Effect of Dietary Tigernut on Acetylcholinesterase, Specific Acetyl - cholinester, and Total Protein Levels in Rabbit Brains of Different Genotypes

This research investigated the impact of different levels of dietary Cyperus esculentus (tigernut) on acetylcholinesterase, specific acetylcholinesterase, and total protein concentrations in various brain regions of three rabbit genotypes. Male and female Dutch belted, Hyla max, and Newzealand white rabbits, totaling 320 at 6 weeks of age, were randomly assigned to diets containing 0, 10, 20, or 30g/kg of tigernut. Each treatment group was replicated five times with 15 rabbits per replicate. The rabbits were provided with ad - libitum feeding and clean water for 8 weeks or 56 days, after which six rabbits per replicate were euthanized. The brains were dissected into different regions including the olfactory lobe, pineal body, optic lobe, cerebellum, and medulla oblongata. The brain regions were homogenized to assess acetylcholinesterase and total protein concentrations, which were further used to evaluate specific acetylcholinesterase levels. The study found no significant differences in acetylcholinesterase activity in the olfactory lobe, pineal body, and cerebellum, except for the optic lobe and medulla. Additionally, dietary tigernut did not affect total protein and specific acetyl - cholinesterase activities in the olfactory lobe and medulla regions. However, tigernut intake had significant effects on total protein levels in other brain regions, leading to significant changes in specific acetyl - cholinesterase levels in the cerebellum. These findings suggest that tigernut added to rabbit diets at levels above 20 g/kg significantly alters brain acetylcholinesterase concentration, total protein levels, and specific acetylcholinesterase, potentially impairing brain functionality.


INTRODUCTION:
The scarcity of animal protein, particularly from animal sources, in the diets of people across Africa and many developing nations is a significant concern (Adedeji et al., 2012).To address this issue, it is crucial to increase the production of non-ruminant species, including rabbits, as they offer a fast and efficient solution for improving animal protein supply in tropical Africa (Adedeji et al., 2012).Despite efforts to meet the demand for animal protein through the consumption of cattle, pigs, poultry, sheep, and goats in Nigeria, the supply remains insufficient (Adedeji et al., 2012).Moreover, the recommended minimum intake of animal protein by the Food and Agriculture Organization (FAO) has not been achieved, largely due to the high cost of meat, which is unaffordable for many Nigerians (FAO, 2011).
Expanding the production of small yet highly prolific livestock is crucial in addressing the low intake of animal protein (Adedeji et al., 2012).Rabbits, in particular, have been identified as an economical livestock option that can help fill the gap in dietary protein intake in Nigeria (Adedeji et al., 2012).These rabbits are micro-livestock, capable of producing approximately 47kg of meat per doe per year, which is sufficient to meet the animal protein requirements of a medium-sized family under small-scale rural farming systems (Adedeji et al., 2012).Furthermore, rabbit meat offers additional nutritional benefits, such as being rich in vitamin B, low in cholesterol and sodium levels, making it an attractive source of high-quality animal protein (Jithendran, 2000;Omole et al., 2005).
The rabbit has gained attention for its potential as an animal that exhibits desirable attributes such as small body size, short generation interval, and high reproductive potential (Adedeji et al., 2012).As an economic source of high-quality animal protein, rabbits are gradually becoming recognized in tropical regions as a means of addressing the nutritional needs of the human population (Adedeji et al., 2012).Known for their prolific nature and efficient conversion of feeds to muscle, rabbits are capable of utilizing diverse forages, which makes them suitable for overcoming the scarcity of cereals in the face of competition from industries and human consumption (Adedeji et al., 2012).To ensure a sustainable balance between population growth and agricultural productivity, it becomes necessary to explore alternative feed options that are lesser known and underutilized (Bamishaiye and Bamishaiye, 2011).One such option is Cyperus esculentus, commonly known as tiger nut, which belongs to the family Cyperaceae (Fasakin et al., 2017;Sharif et al., 2019).Tiger nuts possess essential agronomic and nutritional potentials, comparable to conventionally used energy sources (Fasakin et al., 2017).In Nigeria, there are two main varieties of tiger nut: the yellow and the brown, with the yellow variety being preferred due to its larger size, attractive color, fleshier body, higher milk content, lower fat, and higher protein content, along with fewer antinutritional factors, especially polyphenols (Fasakin et al., 2017).Recent studies have demonstrated that tiger nut residue or the whole plant is rich in energy, fair in amino acid composition, with minimal allergens, making it a valuable ingredient in livestock feed (Ellman, 1961;Reinhold, 1953).However, there is a lack of information regarding the effect of tiger nut meal specifically on rabbits.Hence, this study aims to investigate the impact of varying levels of dietary tiger nut on the acetylcholinesterase (AChE) activities, total protein, and specific acetylcholinesterase (SAChE) of the brain in three different rabbit genotypes.

MATERIALS AND METHODS:
The experiment utilized a total of 320 male and female rabbits, aged 6 weeks, belonging to three different genotypes.The study was conducted for duration of eight weeks at the livestock section of Teaching and Research Farm, The Federal University of Technology, Akure.The rabbits were weighed and randomly allocated to four treatments, comprising diets with tiger nut inclusion levels of 0, 10, 20, and 30g per kg of feed.Each treatment was replicated five times, with 15 rabbits per replicate, following a completely randomized design.After the completion of the experiment, three males and three females from each replicate were slaughtered, resulting in a total of 24 rabbits per treatment.The brains were immediately removed and carefully separated from any attached meninges and blood vessels.Using an ice-cold porcelain tile, the brains were dissected into different regions, including the cerebellum, cerebral cortex, mid brain, and medulla oblongata, following the method described by Adejumo and Egbunike, (2001).The brain and hypophyseal acetylcholinesterase (AChE) activities and total protein concentrations were measured using colorimetric methods as described by Ellmann et al. (1961) and Reinhold, (1953) respectively.Randox commercial kits with catalog numbers TP245 and CE7944 were utilized for the determination of total protein (TP) and AChE, respectively.The brain and hypophyseal samples obtained from each animal were homogenized in an ice-cold phosphate buffer solution containing 0.1% Triton X100 using a Potter Elvehjem homogenizer (catalog number P7734) at a concentration of 1% weight per volume (w/v).The specific acetylcholinesterase (SAChE) activity was calculated by dividing the AChE activity of each sample by its corresponding total protein concentration, expressed in µmole/g protein/min.Statistical analysis was performed using SAS (2008, version 9.2), employing One Way Analysis of Variance (ANOVA) with a significance level of α=0.05.In cases where significant differences were found, means were compared using the Duncan Multiple Range Test based on the guidelines outlined by Snedecor and Cochran, (1980).

RESULTS:
The inclusion levels of tigernut did not significantly affect the acetylcholinesterase concentration in the olfactory lobe, cerebellum, and pineal body of the brain (Table 1).However, there was a significant effect on the optic lobe and medulla oblongata, where the activity of acetylcholinesterase decreased significantly in the rabbits fed diets containing 10 and 30g tigernut/kg compared to the control and 20g tigernut/ kg diet.In terms of breed, there was no significant influence on acetylcholinesterase activity, but sex had a significant effect on the optic lobe, with male rabbits showing higher activity than females.The specific acetylcholinesterase activity in the brain regions (olfactory lobe, pineal body, optic lobe, medulla) did not significantly differ across the varied inclusion levels of dietary tigernut, except in the cerebellum where the activity decreased significantly with increasing inclusion level, with the control group (0g/kg) showing the highest activity (Table 2).Breed had a significant influence on the activity in the olfactory lobe, optic lobe, cerebellum, and medulla, except for the pineal body.Among the breeds, Dutch rabbits had the highest activity in the optic lobe, while Hyla max and Newzealand rabbits showed a similar level of significance.Sex did not have a significant influence on the specific acetylcholinesterase activity in the rabbits' brain.Regarding the total protein concentration in specific brain regions (optic lobe, cerebellum, pineal body), there was a significant effect except for the olfactory lobe and medulla (Table 3).The pineal body showed an increase in total protein at the 30g/kg inclusion level, similar to the control.The effect of tigernut on the total protein of the optic lobe and cerebellum did not follow a consistent pattern, with the 0g/kg inclusion level group showing the highest total protein.Breed had a significant influence on the optic lobe, with Dutch rabbits recording the highest value, while Hyla max and Newzealand rabbits showed a similar level of significance.Sex had a significant influence on the optic lobe, with male rabbits having a higher total protein concentration than females.2004) agreed with the non-significant influence observed in the SAChE activities of the olfactory lob, optic lobe, pineal body, and medulla oblongata regions of the brain.On the other hand, the total protein concentration in the brains of rabbits fed tigernut at inclusion levels of 0-30 g/kg was depleted compared to the control, except for the pineal body where the 20 g inclusion level recorded the highest concentration.However, the total protein concentration in the olfactory lobe and medulla of the experimental rabbits was not affected.The lack of significant difference in total protein concentration in the olfactory lobes and medulla across all the treatment diets indicates that tigernut had no interference with neural mechanisms involved in protein synthesis in the brain of the rabbits.Conversely, the significant decrease observed in the total protein concentrations in the other regions of the brain at higher dosages of tigernut (10 to 30 g/kg) suggests that tigernut disrupts protein synthesis in those regions.Ewuola and Bolarinwa, (2017) explained that the ability of aflatoxin to bind and interfere with enzymes and substrates involved in protein synthesis makes it capable of affecting brain development.If there were a significant difference in total protein in the olfactory lobe, this could have led to retina dysfunction in the rabbits.Protein in the brain functions for repair of worn-out tissues, growth, muscular development, and also binds to some minerals to ensure their bioavailability and proper utilization (Ewuola and Bolarinwa, 2017).

CONCLUSION:
This study explored the potential of tigernut as a dietary component for rabbits.The findings suggest a two-sided effect: It enhances flavor and provides valuable energy (with safe levels at 10-20g/kg diet) due to its carbohydrate, fats and fiber composition.However, high intake appears to be toxic to neurons in the brain, potentially affecting cognitive function.
Further research is needed to understand why and determine the safe limit.The study also suggests excessive tigernut disrupts internal balance (homeostasis).More research is needed to pinpoint the specific effects.Despite its taste and energy benefits, caution is needed regarding its neurotoxic effects.
Unraveling the impact of tigernut on rabbits' brain functions and overall health is crucial to determine safe levels for this intriguing ingredient.This knowledge would significantly improve animal nutrition, making this a novel area for further research.

Table 1 :
Percentage composition of experimental diets fed to matured does and bucks.

Table 2 :
Brain Acetylcholinesterase (AChE) Activities of Three Rabbit Breed Fed Different Levels Tiger nut.

Table 3 :
Brain Specific Acetylcholinesterase (SAChE) Activities of Three Rabbits Breed Fed Tiger nut.
[Values are means ± SEM, Means in a row without a common superscript are significantly (p<0.05)different

Table 4 :
Brain Total Protein (g/dL) of rabbits Breeds Fed Different levels of Tiger nut.

Table 4 .
Adejumo et al. (2005)ce was observed for the cerebellum, with the control (0g) of tigernut recording the highest SAChE activity.Adejumo et al. (2005)did not report any influence of fumonism on SAChE activities in the cerebellum of pigs.The difference in the results may be due to the different species of experimental animals and the different supplements that were fed to them.Adejumo and Egbunike, (