Effects of compound essential oil and oregano oil on production performance, immunity and antioxidant capacity of meat rabbits

Abstract 144 New Zealand weaned rabbits weighing (769.74 ± 67.32 g) were randomly chosen and divided into 3 groups by single factor design: The control group, compound essential oil group and oregano oil group, with 16 replicates in each group and 3 rabbits in each replicate. Different groups were fed with basic diet, basic diet + 300 mg/kg compound essential oil and basic diet + 300 mg/kg oregano oil respectively. The pre-feeding period is 5 days and the formal period is 42 days. The test results are as follows:(1) The feed intake of the compound essential oil group was higher than that of the control group and oregano oil group (p < 0.01), and the daily weight gain and feed/gain (F/G) ratio were not significantly different among the groups (p > 0.05); (2) The diarrhoea index and frequency in the compound essential oil group and oregano oil group were respectively, lower than control group (p < 0.01), and the mortality rate was 31.03% and 41.67% (p < 0.05); (3) The content of immunoglobin G (IgG) in blood of compound essential oil group was 35.39% and 28.57% higher than that of the control group and oregano oil group (p < 0.01), but there was no significant difference between immunoglobulin A (IgA) and immunoglobulin M (IgM) groups (p > 0.05); (4) The activity of superoxide dismutase (SOD) enzyme in oregano oil group was significantly higher than that of the control group and the compound essential oil group (p < 0.01). The content of malondialdehyde (MDA) in the compound essential oil group was 30.81% lower than control group (p < 0.05), but there was no significant difference between the compound essential oil group and the oregano oil group. The contents of glutathione peroxidase (GSH-PX), catalase (CAT) and total antioxidant capacity(T-AOC) were not significant differences among groups (p > 0.05); (5) The economic benefits of the compound essential oil group and the oregano oil group were better than that of the control group. To sum up, both compound essential oil and oregano oil can not only improve the immunity and antioxidant capacity of weaned rabbits, but can also reduce the rate of diarrhoea and mortality as well as improve the economic benefits of farm animal. HIGHLIGHTS Dietary supplementation of compound essential oil or oregano oil improved the immunity and antioxidant capacity of weaned meat rabbits. Dietary supplementation of compound essential oil or oregano oil reduced the rate of diarrhoea and mortality of weaned meat rabbits. Compound essential oil or oregano oil improved the economic benefits, therefore could be used as a new king of feed additive for meat rabbits.


Introduction
It was a frequent occurrence for newborn animals to acquire oxidative stress after birth.Young rabbits were unable to adapt to the changes in the environment and diet in time after weaning, as a result, the accumulation of free radicals in the body eventually led to oxidative damage (Wen and Peng 2017;Sa et al. 2018).Disorder of the oxidation system and antioxidant system caused intestinal inflammation, loss of appetite and increased diarrhoea rate, and when young rabbits were not treated timely, they will die and immense economic losses can't be avoided.The latest research showed that plant essential oils and their active components, such as carvol, eugenol, thymol, cinnamaldehyde, etc., had good antibacterial, antioxidant, anti-inflammatory effects as well as other bioactivities.These findings had attracted wide attention and had been widely used in medicine, food, feed and other fields (Sara 2004;Miguel 2010;Zhu et al. 2016).
The application research of single vegetable oil and compound vegetable oil in livestock and poultry production was mostly focused on the application of pigs, poultry and ruminants (Zhu et al. 2016;Wang et al. 2020;Hu et al. 2021).Feng et al. (2007), Wang et al. (2013) and Sun et al. (2018) reported the application effect of oregano oil in the diet of weaned rabbits; Cardinali and Dal Bosco (2014) reported the effects of oregano and rosemary extracts, and El-Nomeary et al. (2020) reported the effects of cinnamon oil and juniper seed oil on the growth performance and meat quality of meat rabbits; Samar et al. (2015), Zhang et al. (2021) and Cao et al. (2022) studied the effects of single plant essential oil components (cinnamaldehyde, eugenol, thymol) on the production performance, slaughtering performance, immunity and antioxidant capacity and intestinal health of growing meat rabbits; Su et al. (2022Su et al. ( , 2023) ) reported the application effect of adding different combinations of compound essential oils (cinnamaldehyde, eugenol, thymol) in the diet of meat rabbits, and the appropriate amount of compound essential oils in the diet of weaned meat rabbits; Sun et al. (2019) reported the effect of combined use of organic acids (citric acid þ sorbic acid) and essential oil components (thymol þ vanillin); Niu et al. (2022) studied the application effect of oregano essential oil combined with antimicrobial peptides in meat rabbits.
Studies above had shown that both natural plant essential oil and the active ingredients in plant essential oil (used alone, in combination, or in combination with other additives) had adequate antibacterial and antioxidant functions, showed the effects of improving growth performance, reducing diarrhoea rate, enhancing immunity and antioxidant capacity, and were good choices for antibiotic substitutes in animal production.However, the limited source of natural plant essential oil and the complex process pushed up their price; on the other hand, the effective components and their contents in the plant essential oil vary greatly due to the origins, degree of maturity, parts of the extracted plants and the different methods of extraction (Amorati et al. 2013).The main active components in the essential oil, such as cinnamaldehyde, eugenol, thymol, etc., achieved price advantages and stability dusted to the availability of being synthesised chemically and therefore were widely used in animal husbandry.The purpose of this study was to add compound essential oil and oregano oil respectively into the diet of meat rabbits, and compared the effects of the two at the same proportion, on the production performance, immunity and antioxidant capacity of meat rabbits, analysed the economic benefits in order to provide reference for the selection of essential oil products at the breeding end.

Experimental design
One hundred forty-four 35-days weaned New Zealand meat rabbits (half male and half female), with an average weight of 769.74 ± 67.32 g, were randomly chosen and divided into 3 groups, with 16 replicates in each group and 3 rabbits in each replicate, of which 8 2.00 Total(%) 100 a The premix provided the following nutrients(for per kilogram of diets): VA 10000 IU, VD3 2000 IU, VE 10 IU, VK3 1.5 mg, VB1 0.6 mg, VB2 4.8 mg, VB6 1.8 mg, VB12 0.009 mg, nicotinic acid 10.5 mg, D-calcium pantothenate 7.5 mg, folic acid 0.15 mg, Fe (as ferric sulfate) 30 mg, Cu (as copper sulfate) 10 mg, Mn (as manganese sulfate) 35 mg, Zn (as zinc sulfate) 50 mg, I (as Potassium iodide) 0.3 mg, Se (as sodium sulfate) 0.30 mg, Co (as cobalt sulfate) 0.30 mg.b Digestible energy was a calculated value, while the others were measured values.DM: dry materials; DE: digestible energy; MJ: mega joule; CP: crude protein; EE: ether extract; NDF: neutral detergent fiber; ADF: acid detergent fiber; Ca: calcium; TP: total phosphorus.replicates were male and 8 replicates were female.The control group was fed with basic diet (see Table 1), and the diets of experimental groups were added with 300 mg/kg compound essential oil group and 300 mg/kg oregano oil group respectively.The establishment of essential oil concentration was based on consulting relevant literature to find the most suitable concentration (Liu et al. 2022, Cao et al. 2022) and the preliminary experimental had yielded similar conclusions (Su et al. 2022), It was determined to be 300 mg/kg finally.The experimental groups and control group had a five-day pre-feeding period, followed by a formal period of 42 days.Rabbits ate and drank freely, fed twice a day (8:00 and 19:00) in the experimental period.

Growth indexes
On the 0th, 14th, 28th, and 42nd days of the experiment, fasting and water deprivation were observed after 8 pm the previous day, Body weight was measured in the morning 12 h later, and the feed amount of feeding and remains of each replicate were recorded weekly, and the ADG, feed intake and F/G ratio were calculated in turn.

Diarrhoea index and mortality
Observe the faeces and other conditions of meat rabbits every day and score the faeces.See Table 2 for the score standard of faeces, and the determination indices and calculation formula were as follows:

Immunity and antioxidant indexes
After the feeding experiment, 12 meat rabbits from each group (half male and half female) of approximately the average body weight were selected, 10 mL of blood from the heart was taken (Sun et al. 2005) and kept still at 4 C for 2 h, centrifuged at 3500 r/min for 10 min, the serum was then collected, and stored at À20 C. The content of IgA, IgG and IgM in serum was determined by enzyme-linked immune sorbent assay (ELISA).The test kit was purchased from Shanghai Zhucai Biotechnology Co., Ltd; The kit of Nanjing Jiancheng Institute of Biological Engineering was used to determine the levels of superoxide dismutase (T-SOD), malondialdehyde(MDA), glutathione peroxidase (GSH-PX), catalase (CAT) and total antioxidant capacity (T-AOC) in serum.

Statistical analysis
SPSS 26.0 was adopted for statistical analysis.The frequency of diarrhoea and mortality rate were measured with Chi-square test.For the significant indexes with Chi-square test, further Chi-square subdivision was performed to compare the difference between each pair (Ming 2019); the other indexes were analysed by single factor analysis of variance using the GLM model and multiple comparisons using Duncan's method.The results were expressed as the mean ( X) and standard error of mean (SEM), p < 0.01 indicated that the difference was extremely significant, p < 0.05 indicated that the difference was significant, 0.10>p > 0.05 indicated that there was a significant trend.

Growth performance of meat rabbits
It can be seen from Table 3 that the total intake of the compound essential oil group was 13% and 13.09% higher than that of the control group and oregano oil group(p < 0.05).There was no significant difference in the F/G ratio of the whole period among the groups (p > 0.05).

Health of meat rabbits
It can be seen from Table 4 that the diarrhoea frequency and diarrhoea index in the compound essential oil group and oregano oil group were significantly

Serum immune indexes of meat rabbits
Table 5 shows that the content of serum IgG in the compound essential oil group was 35.39% and 28.57% higher than that in the control group and oregano oil group respectively (p < 0.01), but there were no significant differences of the IgA and IgM among groups (p > 0.05).

Serum antioxidant indexes of meat rabbits
It can be seen from Table 6 that the SOD enzyme activity of oregano oil group was significantly higher than that of control group and compound essential oil group (p < 0.01); The content of MDA in the compound essential oil group was significantly lower than   ,b In the same row, values with no letter or the same letter superscripts mean no significant difference (p＞0.05), while with different small letter superscripts mean significant difference (p＜0.05).  b In the same row, values with no letter or the same letter superscripts mean no significant difference (p＞0.05), while with different small letter superscripts mean significant difference (p＜0.05).  b In the same row, values with no letter or the same letter superscripts mean no significant difference (p＞0.05), while with different small letter superscripts mean significant difference (p＜0.05).
that in the control group (p < 0.05), no significant difference between compound essential oil and oregano oil group (p > 0.05).There were also no significant differences of GSH-PX, CAT and T-AOC among groups.

Economic benefits accounting
From Table 7 we can see that the economic benefits of the compound essential oil group (493.46 yuan) and the oregano oil group (490.95yuan) were equivalent, and higher than those of the control group (227.92yuan).

Growth performance of meat rabbits
According to the results of this experiment, when 300 mg/kg of compound essential oil (cinnamaldehyde þ carvol þ eugenol) was added to the diet, the feed intake of 0-42d group was 13.00 and 13.09% higher than that of the control group and oregano oil group, ADG and F/G ratio were no significant difference in the whole period among the groups.As a result of the above results, the author analysed that due to the rapid death of meat rabbits with low feed intake in the control group during the experiment, and the high mortality rate of the control group throughout the entire period of the experiment, when calculated ADG and F/G, the lighter weight meat rabbits had already died due to low feed intake, resulted in no difference in ADG and F/G among the three groups.Based on the above economic benefit analysis, it was intuitively reflected that the economic benefits of the control group differ significantly from those of the experimental group.Similar to the existing research reports, Samar et al. (2015) found that cinnamaldehyde and thymol added to 100 mg and 200 mg/kg diets had positive effects on growth performance, economic benefits and health status of growing rabbits; Zhang et al. (2021) noted when 3300 mg/kg (carvol þthymol) compound essential oil was added to the diet, ADG was significantly increased and the F/G ratio reduced.
In this study, the growth performance of the oregano oil group was not equal to the compound essential oil group but was not significantly different from the control group.This result was inconsistent with some existing research results.Feng et al. (2007) showed that adding 10% oregano essential oil 100 mg/kg to the diet of weaned Rex rabbits could significantly improve the growth performance compared with the control group.Wang et al. (2013) reported that adding oregano essential oil 100 and 150 mg/kg to the diet of rabbits could significantly improve the ADG and decrease the F/G ratio.Sun et al. (2018) reported that 500 mg/kg oregano oil in the diet of meat rabbits can significantly increase the daily weight gain of meat rabbits and reduce the F/G ratio.The inconsistence results from previous ones may be due to the difference of experimental conditions, that is, the source of essential oil, additive concentration, basic diet, experimental environment, etc.

Serum immune indexes of meat rabbits
IgA, IgG and IgM, specific proteins produced by B lymphocytes, are important globulins for the body to participate in the humoral immune response.They play an important role in the body's immune system and their content is an important reference index to measure the body's immunity capacity.IgA is the main immunoglobulin in exocrine, which can inhibit microorganisms and is the first barrier of defense of the body (Gu et al. 2019).IgG is the main immunoglobulin in humoral immunity, and IgM is the main antibody globulin in the response of the body, with antibacterial, antiviral and other immune activities (Friesen et al. 1992).The experiment reported that the serum IgG of The total feed consumption is all the feed consumed by the group, including that consumed by rabbits which were later dead; b The feed unit price is 3.00 yuan/kg; c The sale price of meat rabbit is 19.00 yuan/kg; d The profit is the total income from selling meat rabbits minus the total expenditure of feed, but other costs such as the cost of buying test rabbits are not taken into account.
30-days weaned piglets fed with 200 mg/kg plant essential oil (mainly thymol and cinnamaldehyde) was significantly higher than that of the antibiotic group (Zhou et al. 2017).Wang et al. (2019) reported that adding compound essential oils (thymol, carvol and cinnamaldehyde) in broiler diet significantly increased the content of IgG in serum compared with the control group (basic diet), which could significantly improve the immune function of the body.Ma and Gao (2021) reported that when the different quality of compound essential oils (cinnamaldehyde, thymol, carvol) was added to the basic diet of broilers, the content of IgG in serum was significantly increased compared with the basic diet group, indicating that plant essential oils can improve the immune function of the body.Under our test conditions, the content of IgG in the compound essential oil group was 35.39% and 28.57% higher than that in the control group and oregano oil group respectively (p < 0.01).The results showed that the compound essential oil could effectively improve the immune function of animals.According to the study, it is believed that the mechanism of immune regulation is multifaceted, such as inhibiting inflammatory factors or improving antioxidant capacity, which is also related to individual differences in animals (Zhou et al. 2018;Ma and Gao 2021), and its specific mechanism needs further study.

Serum antioxidant indexes of meat rabbits
The body of young rabbits would produce excessive reactive oxygen free radicals, leading to cell damage, apoptosis and necrosis because they suffered from environmental and psychological stress during early weaning.When the oxidation and antioxidant system in animals are in balance, animals are healthy, when this balance is broken, various diseases even death may fall upon them (Sa et al. 2018).
There are two types of antioxidant systems in animals, enzyme systems and non-enzyme systems.Enzymatic antioxidant systems include superoxide dismutase (SOD), glutathione transferase (GSH-PX), catalase (CAT), and so on.These systems cooperate to complete the intracellular antioxidant function (Ma et al. 2014).When oxidative stress occurs, antioxidants that naturally present in the cell membrane in animals will become active at first, then they can counteract oxidative stress by eliminating free radicals.T-AOC total antioxidant active enzyme is an index reflecting the overall antioxidant capacity of the body while MDA is a metabolite of lipid peroxidation whose content can measure the severity of free radical's attack on the body (Wang et al. 2013).When the level of oxidative stress exceeds the animal's antagonistic ability, the enzyme activity will significantly decrease and the cells will be damaged.Animals' antioxidant capacity can be enhanced when supplied with exogenous antioxidants.Plant essential oil (Sara 2004;Wen and Peng 2017;Sa et al. 2018) and essential oil components (Miguel 2010;Zhu et al. 2016;Hu et al. 2021) have been widely used in livestock and poultry breeding due to their excellent antioxidant properties.Zhang et al. (2021) reported the content of GSH-PX in serum is significantly increased by adding 5% thymol 500 mg/kg; Wang et al. (2013) showed by adding 100 mg/kg or 150 mg/kg oregano essential oil to the diet of meat rabbits, the activities of SOD, GSH-PX and CAT in serum also increased significantly and the content of MDA decreased.Under our test conditions, the SOD in the oregano oil group was 14.36% higher than that in the control group (p < 0.01), and the MDA in the compound essential oil group was 30.9% lower than that in the control group (p < 0.01).The results show that both compound essential oil and oregano oil can effectively improve the antioxidant capacity of the body.It is speculated that the active components in the essential oil, such as phenols and aldehydes, can clear the oxidative free radicals in the body and slow down the oxidative stress of the body at the same time (Sa et al. 2018).

Health and economic benefits of meat rabbits
The mortality rate of the newborn rabbits in this experiment was generally high, with the control group reaching up to 62.5%.Whereas the mortality rates of the compound essential oil group and the oregano oil group were 41.67% and 35.42% respectively.One possible reason was that the experimental rabbit was attacked by coccidian infection on a large scale, Ren et al. (2020) reported that according to the rabbits epidemiological survey of Coccidiosis in Sichuan, the infection rate of coccidiosis in central China (Chengdu) at the age of 1-3 months is 95%, rabbits aged from weaning to 3 months were most susceptible, with a mortality rate of over 50%.In this experiment, diarrhoea and death began on the 10th day of the experiment, and the onset time of coccidia reached.In order to investigate the application effect of the essential oil, no drugs were used for treatment at the beginning.On the 25th day of the experiment, anti-coccidian drug mixture was added for 5 days in a row, and the symptom of diarrhoea and cases of death gradually reduced after a week.Nevertheless, the diarrhoea rate of the compound essential oil group and oregano oil group was significantly lower than that of the control group, and the mortality rate was 21 and 27 percentage lower than that of the control group, respectively (p<0.05).This shows that the compound essential oil group and oregano oil group are resistant to disease, oxidation, and coccidiosis, which was consistent with existing research reports.Feng et al. (2007) reported the diarrhoea rate of oregano oil group was 33.3% lower than that of the control group; Su et al. (2022) claimed the compound essential oil group (cinnamaldehyde þ thymol þ eugenol, 300 mg/kg) significantly reduced the diarrhoea rate and mortality.Cao et al. (2022) added (carvol þ thymol) 300 mg/kg or (cinnamaldehyde þ thymol) 500 mg/kg to the diet, and the diarrhoea rate was significantly lower than the control group.The research of Zhang (2022) and Ke et al. (2016) shows that plant essential oil can alleviate the negative impact of coccidia infection on broilers to a certain extent.
Although the mortality rate of the compound essential oil group was slightly higher than that of the oregano oil group, the growth performance of the compound essential oil group was better than that of the oregano oil group, therefore, the final economic benefit of the compound essential oil group (493.46 yuan) was equivalent to that of the oregano oil group (490.95yuan), but far higher than that of the control group (227.92yuan).The result was consistent with that of Su et al. (2022).It shows that compound essential oil and plant essential oil had good performance in both antibacterial, antioxidant and improving the health of farm animals, while compound essential oil has more advantages than plant essential oil in terms of source, quality stability as well as price.Therefore, the future of promotion and application of compound essential oil may be more promising in production.

Conclusions
Compound essential oil and oregano oil can improve the immune function and antioxidant capacity of weaned meat rabbits and reduce the rate of diarrhoea and mortality at the same time, consequently, they can improve the economic benefits of farm animals.

Table 1 .
Composition and nutrient levels of basal diets (airdry basis).

Table 3 .
Effects of compound essential oil and oregano oil on growth performance of meat rabbits.In the same row, values with no letter or the same letter superscripts mean no significant difference (p > 0.05), while with different small letter superscripts mean significant difference (p<0.05), and with different capital letter superscripts mean significant difference (p < 0.01).The same as below.ADFI: average daily feed intake; ADG: average daily gain; F/G: feed to gain ratio.

Table 4 .
Effects of compound essential oil and oregano oil on health performance of meat rabbits.

Table 5 .
Effects of compound essential oils and oregano oil on serum immunity of meat rabbits.

Table 6 .
Effects of compound essential oil and oregano oil on serum antioxidant indexes of meat rabbits.