks, improved behaviour, performance and blood parameters in the examined broiler chicks under high stocking density.
INTRODUCTION
In broiler production systems, stocking density (or floor space allowance per bird) is believed to have major implications on bird performance and has recently been identified as an important welfare concern in the broiler industry1. A number of studies have investigated the effect of various stocking densities on behavioural traits in broiler chickens as reviewed by Ekstrand and Carpenter2. It is known that increasing the stocking density leads to changes in behaviour in both commercial3 and experimental conditions4. In general, increasing the number of birds per unit area was found to increase the incidence of agonistic behaviour and reduce the amount of time spent lying (resting) behaviour. In recent years, there has been renewed interest in the investigation of bee products (honey, pollen, Royal Jelly (RJ), bee venom and propolis) for their numerous functional, biological and pharmaceutical beneficial effects5,6. The RJ is a honey bee (Apis mellifera L.) secretion component for bee egg, larvae and adult queen nourishment. This secretion is extremely rich in nutrients and has a yellowish-white, creamy liquid appearance and an acidic pH. It is secreted by the hypo-pharyngeal glands of nurse bees. The overall composition of RJ is 60-70% water, 12-16% crude protein, 10-16% total sugar, 3-6% lipids, vitamins and mineral salts. The composition varies according to the source of the exudates, climate and some environmental conditions. The RJ contains many bioactive ingredients. The main bioactive material is 10-hydroxy-2-decenoic (HDA, RJ acid) an unsaturated acid that is only found in RJ in nature. Numerous HDA effects including antibacterial, antifungal, antiviral7,8 and immunoactivating9 effects were reported. The RJ contains major proteins with high levels of essential amino acids and peptides10, possessing immunomodulating11 and antioxidant properties9. Phenols and polyphenols in major protein structures are responsible for significant antioxidant activity7. The RJ is rich in vitamins especially, water-soluble vitamins (vitamins B and C) and minerals such as potassium, calcium, magnesium, iron, zinc, sulfur and copper12. The continuously increasing demand for protein for human consumption has prompted some producers and researchers to search for new studies in animal production and husbandry practices in order to improve production efficiency. Cage density is an important environmental factor affecting production levels and quality in poultry meat13. The goal of poultry producers is to achieve a balance between production efficiency and bird welfare. Increasing the bird number per unit of space (density) reduces housing, equipment and labor costs. It is well documented that chickens housed at high density grow more slowly and have higher mortality and lower production quality14. On the other hand, the RJ has an antioxidant effect that counteracts the lipid per oxidation caused by free radicals under different stress conditions7. The purpose of this study was to examine the potential protective effects of RJ for alleviating the metabolic, hormonal and blood parameters changes resulted from high stocking density.
MATERIALS AND METHODS
Birds and experimental design: The experiment was conducted in accordance with animal welfare. A total of 180 one-day-old unsexed broiler chicks (Ross 308) were randomly divided into six equal groups according to their initial body weights. Each group contained three replicates of 10 chicks obtained from a commercial company were used for this study. The chicks were grown in a separate floor pens with wood shavings and lighting program of 23 h lightning and 1 h darkness day–1constant light. Food and fresh water were provided ad libitum using a two-phase broiler feeding regime. This consisted of a commercial starter crumbles for the first 21 days, followed by a commercial grower/finisher pellets until the conclusion of the study at day 42. The ingredients and chemical composition of the basal diet and calculated energy uptake per kilogram diet are presented in Table 1.
| Table 1: | Ingredients and chemical composition of the basal diet (g kg–1) and energy uptake (kilogram per diet) |
| SBM: Soya bean meal | |
The temperature was maintained at 34°C on day 1 and was gradually reduced to 22°C by day 21. The relative humidity was maintained at 40-65%. The experiment was performed during winter and open windows were used to achieve cross-ventilation. All experimental birds were reared as per the recommendations of the National Research of the US15 in a temperature-controlled room at 24°C. Each broiler chicken was vaccinated against Newcastle Disease Virus (NDV) via drinking water at 6, 14, 21 and 32 days of age and against Infectious Bursal Disease (IBD) at 10, 18 and 25 days of age. The normal stocking density was (10 birds per meter square) and the high stocking density was (15.4 birds per meter square) according to Sorensen et al.16. The stocking densities were coded Low Stocking Density (LSD) and High Stocking Density (HSD) and contained a floor space allowance per meter squar of 8 and 16 birds, respectively. The experimental groups were reared for 1-42 days. Groups were arranged and fed as follows: Group 1: Control group; Low stocking density (LSD; 8 birds per meter square and no supplementation), Group 2: High stocking density (HSD; 16 birds per meter square and no supplementation), Group 3: LSD-RJ 250 (LSD; 8 birds per meter s quare and 2 50 mg kg–1 b.wt.), Group 4: LSD-RJ 500 (LSD; 8 birds per meter square and 500 mg kg–1 b.wt.), Group 5: HSD-RJ 250 (HSD; 16 birds per meter square and 250 mg kg–1 b.wt.) and Group 6: HSD-RJ 500 (HSD; 16 birds per meter square and 500 mg kg–1 b.wt.).
Sample preparation and identification: Chinese Royal Jelly (RJ) was obtained from a commercial firm in China, dissolved in distilled water and kept frozen at -20°C until used. Chemical composition of Chinese Royal Jelly (RJ) was assessed by Gas Chromatography/Mass Spectrometry (GC/MS). The GC/MS analysis was performed at the Analytical Chemistry Unit, Faculty of Science, South Valley University (Table 2). The GC-MS analyses were carried out to detect the main components of RJ with an Agilent GC 6890 gas chromatograph coupled to an Agilent MSD 5973 mass detector in electron impact ionization mode. The gas chromatography column was a Zebron (ZB-1) methyl polysiloxane (30 m×0.25 mm i.d.×0.25 μm film thickness). The components of Chinese royal jelly were characterized by its Retention Time (RT) and determined by considering their areas as percentage of the total ion current (Table 2). The identification compounds were accomplished using computer searches on Wiley and NIST MS data library using MSD Chem Station software G170EA E.02.01.1177.
Broiler performance: The broilers were weighed every week and Feed Intake (FI) was measured weekly during the study. Daily body weight gain (g), daily feed consumption (g) per replication and feed conversion ratio of the chicks for the periods between days 0-21, 21-42 as well as for the overall experiment were calculated.
| Table 2: | Chemical composition of royal jelly bioactive components assessed by GC-MS |
| RT: Retention time, TIC: The ion current generated depends on the characteristics of the compound concerned and is not a true quantitation | |
Dead birds were recorded every day to the end of the study (day 42) to calculate the mortality rate.
Behaviour measurements: Behavioural observations were performed throughout the 6 weeks experimental period using scanning techniques17 in all groups was carried out at 2 min intervals during 2-20 min periods (10:00 to 10:20 and 14:00 to 14:20) for five days per week for six consecutive weeks. Before starting to observe a pen of birds, the observer sat quietly for 5 min to get the birds accustomed to his presence. Instantaneous scan sampling observations of chick’s behavior were performed according to Lee and Craig18. For each scan the number of chicks engaged in eight mutually exclusive activities were recorded every 60 sec (feeding, drinking, resting, walking, standing, foraging, preening and feather pecking). A detailed description of those parameters is presented in Table 3. The percentage of chicks engaged in each behavior was calculated during all scan samples in each pen.
Blood samples collection and analysis: At the end of the experiment (day 42), bird per group were randomly selected from each treatment, slaughtered and then blood samples were collected. Blood samples were divided into two equal parts. The first part of blood was collected with heparin as anticoagulant to be used for counting of Heterophils (H), Lymphocytes (L) and H/L ratio according to Shen and Patterson20 . The second part of each blood sample was centrifuged at 4000 rpm for 15 min to separate blood serum. The obtained serum was kept frozen at -20°C until analyzed. Serum samples were used to estimate serum biochemical by digital-VIS/ultraviolet spectrophotometer (723C visible spectrophotometer, Shanghai Phenix Optical Scientific Instrument Co., Ltd, China). Total serum proteins and albumin (g dL–1) were assayed by a colorimetric method using a commercial kit as previously described21. Serum globulin was calculated by the difference between serum total protein and albumin since, the fibrinogen usually comprises a negligible fraction22. Also, the Albumin: Globulin ratio was calculated.
Statistical analysis: Results were analyzed statistically using SPSS (Statistical Package for the Social Sciences) version 16.0 for windows. The data were described using Means and Standard Deviations (SD) as descriptive statistics. Significant differences among treatment means were measured and compared by One-way analysis of variance (ANOVA) was performed followed by Least Significant Difference (LSD). Statistical difference with p<0.05 was considered as significant.
RESULTS AND DISCUSSION
Effect of Chinese Royal Jelly (RJ) on performance of broiler chicken exposed to high stocking density: As shown in Table 4, daily body weight gain was similar among groups in the beginning of the study. This parameter was significantly lower in the HSD group at day 42 than in other groups (p<0.05). The RJ increased daily body weight gain in experimental groups to levels comparable to the control group. Daily feed consumption and feed conversion ratio were significantly deteriorated by HSD (p<0.05). Daily feed consumption and FCR improved in the HSD-RJ 250 and HSD-RJ 500 groups significantly (p<0.05). Mortality rates were significantly higher in the HSD group at day 42 than in other groups (p<0.05). The RJ supplementation also under HSD conditions, decreased the mortality rates of broilers resulting in levels similar to the control. The effects of poultry rearing under HSD conditions have been studied for years. Similar to the performance results in the present study, HSD was shown to cause decreased daily body weight gain, daily feed consumption and feed conversion ratio were significantly deteriorated by HSD, resulting in deteriorated poultry growth performance and welfare15,23. When the number of birds per unit of space increases, microclimate conditions around the birds deteriorate. The use of different management practices, equipment and several dietary alternatives has been recommended to alleviate such environmental stress5,6. The RJ has many flavonoids, organic compounds, fatty acids and other active ingredients as shown in Table 4.
| Table 3: | Behavioral ethogram of chicks according to Guo et al.19 |
| Table 4: | Effect of different levels of Chinese Royal Jelly (RJ) on broilers performance at 0-6 weeks of age under different stocking densities |
| Each mean represents 3 replicates for each treatment with each replicate containing 5 birds, a-eMeans in the same column and the same effect with different superscripts are significantly different (p<0.05) | |
| Table 5: | Influence of Chinese Royal Jelly (RJ) on total number of birds observed performing various behaviors (Mean%+SD) under different stocking densities |
| Each mean represents 3 replicates for each treatment with each replicate containing 5 birds and a-eMeans in the same column and the same effect with different superscripts are significantly different (p<0.05) | |
These chemical components can improve growth performance of broilers under HSD stress resulting in increased daily body weight gain and daily feed consumption and better feed conversion ratio. The impact of these active ingredients and especially, flavonoids on bird performance under stress conditions was reported previously6.
This result was in accordance with Pourali et al.24 who found that garlic powder improved average daily feed intake in broiler chickens. In the same trend, Javed et al.25 reported that feed intake was improved when broilers were supplemented with aqueous extract of medicinal plants containing garlic (10 mL L–1 of drinking water). Contrary, Aji et al.26 mentioned that administration of 100 mg of garlic not affect feed intake of broiler chickens. Also, Ashayerizadeh et al.27, Onibi et al.28 and Mohebbifar and Torki29 found no effect of garlic powder supplementation on feed intake of broiler chicks.
Effect of Chinese Royal Jelly (RJ) on behaviors of broiler chicken exposed to high stocking density: The effects of stocking density on broilers behaviour are presented in Table 5. The most commonly observed behaviour in all of the densities was resting (lying), the percentage of birds engaged is 39.06±3.48 to 55.20±2.30 in LSD compared to HSD, respectively. The percentage of birds engaged in feeding, drinking, walking, preening and foraging behavior in HSD were significantly lower than the LSD group (p<0.05). Standing and feather pecking behaviour tended to increase in HSD than LSD group of all experimental period. Other studies have shown that as stocking densities increase, chickens spend less time resting, suggesting that an increase in animal density results in decreased opportunities for undisturbed rest3,4,30. Stocking density affected standing behaviour in Table 5, it seems to affect the increase in the level of tension31-33.
The effects of dietary supplementation of Chinese Royal Jelly (RJ) on broilers behaviors are presented in Table 5. A significantly higher proportion of chicks in RJ supplementation, LSD-RJ 250 and LSD-RJ 500 were engaged in feeding behavior 21.51±1.69 and 23.73±1.62 compared to control group14.86±1.29 (Mean±SD, respectively p = 0.001) with HSD group being also increased proportion of chicks engaged in feeding behavior in RJ supplementation but not different from control group (14.75±1.48 and 15.36±1.56) in LSD-RJ 250 and LSD-RJ 500, respectively which in turn was reflected in final body weight and other productive performance of chicks. Results from behavioral observations in the present study indicated that dietary supplementation of Chinese Royal Jelly (RJ) significantly affects the proportion of birds engaged in resting, walking, standing, foraging and preening behaviors (Table 5). Birds in unsuppleminted diet control group were showed significantly less resting behavior 39.06±3.48 compared to birds in LSD-RJ 250 and LSD-RJ 500 groups; 45.09±3.11and 43.18±2.61, respectively (Mean±SD p = 0.001). With HSD group being also increased proportion of chicks engaged in resting behavior in RJ supplementation that differ from control group (52.09±3.36 and 51.66±2.36) in HSD-RJ 250 and HSD-RJ 500, respectively. Results summarized in Table 5 showed non-significant differences of dietary treatment on the percentage of birds engaged in drinking. Increased standing behaviors in control and HSD birds may be related to low feeding behavior that demonstrated in these groups. Hocking et al.34 reported that pacing was negatively related to rate of consumption. Moreover, Hocking35 observed that the proportion of time spent standing and walking was associated with a decrease in the proportion of time involved in eating, scratching and pecking activities.
As shown in Table 5, Percentage of birds engaged in walking behavior, significantly decreased in the HSD group (p<0.001) resulting in locomotion problems and difficulty accessing feeders and drinkers. The RJ supplementation decrease the number of the birds engaged in walking behavior in both doses also under HSD conditions, the number of the birds decreased, resulting in levels similar to the control. The Percentage of birds engaged in standing were significantly higher in the HSD group, HSD-RJ 250 and HSD-RJ 500 groups than in the LSD, HSD-RJ 250 and HSD-RJ 500 groups (p<0.05). The birds have to spend more time standing which results in social anarchy for resting birds36. All of these problems cause physical and physiological stress to the birds37. The total percentage of birds engaged in foraging and preening were higher in the control than HSD, LSD-RJ 250, LSD-RJ 500, HSD-RJ 250 and LSD-RJ 500 groups (p<0.05). The Percentage of birds engaged in feather pecking behavior were significantly higher in the HSD group than in the LSD whereas, RJ supplementation under LSD (LSD-RJ 250, LSD-RJ 500) and HSD (HSD-RJ 250, HSD-RJ 500) conditions significantly decreased the percentage of birds engaged in feather pecking behavior (p<0.001).
Effect of Chinese Royal Jelly (RJ) on serum biochemical parameters of broiler chicken exposed to high stocking density: Results presented in Table 6 indicated that serum total protein, albumin and globulin concentration were similar among LSD and HSD groups. The RJ treatment significantly increased serum total protein, albumin and globulin concentration and this effect was dose-dependent (p<0.05). Adding RJ to the diet of stressed broilers was reflected with significant higher contents of its serum total protein, albumin and total globulin in the HSD-RJ 500 groups (p<0.05).
| Table 6: | Effect of different levels of Chinese Royal Jelly (RJ) on hematological properties portrayed by the chicks at 6 weeks of age under different stocking densities |
| Each mean represents 3 replicates for each treatment with each replicate containing 5 birds, H: Heterophils, L: Lymphocytes and a-eMeans in the same column with different superscripts are significantly different (p<0.05) | |
The results are comparable to Kurkure et al.38 reported an increase in plasma albumin concentration in White Leghorn cockerels, orally treated with RJ at 10 mL bird–1 day–1. The results of Elnagar et al.39 who reported that in stressed male rabbits, RJ led to a significantly increase in serum total protein, albumin and globulin. In contrast, RJ did not influence serum total protein, albumin and globulin in growing rabbits39. Meanwhile, the improvement of serum total protein and its fractions in the group fed RJ may be related to its direct effect as a growth promoter on the haemopoietic tissue and the stimulating effect on the liver exhibiting an anabolic action favoring protein synthesis and also its preserving effect on the body protein from degeneration39.
The present study showed that HSD groups had significantly higher heterophil and H/L ratio compared to control group (p<0.05), while the lymphocytes was not significantly affected by stocking density. Meanwhile, groups fed RJ (LSD-RJ 250, LSD-RJ 500) had significantly lower heterophil and H/L ratio compared to control group (p = 0.001). The HSD groups shown an increase in lymphocytes compared to (HSD-RJ 250, HSD-RJ 500) (p<0.05). The increased percent of lymphocytes in broilers fed diets with RJ may be related to its effect as antibacterial, antiviral and antifungal on their immunity system.
The significantly lower heterophil in RJ supplemented birds, these results indicated that control and HSD groups were more stressed and supplementation of diet with RJ reduced stress in broiler chicks. Maxwell40 mentioned that stressed birds shown an increase in H/L ratio and this ratio may be a more reliable indicator of mild to moderate stress than plasma cortisone concentration. Stress in HSD birds was reflected on its productivity. These results were in accordance with Saxena and Madan41 who reported that stress evokes harmful responses that interferes with the general health, productivity and resulted in immunosuppression. Results of this study were in agreement with Al-Kassie42 and Najafi and Torki43 they reported that groups fed oil extract derived from thyme had significantly lower H/L ratio. Also, Ali44 found supplementation of the broiler chickens diet with Thymus vulgaris leaves powder from 0-8 weeks of age resulted in significant (p<0.01) decrease in heterophil to lymphocyte ratio.
CONCLUSION
In conclusion, the RJ is regarded as a valuable food supplement because of its functional, biological and pharmaceutical properties. This study suggest the potential protective activity of this bee product on performance, behaviour and blood parameters of broilers under HSD stress.
ACKNOWLEDGMENT
We acknowledge all staff at the Faculty of Veterinary Medicine. All workers in the farm of the Faculty of Veterinary Medicine are gratefully acknowledged for their efforts and help.