Effects of dietary phytogenic supplementation on performance, semen quality and serum biochemical of rabbits

Recently, restrict the use of antibiotic growth promoters by several countries and the menace of antibiotic resistant bacteria has forced to look for alternatives in rabbits. This study aimed to assess the eciency of thyme essential oil (TEO) as a novel feed additive to dietary antibiotic to improve productive, reproductive performance and serum biochemical of male rabbit. Methods A total of one hundred and fty 70-day-old male Californian rabbits were assigned to ve dietary treatments including a basal diet as a negative control (NC), a basal diet was supplemented with an antibiotic as positive control (PC) and the basal diet was supplemented with 60, 120 or 180 mg/kg of TEO. The experiment period lasted for 60 days. Results


Introduction
Recently, rabbits industry becomes an important role to meet the high requirements of animal protein for human consumption and a source of the national economies in Egypt [ 1,2]. Rabbit meat is characterized by a high protein and low fat and cholesterol content [ 3], these nutritional meat qualities are of great value for the meat industry and consumers. Ban on antibiotic growth promoters by several countries and the menace of antibiotic resistant bacteria has forced to look for alternatives for improving e ciency in animal production [ 4]. Due to this ban, much research has been conducted to explore the use of phytogenic as alternate feed additives in animal nutrition [ 5,6]. Phytogenic substances are generally regarded as safe and are frequently used in the food and feed industries [ 7]. The impact of phytogenic on the intestinal health, antioxidant status and antimicrobial activity is considered essential for the biological activities. Moreover, the thyme oil as phytogenic feed additives can have a bene cial impact on rabbit performance, health status, and welfare [ 8]. Furthermore, dietary Thyme extract supplementation at 0.5 g/kg signi cantly improved gut integrity and antioxidant status of rabbits [ 9] which mainly due to thyme active components. The main components of thyme essential oil (TEO) are thymol, carvacrol, γterpinene, p-cymene, β-myrcene, linalool and terpinen-4-ol [ 2,10]. These active components had the best oxidative status [ 11] and may decrease serum cholesterol [ 12] which could enhance reproductive performances of rabbits.
El-Ratel et al. [ 13] reported that oral administration of phytobiotic improved liver function of rabbits compared with the control. Likewise, supplementation of 2.5% of thyme leaves to rabbit diets signi cantly improved kidney function by decreased urea and creatinine levels [ 14]. However, no information is available about effects of TEO as alternatives to dietary antibiotics growth promoters on performance semen quality, kidney and liver functions and testosterone levels in male rabbits.
To explore the effects of TEO on productive and reproductive performance of male rabbits, we investigated the e ciency of TEO as alternatives to dietary antibiotics for improving productive, reproductive performances, liver and kidneys functions of male rabbit.

Materials And Methods
This trial was carried out at Agricultural Research Centre, Rabbit Farm, South Valley University, Egypt. The Institutional Ethics Committee of the South Valley approved the experimental procedures regarding the handling and care of rabbits. All procedures by this study were in accordance with international ethical standards.

Experimental Animals, Design and Management
A total of 150, seventy-day-old male Californian rabbits (BW 1250±30 g) were assigned to ve dietary treatments (n = 30 per dietary treatment). Dietary treatments included a basal diet as a negative control (NC), a basal diet was supplemented with an antibiotic at 500 mg/kg oxytetracycline as a positive control (PC) and the basal diet was supplemented with 60, 120 or 180 mg/kg of TEO. The experimental rations ingredients and chemical composition are given in Table 1. The diets were formulated to contain adequate levels of nutrients for rabbits according to National Research Council (NRC) [ 15].
Rabbits were housed separately in cages of galvanised wire net (width × length × height: 60 cm × 60 cm × 40 cm), equipped with an automatic drinker and a manual feeder. Farm temperature was maintained at 23°C and cycle of 16 h of light and 8 h of dark was used during the experimental period.
Animal initial and nal body weights (BW) was recorded and BW gain was calculated. The daily feed intake (g/rabbit) of male Californian rabbits was measured. The feed conversion ratio (FCR) was calculated by dividing feed intake, g/kg by average BW gain g/kg. Mortality was recorded as it occurred.

Thyme Essential Oil Preparation and Analysis
The chemical composition of hydrodistilled TEO (Table 2) was analyzed using a GC/MS system according to Abozid and Asker [ 16]. The TEO was analyzed by gas chromatography (Delsi 121C gas chromatograph). Constituents were identi ed by coupling gas chromatography with mass spectrometry (GC/MS), using a Sigma 300 apparatus attached to a HP 5970 300 mass spectrometer.

Chemical analysis
The feed were analyzed for moisture by oven drying (Method no. 930.15), ash by incineration (Method no.

Semen Characterizations
Semen characterizations including volume of each ejaculate, sperm livability, sperm motility, abnormal sperm and sperm forward motility of sexually mature and healthy rabbits (130 days of age) of the male rabbits were assessed as described previously by by Abdel-Wareth et al. [ 5] and El-Desoky et al. [ 18].

Blood biochemical
At the end of the experiment, fteen rabbits were used to collect blood samples from ear vein of in each treatment in non-heparinized sterile tubes. The samples were allowed to coagulate at room temperature for 30 min and then centrifuged at 3000 rpm for 15 min and the serum was harvested and stored at -20 C until used in the biochemical analysis as described previously by Abdel-Wareth et al [ 5]. Serum testosterone, aspartate transaminase (AST) and alanine transaminase (ALT), urea and creatinine were assessed conferring to the manufacturer's instructions by the spectrophotometric technique using commercial diagnostic kits (Monobind Inc. Lake Forest; CA 92630, USA).

Statistical Analysis
The statistical analysis was analysed by ANOVA followed by Duncan's test using SAS software [ 19]. The cage was the experimental unit for each parameter. The signi cance effects were declared at (P<0.05).
Orthogonal polynomial contrasts were also used to determine linear and quadratic effects of levels of TEO inclusion considering only negative control (0 mg/kg TEO) as a control, and positive control was not included in this analysis.

Growth Performance and Health Status
The effects of TEO levels as alternatives to dietary antibiotic on growth performance are presented in Table 3. TEO levels signi cantly increased (P<0.01) BW gain and improved FCR of male Californian rabbits compared to PC and NC groups during the periods of 70-100, 100-130 and 70-130 days of age. On the other hand, the supplemented TEO groups did not affect feed intake (P≥0.05) compared to control groups.

Semen characteristics
Effects of TEO on semen characteristics were presented in (Table 4). Results showed that TEO increased the sperm livability, sperm motility and ejaculate volume compared to PC and NC groups at the end of treatments. Abnormal sperm was decreased (P<0.01) with increasing TEO, compared to PC and NC groups. Moreover, supplementation of TEO up to 180 mg/kg to male rabbit diets signi cantly increased (P<0.001) sperm forward motility% and sperm livability% compared to control groups, however there was no signi cant difference in the semen pH value between treatments. Over all, the PC (oxytetracycline) exhibited a signi cant increase in the semen quality compared to NC.

Blood biochemical constituents
The effects of dietary supplemental TEO on blood serum constituents of rabbit male are showed in Figure 1, 2 and 3, respectively. Rabbits fed the diets supplemented with TEO at 60, 120 and 180 mg/kg signi cantly (P<0.001) decrease serum urea and creatinine compared to PC and NC groups. Moreover, activity of serum ALT and AST were signi cantly (P<0.05) decreased by supplementation of TEO to male rabbits diets compared with PC and NC groups. Furthermore, male rabbits fed diets supplemented with the TEO up to 180 mg/kg showed the highest improvement serum testosterone concentrations (P<0.05) compared to PC and NC groups.

Discussion
The TEO phytogenic feed additive as alternatives to dietary antibiotics high safety and could be used to improve semen quality and health conditions. The impact of phytogenic on the intestinal health, antioxidant status and antimicrobial activity is considered essential for the biological activities; however, studies lack determination of active components and explore their mechanisms are still not clear [ 20,21,22]. Therefore more studies under standardization are needed to explore the mechanisms of herbal essential oils on rabbit production and reproduction. In current study, the main active component of TEO was thymol which constitutes 40% relative of its analysed composition. The composition of TEO used in the present study was consistent with those reported in the literature [ 2,10]. The current study showed that BW gain and FCR were improved in rabbits fed a control diet supplemented with TEO up to 180 mg/kg compared to PC and NC groups. These improvements may be due to the active compounds stimulated digestive enzymes and therefore leads to improve nutrient digestibility [ 6,23]. Similar results were reported supplementation of TEO with olive oil improved growth performance of male Californian rabbits under high temperature environments [ 2]. Likewise, oral administration of aqueous thyme extract with 50 mg/kg BW improved (P < 0.001) BWG, feed intake and FCR of rabbits compared to control [ 24].
The TEO levels signi cantly increased reproductive performance of rabbit male at the end of the treatments period (Table 4). Interestingly the improvements in the reproductive performance of rabbit male were in parallel with testosterone concentration (Fig. 1). Ruiz-Olvera et al. [ 25] reported that semen volume and sperm motility re ects to serum testosterone concentrations. Semen quality of rabbit was signi cantly increased by supplementation of thyme aqueous extracts [ 26]. Also, rabbit fed on extracts of thyme signi cantly increased (P < 0.05) semen volume, sperm motility, sperm concentration and sperm livability compared with the control [ 24].
In the current study, supplementation of TEO to male rabbits signi cantly decreased serum urea, creatinine, ALT and AST activity, as well as increased serum testosterone levels compared to PC and NC groups. These results are in accordance with those of El-Ratel et al. [ 13] reported that the activity of AST and ALT of rabbits were signi cantly improved by oral administration of phytogenic (5 or 10 mg allicin/ BW) compared with the control. Supplementation of 2.5% of thyme leaves to rabbit diets signi cantly decreased urea and creatinine [ 14]. Likewise, Abdel-Gabbar et al. [ 27] reported that thyme extracts at 100 mg/kg induced signi cant (P < 0.001) decrease in creatinine and urea as well as ALT and AST of rabbits compared to control group. Therefore, the effect of the TEO on productive and reproductive of rabbits is better known. It increased both productive and reproductive production, as well as the health status, as observed in this study.

Conclusions
In view of the above ndings, thyme essential oil levels up to 180 mg/kg can play an important role, as an alternative to dietary antibiotic, to improve productive performance, semen quality and testosterone levels as well as kidney and liver functions in California male rabbits.  Tables   Table 1 Ingredient Figure 1 Testosterone ng/ml of male rabbits in response to dietary treatments included a basal diet (negative control), a basal diet was supplemented with an antibiotic at 500 mg/kg oxytetracyclin (positive control) and the basal diet was supplemented with 60, 120 or 180 mg/kg of TEO at 130 days of age.

Figure 2
Liver enzyme (ALT and AST) levels of male rabbits in response to dietary treatments included a basal diet (negative control), a basal diet was supplemented with an antibiotic at 500 mg/kg oxytetracyclin (positive control) and the basal diet was supplemented with 60, 120 or 180 mg/kg of TEO at 130 days of age. Serum creatinine mg/dl and urea mg/dl of male rabbits in response to dietary treatments included a basal diet (negative control), a basal diet was supplemented with an antibiotic at 500 mg/kg oxytetracyclin (positive control) and the basal diet was supplemented with 60, 120 or 180 mg/kg of TEO at 130 days of age.