Effects of  Dietary Supplementation of Vitamin E, Moringa Oleifera Leaves and Carrots on growth performance and haemato-biochemical parameters of rabbit

doi:10.21203/rs.3.rs-4641909/v1

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Effects of Dietary Supplementation of Vitamin E, Moringa Oleifera Leaves and Carrots on growth performance and haemato-biochemical parameters of rabbit

https://doi.org/10.21203/rs.3.rs-4641909/v1

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Rabbit meat has excellent nutritional and dietetic properties because it contains high protein contents, enriched with poly unsaturated fatty acids (PUFA), vitamin B complex and low cholesterol level which meet the requirements for modern consumer healthy lifestyle. Rabbit farming preferred over poultry farming due to low risk of diseases and no special care. The aim of this study is to evaluate the effect of dietary supplementation with vitamin E, Moringa oleifera leaves (MOFL) carrot on growth performance and haemato-biochemical parameters rabbit. A total of 60 rabbits with equal weights were randomly divided into four groups each contain 10 does and 5 buck and their dietary supplementation continued for 60 days. Animals were feed two times a day with basal diet. Group A (T1) kept as control, Group B treatment (T2) with 50mg/kg of carrot, Group C treatment (T3) with 50mg Vitamin E/kg B, and Group D (T4) 50gm/kg BW with Moringa oleifera leaves. Blood samples were collected from jugular vein at every 20-day interval and carried to laboratory for analysis. The result of the experiment showed that the group feed with MOL and Vitamin E improved parameter of hematology and lipid profile as well as growth parameters.

Carrots

Hematology

Lipid

Moringa oleifera

Rabbit

Reproduction

Vitamin E

During the past few decades the potential contribution of diet to the health has been empirically documented (Kapp & Sumner, 2019). In the developing countries, there is a dire need to search for alternative sources of protein to meet up the food demand of the population. Among such substitutions is the use of those livestock species that play a major role in animal production. Livestock species with short generation interval like rabbits bears a number of characteristics that could have a benefit in the subsistence of small farmers and also plays a vital role in building the economy of the country. Rabbits are favorable to meet this demand because of their high reproduction and fast growth rate, early maturity, vast feed diversity and excellent meat quality (Marai et al., 2014). Rabbit meat has good nutritional value for instance high contents of poly-unsaturated fatty acids, essential amino acids and proteins(Oonincx & Finke, 2021). Even so, moderate high energy values, low cholesterol level and low fat adds to its desirable composition. Moreover, good meat to bone ratio is attracting feature for the consumers (Amaefule & Obioha, 2005). Small body size of rabbit compared to other frequently consumed farm animals like cattle and goat, gives a sufficient amount of meat that might be very easily ingested in a single meal without any requirement for more processing to save excess quantities. Rabbit meat is also used in the production of some functional foods due to the presence of different minerals, vitamins and fatty acids (Dalle Zotte & Szendrő, 2011). In spite of different benefits, rabbit farming and its production is not achieving its full potential as a cheap source of animal protein in the tropical areas. The survival rate of their kittens and unsatisfactory reproductive performance act as a main problem in this production (Marai et al., 2014).

Nutritional approach including vitamins, minerals and various plants leaf meal has been used for remodeling productive and reproductive performance in livestock. Supplementation with different vitamins and trace elements having the property of antioxidant attempts to minimize the various health problems associated with productive as well as reproductive efficiency. Antioxidants like vitamin E is an important class of bio-molecules that provides the protection against the damaging effects of free radicals in the animals (Ebeid et al., 2012). Vitamin E is essential for different body functions like growth, immune function enhancement, tissue integrity, reproduction, disease prevention and antioxidant function in the biological systems (Dalle Zotte & Szendrő, 2011). Several studies showed that rabbit meat could be fortified with vitamin E using dietary supplementation of α-tocopherol acetate (Mancini et al., 2015). Moreover, feeding of rabbits with supra-nutritional levels of α-tocopherol acetate improved the oxidative stability of uncooked and meat during refrigerated and frozen storage, while lowering the per-oxidation processes which resulted in improving its nutritional value (Dalle Zotte & Szendrő, 2011). Dietary vitamin E may enhance immune competence and increase the antibody production in broiler chicks (Singh & Sikka, 2006)and mammals (Rooke et al., 2004).

Moringa oleifera leaf is a good natural source of protein and can be conveniently used to replace soya bean or ground nut cake in the livestock diet (Ozumba & Jiburum, 2000). Moringa leaves contain four times more calcium than in milk, seven times vitamin C in orange, three times potassium in banana, two times protein in milk and four times vitamin A in carrot (Tambe).The leaves contain magnesium, potassium and all the essential amino acids like cysteine, histidine, arginine, methionine, lysine, phenyl alanine, leucine, isoleucine and valine. They contain very strong concentration of vitamins A, C and B–complex vitamins, iron, protein, zinc, selenium which is unusual for a plant source (Assessment, 2001).

Carrot (Daucus carota) is rich in antioxidants, its less woody-textured taproot rich in Beta-carotene. Beta-carotene belongs to a group of substances called carotenoids. All carotenoids including beta-carotene also possess antioxidant abilities (Ewuola & Odefemi, 2019). Eating food like carrots that contain several carotenoids may improve overall health, growth, reproductive potential, antioxidant protection and lowering the plasma cholesterol concentration (Arumugam et al., 2021).

Ethical approval

and study area

This experiment was approved from the Animal Ethical Committee (AEC) Faculty of zoology, University of Central Punjab with approval number Zol-103-R/AH The experiment was conducted at animal house, Faculty of Zoology, University of Central Punjab Bahawalpur.

Animal feeding and management

A total of 60 healthy rabbits of mix breed were selected for the experiment. The rabbits were 8-to-9-week age and weighing between 800-900g. Fresh carrot and moringa leaves were obtained from the local market of Bahawalpur. Vitamin E capsules (50mg) were purchased from pharmacy with purity of 99.9% α-tocopherol acetate. Each group of treatment was kept in hutch which is prepared with iron and gauze of stainless steel. Canopy of hutch is prepared with hard plastic sheet. The length of the experiment was 2 months.Throughout the experiment all the animals were kept under closed observation. Fresh clean water and basal diet were provided ad libitum to all the experimental groups. All these animals were housed under similar conditions.

Table 1

Composition of the basal diet
Ingredient	Diet (%)
Fresh Berseem	30.05
Barley grain	23.00
Yellow corn	1.60
Wheat barn	21.50
Soybean meal	17.50 (44%CP)
Molasses	3.00
Limestone	0.95
Di-calcium phosphate	1.60

Study design

Rabbits were equally divided in to four experimental groups that is group 1 to group 4 with 15 rabbits in each group. The group A is kept as control (basal diet Table 1) without additional supplementation, group B was served with carrots (50g) as supplementation, group C was provided with moringa fresh leave (50g) while vitamin E (50mg) was given to group D. (1) control (basal diet without any supplementation); (2) vitamin E (basal diet + 50 mg α-tocopherol acetate/kg diet); (3) Moringa fresh leaves (basal diet + moringa fresh leaves 50 gm/kg diet) and (4) Carrot (basal diet + Carrot 50gm/kg diet).

Blood and serum analysis

At the start of experiment and every 20 days interval, blood samples were collected from each rabbit for biochemical analysis. A 2.0 ml blood was collected into 2 different labeled sterile vacutainer bottles containing Ethylene-Diamine-Tetra Acetic acid (EDTA) as anticoagulant and serum separating bottles containing clot activator. The blood samples were analyzed for complete blood count (CBC) including hemoglobin, differential leukocytic count (DLC), total red blood cell count (RBC), mean corpuscular volume (MCV), hematocrit (HCT), platelets, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), lipid profile including high density lipoprotein (HDL), low density lipoprotein (LDL), triglyceride and total cholesterol. The selected parameters were performed according to standard procedures described (Khalil et al., 2020).

Growth performance

The rabbits were weighed individually biweekly, and the live body weight change was taken as the measure of growth. A digital weight balance used with error of ± 5 g. Body weight gain (expressed in grams) was calculated.

Statistical analysis

Analysis of variance (ANOVA) was carried out using prism graph pad Software. Data were presented and expressed as means ± standard error of mean (SEM). Values of P < 0.05 were considered significant.

HDL

The rabbits supplemented with vitamin E and carrot showed the significantly enhanced in HDL level while Group 1 rabbits have also high level of HDL after 20 days of supplementation when compared with control group (Fig. 1). At 40 days, HDL concentration significantly enhanced in vitamin E and group M while in group carrot HDL concentration also increased as compared to control group. At 60 days vitamin E and moringa group showed significantly enhancement in HDL level while group treated with carrot also increased HDL level as compared to control group.

LDL

At 20 days the rabbits Supplemented with Carrot shows significantly decrease as compared to control followed by moringa and vitamin E group (Fig. 2). At 40 days the value of LDL significantly increases in control group while in carrot group LDL level remain same as on 20 days but the group treated with moringa and vitamin E shows decrease level of LDL. At 60 days the concentration of LDL significantly decreases in group supplemented with vitamin E followed by carrot, moringa and control groups, respectively.

Triglycerides

At 20 and 40 days the concentration of triglycerides is not significant in supplemented group as compared to control group (Fig. 3). At 60-day, concentration of triglycerides is decreased as compared to all supplemented group which has same triglycerides concentration.

Total Cholesterol

At20 and 40 days there was no difference in concentration of cholesterol level (Fig. 4). After the 60 days of experiment the value of total cholesterol slight lower in supplemented group on comparison with the start of the experiment.

Hb level

At 20 days Hb level increased in moringa and Vitamin E group supplementation while in carrot and control group it is decreased (Fig. 5). At 40 day, moringa and vitamin E supplemented groups has enhanced in Hb level while Carrot supplementation has also enhanced the level of Hb as compared to control group. At 60 day, moringa supplemented group has further increased in Hb level; vitamin E has no further increase in Hb level while carrot Hb level increased and control group Hb level decreased. Overall, the results of Hb showed a significant p < 0.05 results as compared with control group.

RBC count

At 20 days the concentration of RBCs in blood enhanced in group supplemented with moringa leaf as compared to control group (Fig. 6). The value of RBCs in supplemented group of carrot show decreases as compared with control group. At 40 days the value of RBC counts equal in group supplemented with moringa and vitamin E and significantly enhanced as compare with control group. At 60 days the value of RBCs significantly enhanced in group supplemented with moringa followed by carrot, vitamin E and control group respectively.

WBCs

At 20 days Carrot and moringa group has increased WBC count while Vitamin E group has also increased WBC count as compared to control group (Fig. 7). At 40 day, WBC count of moringa group is decreased while group supplemented with Vitamin E and carrot value is sustained as compared to moringa and control group. At 60 days group supplemented with carrot and moringa has enhanced WBC count as compared to control group; WBC count in vitamin E group is also sustained if compared with 40 days sampling. Overall, the results of WBCs showed a non-significant p < 0.05 results as compared with control group.

PCV(HCT)

The rabbits supplemented with Moringa showed the significantly enhanced in HCT level while in group supplemented with carrot and vitamin E rabbits have also high level of HCT after 20 days of supplementation when compared with control group (Fig. 8). At 40 days HCT concentration increase in Vitamin E group while decrease in group supplemented with moringa and carrot as compared with control. At 60 days HCT concentration decrease in all supplemented group compared with control group.

MCH

At 20 days the value of MCH concentration in group supplemented with carrot significantly enhanced as compared with control group (Fig. 9). The vitamin E concentration in blood is also enhanced as compare with control. At 40 days the group supplemented with moringa show significantly enhanced as compared with control followed by carrot and vitamin E. At 60 days the concentration of MCH in blood is highly significant enhanced in group supplemented with moringa followed by vitamin E, Carrot and control respectively.

MCV

At 20-day, group supplemented with carrot and vitamin E have enhanced in MCV value while group Moringa has also enhanced MCV value as compared to control group (Fig. 10). At 40 days group supplemented with moringa has enhanced further MCV value while decrease in MCV value was observed in Carrot followed by vitamin E and control group. At 60 days group supplemented with carrot have enhanced MCV value while decrease in the value of MCV is examined in moringa group followed by vitamin E and control group.

MCHC

At 20 and 40 days the concentration of MCHC remains same in all supplemented group (Fig. 11). At 60 days the value of MCHC concentration in group supplemented with moringa show significantly enhanced as compared with control group followed by vitamin E and carrot.

Eosinophil

At 40 day, carrot and control group increased eosinophils counts while group supplemented with vitamin E has decreased in eosinophils count and no change in moringa supplementation (Fig. 12). At 60 days decreased in eosinophils count in group supplemented with carrot as well as in control group and no change in eosinophils count in groups supplemented with moringa and Vitamin E.

Neutrophil count

At 20 days the neutrophil count is enhanced in carrot, vitamin E and moringa supplemented group (Fig. 13). At 40 days, the neutrophil count enhanced in the group supplemented with carrot while other remain same. At 60-day, neutrophil count in all supplemented group remained the same. But previously sampling group supplemented with moringa significantly enhanced.

Lymphocytes count

At 20 days rabbits supplemented with vitamin Ehave enhanced lymphocyte count while moringa, carrot and control group have no change in Lymphocyte count (Fig. 14). At 40 and 60 days no change observed in lymphocytes count. Overall, the results of lymphocytes showed a non-significant p < 0.05 results as compared with control group.

Monocytes

At 20 days decreased in monocytes count in group supplemented with carrot, vitamin E and moringa was observed as compared to control group (Fig. 15). At 40 days a significantly decrease in group supplemented with vitamin E was observed while group supplemented with carrot and moringa also decreased as compared to control group. At 60 days a significantly decreased in group supplemented with moringa and carrot. Animals supplemented with vitamin E showed increased in monocyte count.

Body weight

The difference in body weight between the groups treated with carrot, vitamin E, moringa leaf and control group was observed (Fig. 16). Group treated with moringa leaf showed a significant increase (P < 0.05) in body weight as compared with the control group. Group treated with vitamin E and carrot show non-significant increase as compared with control group.

The rapid increase in demands for dietary product like meat is due to rapid increase in population of humans while the resources are limited in developing countries which have triggered the growing researcher to find the potential of animal with low input production system. Furthermore, rabbit farmers have many production advantages like rapid growth, high prolificacy and short generation period, and can also get energy and nutrients from the less cost of fiber rich feed and waste from organic with low competition for the feed with human and animals which are monogastric. There is limited work has done on the local breed of rabbits to find the potential of their meat.(Hoffman et al., 2004); the tree, moringa oleifera is cultivated widely around the world (Osman et al., 2012) and used for various purposes one of which is as a feed supplement to livestock (Martin et al., 2007; Osman et al., 2012). In this study, rabbits were used to test the nutritional values of moringa oleifera via its effect on blood parameters as well as on changes in the animals ‘body weights. Dietary components of moringa oleifera were reported to have measurable effect on blood constituents (Amaefule & Obioha, 2005). With the exception of MCH, Hb, RBCs and MCV in rabbits, the other blood parameters did not change significantly with inclusion of moringa oleifera fresh leaves. However, mean values of each parameter were within the normal range (Piriou et al., 2002b). In contrast, the body weights of rabbits increased significantly with moringa oleifera fresh leaves (Anwar et al., 2007), (Moseby et al., 2009). The increase in the body weight of rabbits might be due to the fact that moringa oleifera is rich in amino acids, vitamins and minerals particularly iron (Abd Elhamid et al.). The significant increase in body weights of rabbits might also be attributed to captivity, where energy expenditure is minimal (Osman et al., 2012). Growth performance of other animal species fed Moringa oleferia leaves have also been documented but (Vetere et al., 2015) reported no significant of using Moringa oleferia leaves at 5, 7.5 and 10% in boiler diets on growth performance. The observation was also made by (Ayodele et al., 2014) who reported significant increase in hemoglobin concentration in rabbits fed graded levels of MOLM.

Vitamin E is a good antioxidant. In present study, it show a result increased in body weight but not much significant as compared to group treated with moringa but effect of Vitamin E on growth performance in rabbits (Ebeid et al., 2013) reported that there were increase in body weight as compared to control group due to supplementation as same in our study. Furthermore, our study show similar result with (Hashem et al., 2017) reported that there were no significant difference of Vitamin E supplementation on hematological parameters including RBCs, HCT, MCV, MCH and MCHC in rabbits, (R Cardinali et al., 2015) also reported that there were no effect on growth performance of rabbits with vitamin E supplementation.

In this study, group treated with carrot do not show any significant effect of growth performance as well as hematological parameter (Salisu et al., 2012) also reported that there was no significant effect of carrot on growth performance. In vitamin E group, neutrophils count was not significantly differing in 2nd sampling although it was decline in 3rd sampling and again increased in 4th sampling. In moringa group neutrophil count was declined in 2nd and 3rd sampling but it is increased in 4th sampling. Group feed moringa and vitamin E has improved neutrophils count in 2nd and 3rd sampling and showed best performance during whole research. Neutrophils counts was observed higher in 4th sampling in all groups. It is reported that the high values are observed during illness in rabbits and when they are under stress (Abdull Razis et al., 2014; Poljičak-Milas et al., 2009). The tested rabbits were active and in good health and under continues veterinary care, the determined high count of neutrophils in rabbits was probably the result of a stress induced due to blood sampling. Similar changes in neutrophils caused by stress during the blood sampling of animals are also found in other research (Ward & Surgery, 2006). Even though the neutrophils count obtained in this study differs statistically among treatments, they were however within the acceptable normal range given for physiologically active rabbits (Ogunlade et al., 2019; Salem et al., 2020). It is reported moringa, carrot and vitamin E can increase lymphocytes count in bone marrow (Al-Majali et al., 2017; Astley et al., 2004; Puthpongsiriporn et al., 2001) but over result show that there is no significant effect of antioxidant treatment on Lymphocytes count in rabbits. An increase or decline in the lymphocyte count has a clinical significance. The increase in lymphocyte number is known as lymphocytosis. It is an indication of viral infection and also can be due to leukemia. The decline or decrease in the lymphocyte number from the normal range is known as lymphocytopenia. It is associated with immune deficiency diseases, malnutrition, viral or bacterial infection. Lymphocyte count is increased in biological system where an intracellular infection is observed. Monocytes play a vital role in Inflammation however increased number of monocytes in biological system can lead to oxidative stress by the production of nitric oxide and with the patho-physiology of numerous diseases such as septic shocks, atherosclerosis and chronic inflammation. Even though the monocytes count obtained in this study differs statistically among treatments, they were however within the acceptable normal range given for physiologically active rabbits (Ogunlade et al., 2019; Salem et al., 2020). Eosinophils are granulocytes white blood cells and role in major allergic and viral infection. It releases peroxidase which helps in eliminating foreign pathogens. If an excess peroxidase release from eosinophils it can lead to increase in oxidative stress due to peroxidase oxidation. Moringa oleferia leaves group show significant result only in 4th sampling and not significant in 1st, 2nd and 3rd sampling. The overall value observed during whole experiment is within normal range for normal function of rabbits (Salem et al., 2020).

The moringa oleifera is cultivated widely and used as a feed supplement to livestock. In this study, rabbits were used to test the nutritional values of moringa oleifera via its effect on the lipid profile on rabbits (Amaefule & Obioha, 2005). MOL treatments decreased blood total lipid profile and Meanwhile HDL under dietary MOL increased (P ≤ 0.05) while the triglyceride and LDL concentrations showed non-significant changes under different dietary MOL levels.

The change in lipid profile with moringa oleifera may help to the decreased incidence of atherosclerosis. Low-density lipoprotein is a major component of the total cholesterol and is directly related to coronary heart disease as being a major atherogenic lipoprotein and hence, should be the main target of any lipid lowering agent, such as MOL, as indicated in the current study. The mechanism of current cholesterol reduction is thought to occur through lowering plasma concentrations of LDL by B-sitosterol, a bioactive Phyto-constituent isolated from moringa oleifera. Dietary components of moringa oleifera were reported to have measurable effect on lipid profile (Abdelhamid et al., 2018). With the exception total high-density lipoprotein and low-density lipoprotein in rabbits, the other blood parameters did not change significantly with inclusion of moringa oleifera fresh leaves and the mean values of each parameter were within the normal range (Piriou et al., 2002a). MOL is a good source for ᾶ-carotene (precursor of vitamin A) and vitamins B-complex, C, D and K, in addition to some important such as calcium, potassium, zinc, iron which also effect on the body weight of the rabbit and the HDL level increases (Abdelhamid et al., 2018). Rabbits also showed decrease in the lipid profile of liver, heart and aorta as compared to the corresponding control group while similar treatment of normal animals did not produce significant reduction in the heart (Mehta et al., 2003).

Vitamin E is considered as anti-atherogenic due to its antioxidative potency, consider as good antioxidant. In present study, it shows a result increased in body weight but not much significant as compared to group treated with moringa but effect of Vitamin E on growth performance in rabbits (Fathi et al., 2019) reported that there was increase in body weight as compared to control group due to supplementation as same in our study. Furthermore, our study show similar result with (Hashim et al., 2017) reported that there were no significant difference of Vitamin E supplementation on lipid profile including HDL, LDL and total cholesterol and triglycerides in rabbits (Raffaella Cardinali et al., 2015) also reported that there was no effect on growth performance of rabbits with vitamin E supplementation. In our study, when we compare all the treatments the HDL and LDL shows the significant result due to moringa diet and slightly significant with Vitamin E as compared to total carrot and control. The total cholesterol and triglycerides show the non-significant results.

In conclusion, our results demonstrated a clear positive effect of Vitamin E and MOFL on hematological indices and lipid profile of rabbits. MOFL shows significant increase in body weight of experimental animals. Based on the data presented above it is possible to suggest supplementation of Morinaga leaf and vitamin E involved in enhancing growth, as well as hematology and lipid profile.

EAA	Essential Amino Acid Levels
EDTA	Ethylene- Diamine-Tetra Acetic Acid
MOLM	Moringa Oleifera Leaf meal
PCV	Pack cell Volume
SFA	Saturated Fatty Acids
SOD	Superoxide Dismutase
WBC	White Blood Cells
RBCs	Red Blood Cells
Hb	Hemoglobin
MCV	Mean corpuscular volume
HCT	Hematocrit
MCH	Mean corpuscular hemoglobin
MCHC	Mean corpuscular hemoglobin concentration
HDL	High density lipoprotein
LDL	Low density lipoprotein

Acknowledgements

Not applicable

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors

Data Availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contributions

Muhammad Muzammil Nazir and Mohammad Rameez Hashmi performed the lab work and collected data. Muhammad Mubashir Nazir, Orooj suriya, Nasir Mahmood, and Asma asharf organized the literature, analyzed the data, and drafted the manuscript. Muhammad Muzammil Nazir, Iqra Farzeen, Asim Abbasi and Esha Gulfreen conceptualized the study, edited the final version, and obtained funding for the study. All authors have approved the final version of the manuscript for submission.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Effects of Dietary Supplementation of Vitamin E, Moringa Oleifera Leaves and Carrots on growth performance and haemato-biochemical parameters of rabbit

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