20 | Effect of Alchornea Cordifolia and Costus Afer based diet on Gut Microbes of …

The gut microbiome of animals affects the health and performance of the animal. This study was carried out to evaluate the effect of Alchornea cordifolia and Costus afer leaf meal on gut microbiome of young male rabbits. Twenty-four (24) 5-6 months old rabbit bucks with an average weight of 5.3kg were used for this study. The completely randomized design was used and the rabbit bucks were distributed into four (4) feeding /treatment groups (T) designated as T1 (the control fed with regular rabbit feed only), T2 (fed with Alchornea cordifolia ), T3 (fed with Costus afer ) and T4 (fed with a mixture of Alchornea cordifolia and Costus afer ). The rabbits were fed the experimental diet for eight weeks and samples for microbiological analysis were collected from the different sections of the rabbit gut (stomach, ileum, caecum and colon) and droppings and analyzed for total heterotrophic bacteria (THB) and total coliform counts (TCC). The population density of THB was highest in T4 at the stomach (5.03±0.87 x 10 5 cfu/g) and ileum (5.32±1.19 x 10 5 cfu/g), but in T3 at the caecum (4.83±0.95 10 5 cfu/g), colon (4.15±0.60 x 10 5 cfu/g) and droppings (3.35±0.65 x 10 5 cfu/g. The population of TCC was highest in T4 in the stomach (i.e. 2.72±0.40 x 10 5 cfu/g) and ileum 2.67±0.53 x 10 5 cfu/g), in T3 at the caecum (3.53±0.69 x 10 5 cfu/g) and droppings (2.02±0.37 x 10 5 cfu/g, but T1 in the colon (2.77±0.60 x 10 5 cfu/g). The feed intake was highest in T4, followed by T3 and T1, but least in T2 (P <0.05), whereas there was no significant difference (P >0.05) in the weight gain of the rabbits (1.75 kg). We found that while Alchornea seemed to be reducing the population, Costus afer was enhancing their population and the overall effect on weight gain was insignificant.


Introduction
Due to poverty, protein malnutrition is common in many developing countries including Nigeria, where the source of animal protein is mostly beef, chicken and wildlife.Rabbit is a source of protein that is underutilized.Rabbit farming is not common in the country.Two of the major challenges in animal farming is the availability of good quality feed (Ohimain and Ofongo, 2014) and problem of microbial infections (Ofongo and Ohimain, 2019;Ofongo and Ohimain, 2015), which prompted the use of antibiotic growth promoters (AGP) in animal farming (Ohimain andOfongo, 2012, Ofongo andOhimain, 2019).But due to the problems of antibiotic resistance in humans and animals, the use of synthetic antibiotics is either restricted or banned in several countries.Without antibiotics, microbial infections would threaten animal farming.Common microbial infections in animal farming are caused by bacteria (Salmonella, Clostridium perfringes, Escherichia coli) and parasites particularly Eimeria leading to diseases, poor growth and even mortality (Ohimain and Ofongo, 2012).This has prompted the scientific community to research into alternatives to AGP.Several alternatives to AGP have emerged, which could be classified as photogenics and phytobiotics, probiotics, prebiotics and enzymes (Ofongo- Abule et al., 2016, Abule andOhimain 2016).
Meanwhile, two herbs common in traditional African medicine are Alchornea cordifolia and Costus afer.The phytochemical analysis of the leaves of these herbs revealed the presence of several bioactive substances of medical importance including Alkaloids, Saponins, Tannins, Flavonoids, phenols, glycosides and terpenoids (Momoh et al, 2011;Ukpabi et al, 2012 andAnaga et al 2004).These plants have been used in diverse forms.Due to its high anti-inflammatory properties (Osadabe and Okoye, 2003), Alchornea cordifolia have been used for the treatment of chancre, yaws, wounds and ulcers, gum inflammation and conjunctivitis (Neuwinger 2000).Costus afer have been reported to have hypoglycaemic property (Momoh et al. 2011), and has ability to alleviate hepatic oxidative stress and toxicity (Ukpabi et al. 2012).
Olawosulu and Ibrahim (2006) screened five medicinal plants and reported that they can be explored as alternative feed stuffs of protein for the formulation of animal diets in order to reduce the competition and cost of protein in animal feeds.Several other researchers have used different plant species to supplement for protein and as alternative to antibiotics in the diet of animals especially the monogastrics (Karsin et al, 2008, Edeoga andOkoli (2000) Egena et al, 2007;Obun and Ayanwale, 2008).Alchornea cordifolia have been used as protein supplements in feed to boost the performance of many reared animals including rabbits (Alikwe et al., 2014a), rats (Alikwe et al., 2014b), chicken (Alikwe et al., 2014c) and Snail (Alikwe et al., 2014d).However, the use of Alchornea in rabbit diet have led to various outcomes.Timibitei et al (2014a) showed that A cordifolia in the diet of rabbits resulted in the decrease in the weight of some organs including liver, kidney and heart of rabbits, while Timibitei et al., (2013) claimed that there was no difference in organ weight including the gonads when feed with Alchornea cordifolia leaf meal up to 5-15g/kg feed.Alchonea seemed to affect the reproductive health of rabbits.Timibitei et al., (2014b) reported that Alchornea in feed caused a progressive decrease in estrogen and progesterone levels of rabbits, while it was recently demonstrated that it can boost the reproductive health of male rabbit (Nodu et al., 2020).But studies on the effects of Alchornea meal on gut microbes of rabbit are not common, Kigigha and Atuzie (2012) tested the antibiotic activities of extracts of Alchornea cordifolia in-vitro and found it to be effective against Staphylococcus aureus and Escherichia coli, which appears to justify their use in traditional medicine.Hence, this study was aimed at investigating the effect of feeding Alchornea cordifolia, Costus afer and their mixture on the gut microbial densities of young male rabbits.

Materials and Methods
The study was conducted in the rabbitry unit of the Teaching and Research Farm, Faculty of Agriculture, Niger Delta University.Twenty-four (24) domestic mixed breed adult (Oryctolagus cuniculus) rabbits with a mean weight of 5.3kg were assigned randomly to four treatments (T=4) with six rabbits (n=6) per treatment and were further subdivided into three replicates (r=3) per treatment in a completely randomized experimental design (CRD).Alchornea cordifolia and C. afer leaves were harvested fresh from the Teaching and Research Farm, the leaves were plucked, washed and dried, ground and fed along with growers' mash according to the different treatments diets (T).Treatment 1 (T1) i.e. the control group was fed with concentrate feed as shown in Table 1 and other forages such as Puereria, Ipomea batata leaf and Aspillia africana, Treatment 2 (T2) was fed with 50% concentrate feed and 50% Alchornea cordifolia, Treatment 3 (T3) with 50%concentrates and 50% Costus afer, and treatment 4 (T4) with concentrates (50%) and a mixture of Alchornea cordifolia and Costus afer at 25:25 ratio.The rabbits were fed ad libitum for a period of 8 weeks.

Performance
Feed Intake and weight gain were measured and used to assess performance.The quantity of feed consumed by the rabbits in the different experimental groups was measured.The feed intake was measured by subtracting the leftovers from the total feed administered.The weight of the rabbits was weighed at the beginning and ending of the experiment and the difference is the weight gain.

Microbiological Analysis
At the end of the experiment, the rabbits were slaughtered and the contents were collected aseptically at different sections of the gut (ileum, caecum, stomach and colon) and droppings and analyzed for total heterotrophic bacteria counts (THB) and total coliform counts (TCC).The samples were serially sterilized diluted up to 10 7 using distilled water and plated on nutrient agar and MacConkey agar using pour plate method respectively for THB and TCC.MacConkey Agar was used as selective and differential medium for cultivation and presumptive identification of coliform organisms.The plates were incubated at 35 o C for 24-48 hours.Colonies of coliforms appears red or pink on MacConkey Agar.The colonies were counted using a conolony counter.Only colonies within the range of 30 -300 were counted as valid and was recorded as colony forming units per gram (cfu/g).

Statistical Analysis
Data obtained from the laboratory analysis were subjected to analysis of variance (ANOVA) and means were separated using Duncan Multiple Range Test (DMRT) using SPSS version 17 (SPSS Inc, Illinois, Chicago).

RESULTS
The population density of THB in the stomach was highest (5.03±0.87x 10 5 cfu/g) in Treatment 4 i.e. when both Alchornea and Costus were incorporated in the diet of the male rabbits, though, not significantly different (P>0.05) with the other treatments including the control (Table 2).At the ileum, the highest THB population of 5.32±1.19x 10 5 cfu/g was also reported in Treatment 4, which was significantly (P<0.05)different from the other treatments, followed by Treatment 3 and least in Treatment 2. At the caecum, the highest THB density of 4.83±0.95 10 5 cfu/g was recorded in Treatment 3, which was significantly different (P<0.05) from the other treatments.At the colon, the highest THB population of 4.15±0.60x 10 5 cfu/g was recorded for Treatment 3, which was not significantly different (P>0.05) from the others.The population of THB in the droppings was also highest in Treatment 3 being 3.35±0.65x 10 5 cfu/g, but was also not significantly different (P>0.05) from the others.Along the column, mean ± standard error (n=6) with the same alphabets are not significantly different (P>0.05) according to Duncan Statistics The population of TCC in the stomach of the rabbits was highest in Treatment 4, i.e. 2.72±0.40x 10 5 cfu/g, which was not significantly different (P>0.05) from the other treatments (Table 3).At the ileum, the highest population of 2.67±0.53x 10 5 cfu/g was reported in Treatment 4, which was significantly different (P<0.05) from the values recorded in Treatments 1 and 2. At the caecum, the highest TCC of 3.53±0.69x 10 5 cfu/g was recorded in Treatment 3, which was significantly different (P<0.05) from the other treatments.In the colon, the highest population of coliforms of 2.77±0.60 x 10 5 cfu/g was recorded, which was significantly different (P<0.05) from the control, but not from the other treatments (P>0.05).In the droppings, the highest coliform density of 2.02±0.37 x 10 5 cfu/g was recorded in Treatment 3, which was not significantly different (P>0.05) from the other treatments.Along the column, mean ± standard error (n=6) with the same alphabets are not significantly different (P>0.05) according to Duncan Statistics Feed intake and weight gain was used to assess performance (Table 4).The feed intake was highest in T4, followed by T3 and T1, but least in T2 (P <0.05).Notwithstanding the differences in feed intake, there was no significant difference (P >0.05). in the weight gain of the rabbits.The weight gain of 1.75 kg was the same in T1, T3 and T4 but slightly lower in T2.

Discussions
Overall, the results tend to show that Alchornea extracts results in the reduction in the population of heterotrophic bacteria, while Costus tend to enh`ance it.The reason for these differences is not known, but it could be due to the antibiotic property of Alchornea cordifolia, which have been established by Kigigha and Atuzie (2012).It should be noted that heterotrophic bacteria are not pathogens, they mostly consist of the normal flora/ microbiome of the intestinal tract that protects the gut from pathogenic species.They compete with pathogens for food and attachment surface and become beneficial to the animal.
The results do not seem to show that the herbs result in any significant reduction of coliforms in the rabbits.Coliforms, though, a normal flora in the intestinal tract, could become pathogenic especially when the immunity of the animal is weakened, hence, they can easily become opportunistic pathogens.
The results of the performance show that that the presence of Alchornea cordifolia in the diet tend to result in decreased feed intake significantly and weight gain insignificantly.The reason for this pattern of growth is not fully understood.But it has been observed that Costus afer is sweet, while Alchornea is not.Hence, the rabbits'

Table 1 :
Percentage composition of experimental diet

Table 3 :
Changes in coliform in the gut of male rabbits fed with Alchornea cordifolia and Costos afer based diets, x 10 5 cfu/g

Table 4 :
Performance of male rabbit fed with Alchornea codifolia and Costus afer based diets (P<0.05) Means on the same row with different superscript are significantly different.