MATERIALS AND METHODS

This study was carried out at the Rabbits Farm of Sakha Station, Animal Production Research Institute, Agriculture Research Center, Egypt.

Preparation of aqueous M. oleifera leaves extract (AMOLE): Fresh leaves of M. oleifera were collected early in the morning at Dokki area of Giza Governorate. The M. oleifera leaves were manually removed from the stem, cleaned and made free of sand and other impurities using distilled water. The fresh leaves were blended into powdered using an electric kitchen blender. Finely pulverized M. oleifera leaves weighing 300 g was poured into a 2.5 L macerating flask and 1.5 L of distilled water was added. The resulting mixture was thoroughly homogenized and sieved with a cheese cloth and then filtered using Whatman filter paper (24 cm). Resulting filtrate was stored in the freezer (4 or -20°C) until further analysis.

Experimental animals and management: Sixty four APRI line rabbits (Egyptian line selected for litter weight at weaning according to Khadiga et al.¹² were divided and assigned randomly into four experimental groups of 16 rabbits each (8 males+8 females) of 5 weeks of age with an average live body weight of 661.8±8.08 g. The four experimental groups were as follows: The control group (G₁) received basal diet and water without any supplementation, while groups 2, 3 and 4 received basal diet and water supplemented with 30 (G₂), 60 (G₃) and 90 (G₄) mL AMOLE/L drinking water, respectively. Basal diet was formulated to cover all essential nutrient requirements for growing rabbits according to NRC¹³. Table 1 shows the formulation and nutrient composition of the basal diet.

All rabbits were kept under the same managerial conditions. Feed and water were offered ad libitum throughout the experimental period (5-13 weeks of age).

Experimental procedure: Live body weight (BW, g), daily feed intake (DFI, g) and number of dead rabbits were recorded weekly. Daily weight gain (DWG, g), feed conversion rate (FCR, g g^–1) and mortality rate (MR, %) were calculated weekly. Economic efficiency (EE, %) was calculated according to price marketing during 2016. Also, relative growth rate (RGR, %) and performance index (PI, %) were calculated on a group basis¹⁴:

Whereas:

Table 1:	Composition and chemical analysis of basal diet
*One kilogram of mineral-vitamin premix provided: Vitamin A: 150,000 UI, Vitamin E: 100 mg, Vitamin K3: 21mg, Vitamin B1: 10 mg, Vitamin B2: 40 mg, Vitamin B6: 15 mg, Pantothenic acid: 100 mg, Vitamin B12: 0.1 mg, Niacin: 200 mg, Folic acid: 10 mg, Biotin: 0.5 mg, Choline chloride: 5000 mg, Fe: 0.3 mg, Mn: 600 mg, Cu: 50 mg, Co: 2 mg, Se: 1 mg and Zn: 450 mg. **Calculated according to NRC (1977). ***Digestible energy (kcal kg–1 DM)= 4253-32.6 CF (% DM)-114.4 Ash (% DM). According to Fekete and Gippert,15

At the end of growing period (13 weeks of age), four rabbits were taken randomly from each treatment, fasted for 12 h, weighed and slaughtered to estimate some of carcass traits according to the method described by Blasco et al.¹⁶. Carcass parts were presented as a percent of live body weight. Samples of cecum content were taken individually from rabbits of each group and filtrated to estimate pH and cecum microflora. Total anaerobic bacteria count and Escherichia coli (E. coli) were estimated according to the method described by Collins et al.¹⁷ and lactobacilli bacteria count according to Kim and Goepfert¹⁸. In addition, cecum pH was measured by using pH meter in filtrate cecum content. Ammonia nitrogen concentration was determined as described by Conway¹⁹.

Statistical analysis: Data were statistically analyzed according to SAS²⁰ computer program using the following fixed model:

Y_i = μ+T_i+e_i

Where:

Data presented as percentages were transformed to the corresponding arcsine values²¹ before being statistically analyzed. The differences among means were tested using Duncan’s new multiple range test²². All data are presented as least square means.

RESULTS AND DISCUSSION

Productive performance: Effect of different levels of AMOLE on final body weight (BW), DWG, DFI, FCR, RGR (%) and PI (%) of APRI line rabbits are presented in Table 2. Values of final BW, DWG and FI at 5-13 weeks of age were higher significantly (p<0.01) for rabbits in G₂, G₃ and G₄ than those in control group. The proportional increments were 3.5, 7.7 and 8.3% for final BW; 5.6, 11.7 and 12.4% for DWG and 3.4, 8.0 and 8.2% for FI, for the three levels, respectively. These results are in agreement with El-Gindy et al.²³. While, Alabi et al.²⁴ showed negative effect of AMOLE for final BW and DWG in broilers. Also, present study showed that values of final BW and DWG in G₃ group (60 mL AMOLE/L) was not different from G₄ group (90 mL AMOLE/L).

Results show that DFI significantly increased as AMOLE levels increased. The improvement in FCR at 5-10 weeks was significantly high among the three levels of AMOLE; the best FCR was recorded in G₃ (3.6%) and G₄ (3.8%) compared to control (G₁) which means better returns on investment. These findings were confirmed by Alabi et al.²⁴, who found that addition of 90 mL and 120 mL of AMOLE/L in broilers diet produced better FCR than control. It has also been documented that high packed cell volume (PCV) and high hemoglobin content (Hb) "as mentioned in Table 5" are associated with high feed conversion ratio²⁵.

Results indicate that addition of AMOLE caused significant differences (p<0.05) in the values of RGR and PI. The highest values of RGR and PI (%) were observed in G₃ and G₄, without any significant differences between G₁ and G₂.

The antimicrobial (lipophilic compounds) and antioxidant (polyphenols, tannins, anthocyanin, glycosides compound) present in AMOLE may attach to the cytoplasmic membrane and remove free radicals, activate antioxidant enzymes and inhibit oxidases thus, making this elements more available for the poultry to use²⁶. Furthermore, the synergy between individual bioactive compounds in AMOLE may be an important feature of their action which may affect broad aspects of physiology, such as nutrient absorption^27,28.

Table 2:	Effect of Moringa leaves extract water supplementation on growth performance of growing APRI-line rabbits from 5-13 weeks of age
SEM: Standard error of means, Sig.: Significance, ***Significant at 0.1% level of probability, **Significant at 1% level of probability, *Significant at 5% level of probability, NS: Non-significant. a, b, c, Means in the same row the different superscript are significantly different (p<0.05). #Chi-square test

Table 3:	Effect of Moringa leaves extract water supplementation on carcass traits of growing APRI-line rabbits
SEM: Standard error of means, Sig: Significance, A, B, C, Means in the same row the different superscript are significantly different (p<0.05). ***Significant at 0.1% level of probability, **Significant at 1% level of probability, *Significant at 5% level of probability, NS: Non-significant, GIT: Gastrointestinal tract

Some carcass characteristics: Table 3 shows that dietary AMOLE levels increased the values (p<0.05) of carcass and total edible parts percentages as compared to control group. However, liver, heart, abdominal fat and GIT percentages of the rabbit received AMOLE significantly decreased compared with that of the control group (G₁). Also, the results showed that the effect of dietary treatments on kidney was not significant. These results are in agreement with Nuhu²⁹.

The increase in carcass for treated groups may be related to the increase in growth performance. Therefore, pre-slaughter weight is considered to be one of the most important factor affecting carcass traits in rabbits. Szendro et al.³⁰ reported the important effect of pre-slaughter body weight on carcass traits. It seems that the literature is still sparse on the effect of AMOLE on rabbit’s carcass traits.

It is a common practice in feeding trials to use weights of some internal organs like the kidneys as indicators of toxicity if there was any serious effect of anti-nutritional factors on them being major detoxification organs³¹. It was obvious in this study that the weight of organs such as kidney was insignificantly affected by AMOLE treatments as a result of absent of anti-nutritional factors. On the other hand, significant decreased of liver and heart weights as increased of AMOLE concentration need histological study to discuss this point.

Gastrointestinal tract and abdominal fat percentages were decreased (p<0.01 and p<0.05, respectively) by the addition of AMOLE. The lower gastrointestinal tract percentage could be explained by the increase in the carcass percentage. This was also observed by Amber et al.³², who reported the lowest abdominal fat percentage value for poultry treated with prebiotic. No clear mechanisms have been reported responsible for the reduction of lipid synthesis by prebiotics and herb oligosaccharides. It might be due to increase in beneficial bacteria such as Lactobacillus that decrease the activity of acetyl-CoA carboxylase, which is the rate-limiting enzyme in fatty acids synthesis³³.

Table 4:	Effect of Moringa leaves extract water supplementation on caecum content and microbial activity of growing ARPI-line rabbits
SEM: Standard error of means, Sig: Significance, ***Significant at 0.1% level of probability, **Significant at 1% level of probability, a, b,….e, Means in the same row with different superscripts are significantly different (p<0.05). Germ counts expressed in CFU g–1 caecal digesta

Table 5:	Effect of Moringa leaves extract water supplementation on economical traits of APRI LINE rabbits at 13 weeks of age
Other conditions like management are fixed. Ingredients price (L.E. per ton) at 2016 were: 4100 yellow corn, 4000 barley, 2000 berseem hay, 3600 wheat bran, 7000 soybean meal (44%), 250 limestone, 12000 premix, 60000 methionine, 40000 lysine, 1000 di-calcium phosphate, 3500 molasses, 250 salt. Adding 100 L.E./ton for pelliting. *Price of kg live body weight was 25 L.E., #Net revenue = Selling price-total feed cost

Caecum content and microbial activity: Results indicate that dietary AMOLE levels had some effects on the microbial ecology of the gastrointestinal tract in growing rabbits. Table 4 shows that supplementing AMOLE in drinking water of rabbits significantly affected caecum content. Ammonia content was significantly decreased (p<0.001) by supplementing AMOLE in drinking water. Total count of bacteria increased in caecum content for rabbits in control group (G₁) as compared to those received AMOLE in drinking water. Pathogenic bacteria (Escherichia coli and Clostridium sp.) decreased (p<0.001) by supplementing AMOLE in drinking water. Similarly, Mateos et al.³⁴ and Amber et al.³² indicated that supplementation of rabbit feeds with certain prebiotics increased volatile fatty acids in the caecam and decreased the caecal ammonia concentration. In addition, to stimulate the beneficial microflora of the gut, prebiotics may prevent the adhesion of mucosa pathogens and stimulate the immune response³⁵. These results are in agreement with the results of Amber et al.³², who found that addition of some prebiotics in rabbit diets reduced number of total bacterial count (especially pathogenic bacteria) in caecum content of rabbits. The most susceptible organisms to the antibacterial activity of M. oleifera was E. coli ³⁶. Therefore, M. oleifera could be a promising natural antimicrobial agent.

On the other hand, the antioxidant properties of AMOLE may affect the alimentary canal through antimicrobial activity.

The antimicrobial activity of M. oleifera leaf may be due to the presence of an array of phytochemicals³⁶. Bukar et al.³⁷ identified, most importantly, the presence of a short poly-peptide named 4 (ά-L-rhamnosyloxy) benzyl-isothiocyanate in M. oleifera which may act directly on microorganisms and result in growth inhibition by disrupting cell membrane synthesis or synthesis of essential enzymes.

Economical evaluation: Data concerning economical evaluation indicate an increase of net and relative revenue for rabbits treated with AMOLE levels compared to those untreated (Table 5). The highest increase (16.8%) in net revenue was observed for rabbits of G₄ (90 mL AMOLE/L). This result is in harmony with those of Abou Sekken³⁸ who used AMOLE in the drinking water of broilers. Also, results showed that total feed cost increased by the addition of AMOLE, as a result of increased in feed intake. Also, selling price was increased by the addition of AMOLE in drinking water. This increase in selling price in treated groups may not only due to increase in average weight gain (kg/head), but also reduction in mortality rate in the same groups.

CONCLUSION

It is concluded that AMOLE can successfully be incorporated into the drinking water of growing rabbits up to the level of 90 mL L^–1. However, the addition of 60 mL of AMOLE/L in drinking water, improved the production performance and microbial ecology of the gastrointestinal tract with high profitability of growing rabbits, under Egyptian environmental conditions.