Comparative Screening of the Nutritive Composition and Serum Nutrient Levels of Rats Weaned with Rice ( Oryza spp ) - Beniseed ( Sesame indicum ) Flour Blends

: Weaning foods should complement the nourishment the baby receives from milk. This study was carried out to evaluate the nutritional value of Rice-Beniseed (R-B) flour-blends in rats’ pups. The objective of the study was to determine the Nutritive value of Rice-Beniseed flour blends of rats’ pups weaned with different ratios of the flour-blends. First, mature female and male rats in the ratio of 2:1 were mated to produce pups used in the analyses. The pups were randomly distributed into five groups of five rats and were fed with the experimental (Rice-Beniseed) blends. Group 1 was fed with a standard weaning meal, groups 2, 3, 4 and 5 were fed ad lib with Rice-Beniseed blends (after proximate analysis for nutritive content) in the ratio of 30:70, 50:50, 60:40 and 70:30 respectively. All treatments lasted for 21 days, after which their blood was collected for haematological and biochemical analyses. The results showed carbohydrate (71.76 %) and moisture content (14.75 %) of rice to be higher than beniseed with values of 18.74 and 8.24 % respectively. The crude proteins (23.19 %), crude fibre (11.47 %) and fats (36.40%) content in beniseed was more than doubled compared to that in rice with 9.38 %, 1.339 % and 2.41 % for crude protein, crude fibre, and fats contents respectively. Percentage weight change in pups was highest (26.31 ± 1.03%) and least (8.69 ± 2,32%) in the 50:50 and 60:40 rice-beniseed groups on day 7. Percentage weight change of pups on day 14 showed that groups fed with 70:30 and 50:50 ratios with the highest (37.24 ± 0.47%) and least (6.16 ± 1.14%) weight gain respectively. Hence, 70:30 (Rice-Beniseed blend) meal produced the best growth index (weight increase) and was potentially viable for the formulation of infant weaning formula amidst appreciable performance of all other ratios.


Introduction
The growth and survival of infants after the recommended period of exclusive breastfeeding of Six (6) months depend on the nutritional quality of the weaning food (Yomin, 2020). Breast milk is a sole and sufficient source of nutrition during the first 6 months of infant life. It contains all the nutrients and immunological factors that infants require to maintain optimal health and growth. Thus weaning foods should complement the nourishment the baby receives from milk, rather than replacing it.

Malnutrition
such as protein-energymalnutrition (PEM) is a serious health problem in developing countries like Nigeria. In many indigent communities in Nigeria, low-cost highprotein diet is still a challenge due to the high level of poverty and hunger (Akinyetun., et al 2021). Hence the high cost of commercial feeding formulae is a disincentive to acquire a nutritious weaning meal which could be a major cause of infant malnourishment and mortality in post-neonatal infants and pre-school children (Zakpaa, 2010) Therefore, a decline in the lactation coupled with early supplementation or replacement of breast milk with wrongly and un-hygienically constituted meals could have serious implications on the nutritional status of infants. (Umar & Oche, 2013) The major criteria for a good-quality weaning food are high balancedprotein content, high caloric value per unit of food volume, soft texture with low fiber content, adequate vitamin and mineral contents, and absence of anti-nutritional factors (Umar & Oche, 2013). With these requirements kept in mind, weaning foods are usually formulated using a mixture of cereals and legumes which guarantee a proper balanced diet. Thus, cereals such as rice and beniseed whose nutrient compositions are recommended as foods for fighting hunger and infant malnutrition can be blended by mothers as alternative weaning formula for infants. Therefore, the aim of this study was to evaluate the nutritional value of Rice-Beniseed flour blends as a weaning formula in rat pups.

Rice, Beniseeds and standard feed
Rice, Beniseed and standard formula (Cerelac ® ) were purchased at the Wadata market of Makurdi metropolis.

Experimental Animals
The breeding rats were sourced from the local breeder in Makurdi, Benue State, Nigeria, and bred for 28 days in plastic cages that was maintained at room temperature of 25±2 o C with a 12-hour light/dark cycle where the pups were produced. Twenty-five (25) albino rat pups of both sexes used for the experiments were allowed to attain weaning age of 21 days before they were separated from the dam and employed for the experiment. The experiment was conducted according to international guiding principles for biomedical research involving animals (CIOMS, 1985).

Preparation of Rice-Beniseeds Rations
The rice grains were cleaned and sorted out, washed and dry-milled and sieved to obtain a fine rice flour and packaged in a polythene bag and stored until needed. The beniseeds treated similarly with rice except roasting before milling. The rice-beniseed flour blends were thereafter mixed into the following 4 different rice to beniseed ratios 50:50, 70:30, 60:40 and 30:70 respectively.

Proximate Analysis
Proximate analysis of the commercial standard formula (Normal Control Group -Group 1) and the experimental blends were carried out using the standard procedure of AOAC (2002) to ascertain their individual nutritional values.

Experimental Design
Twenty-five rat pups were grouped into 5 groups (1-5) of 5 pups each. Group 1 pups were fed standard commercial weaning formula, whilst groups 4 to 5 were fed rice-beniseeds flour blends ratios 50:50, 70:30, 60:40 and 30:70 for groups 2, 3, 4 and 5 respectively. Feeding lasted for 21 days where weekly weights were taken. Twelve (12) hours after the last feeding, all rats were bled into sample bottles for the analysis of haematological and biochemical growth index parameters.

Determination of Haematological Parameters
The haematocrit or PCV was determined by the standard microhaematocrit method as described  (1986). The RBC Count was determined using the haemocytometer method as described by Schalm et al. (1975). The Haemoglobin concentration was determined by Drabkin's cyanomethaemoglobin method as described by Chessbrough (2005). The WBC count was determined by the improved Neubauer method as described by Chessbrough (2005) and Differential WBC counts were determined by method described Chessbrough (2005).

Determination of Biochemical Parameters
Serum potassium, sodium, chloride, bicarbonate, total and ionized calcium were measured with ion selective electrode principle using XI-921D electrolyte analyser-(Caretium medical instrument Co., Ltd, Shenzhen China.). Colorimetric method was applied to measure serum Total protein, albumin, Magnesium, inorganic phosphate and glucose levels following the insert methodology of commercially prepared chemistry kits by Agappe diagnostics Switzerland. Absorbance was read using NB201-C semi-auto chemistry analyser, Caretium medical instrument Co., Ltd, Shenzhen China).

Determination of Feed Efficiency Ratio (FER)
Feed efficiency ratio was calculated using the following formula.

Effects of Feeding Rat Pups with Rice-Beniseed Flour Blends on Weekly Digestibility Index
At week one the digestibility result revealed that the RB 30:70 (39.22%) digested better than other food ratio groups as compared to the control group (30.65%), this pattern seemed consistent with weeks 2 and 3 as shown in Table 3.

Effect of the Different Food Ratios on the Body Weight of Rats
The percentage change in weight was significantly increased (p<0.05) in the 50:50 food ratio group (26.31±1.03%) compared to the control group (8.48±1.21) after 7 days of feeding the formula whereas, the 60:40 food ratio group had a significantly decreased (p<0.05) weight gain (8.69±2.32%) as represented in Table 4 (Appendix 1). However, the weight gain of the 50:50 ratio was significantly decreased (p<0.05) (6.16±1.41%) at day 14 of feeding the food ratio, where ratio 70:30 feed formula significantly increased (p<0.05) by 37.24±0.47% higher than other food ratio group but less compared to the percentage weight gain of the control group (46.86±1.53%). The percentage change in weight at day 21 of all the food ratio groups was significantly lower (p<0.05) than the control group (22.84±1.16%). However, an overall 95.61±1.78% weight gain between days 0-21 was observed in the control group, meanwhile, 70:30 food ratio group gain an overall weight of (63.53±1.53%) which was close in percentage weight gain in the control group than in other food ratio groups.
Haematological parameters also revealed lymphocytes values to be significantly different between the food ratio groups and control.

Effects of the Different Feed Rations on the Biochemical Parameters of Rat Pups
Biochemical parameters (( 1104 (5.42 ± 0.07 mg/dL) and other Food Ratio Groups (see Table 6 -Appendix 1).
Analysis of macromolecules like glucose and total protein revealed thus, for Glucose Group 2 ( with 6.52±0.57 mmol/L) showed a statistically significant increase (p<0.05) when compared to the Control Group (4.32 ± 0.75 mmol/L) and other Food Ratio groups of Group 4 (4.12±0.51 mmol/L), and Group 5 (3.22±0.68 mmol/L), whilst Total protein values of Group 4 (69.68 ± 1.74 g/L) and Group 5 (74.00 ± 3.14 g/L) showed a scientifically significant increase (

Discussion
The result of this work showed that the protein contents for all the feed formulation meet the normal required standards set for infant diet (FAO/WHO, 1992). The high fat content obtained in R-B 30:70 and R-B 50:50 may be due to the fact that beniseed is an oilseed, and both food ratios has a higher percentage ratio of beniseed content and the fat content in the diets meets the normal required standards recommended for infant diet (FAO/WHO,1992).
The carbohydrate content of all the food ratio groups were higher when compared to the control, with the 5 th group (R-B70:30) having the highest percentage of carbohydrate (71.98 %), this may be due to the increased ratio of the rice in the feed formulation, as the higher the rice content the higher the carbohydrates level, since rice is very rich in carbohydrate (Fri and Backer, 2004). However, the percentage level of carbohydrate found in R-B 30:70 (60.47 %) meets the normal required standard for infants, as the Institute of Medicine encourages infants up to 6 months of age to consume at least 60 grammes of carbohydrates (Erin Coleman, 2018). The crude fibre contents of the R-B 30:70, R-B 50:50, R-B 60:40 and R-B 70-30 are 4.60%, 2.44%, 2.32% and 1.75% respectively. These values are low when compared to that of the control diet. A very low level of fibre content in weaning food has been recommended (Imtiaz, 2011). The low fibre content will encourage high digestibility and absorption of the diets by the infants. For moisture, and ash contents, the moisture content of all the diets were lower than that of the control diet. However, these values still fall within the expected range for weaning diet which must not exceed 10%. The ash content of the diets was lower than that of the control diet but falls within the recommended value for weaning food which must not exceed 5%.
During the 21-days experimental period the adaptation of the animals fed on each dietary sample and utilization of each diet were studied. Food and water were given to them ad libitum. The animals that fed on the R-B 50:50 diets were found to become leaner and lost weight at the 2 nd week of been fed with the Rice-Beniseed 50:50 food ratio, Changes were observed on their skin and in their consumption rate. Loss of weight was dramatic from average weight gain of 26.31% at day 7 to 22.34% at day 21, though no death was recorded. On the other hand, the animals fed with other diets increased in weight especially in the commercial diet group followed by the Rice-Beniseed 70:30 food ratio which did better in weight gain and in the consumption of the feed when compared to other food ratio group, this could be because of the low fibre (1.7%) and fats (4.18%) content in the R-B 70:30 food ratio, as low fibre aids proper digestibility and absorption of consumed food. The high carbohydrate 71.98 content also contributed to the level of weight gain in that group as its carbohydrate level is higher than the other food ratio groups, then by the R-B 60:40 and lastly by R-B 30:70 as shown in table 4.
After the animals were fed for 21 days with the weaning meals of different ratios for the different groups, their blood samples were collected for haematological analysis, all the www.ejtas.com EJTAS 2023 | Volume 1 | Number 4 1105 groups did well with the haematological parameters though the group 3, which is R-S50:50 had the lowest values of PCV (37.93%), other RBC (6.61%), Hb (12.64%), and WBC (7.19%), when compared to the control group and food ratio groups, but their haematological parameters all met the standard of that of children of the weaning age bracket. This shows that the Rice-Beniseed weaning meal supports the need and development of children of the weaning age. Blood examination is a good way of assessing the health status of animals as it plays a vital role in physiological, nutritional and pathological status of an organism (Ehrlich, et al., 2021). Both muscle tissues and neurons are considered electric tissues of the body. Muscles and neurons are activated by electrolyte activity between the extracellular fluid and intracellular fluid and the balance of these electrolytes in the body is essential for normal function of the cells and organs (Coso et al., 2008). Electrolytes occur in large quantities in both extracellular and intracellular fluids, with respect to serum sodium and chloride levels. Muscle contraction is dependent on the presence of electrolytes like; calcium (Ca 2+ ), sodium (Na + ), and potassium (K + ), without sufficient levels of these key electrolytes, muscle weakness or severe muscle contractions may occur in children (Gałęska et al 2022). However, for potassium, only group 2 and 5 showed a scientific significant decrease (p<0.05) when compared to the control group, the rats in group 3 and 4 showed no significant difference as compared to the control group, while for sodium and chloride, the group 5 had no significant difference when compared to the control though lower in value, thereby showing they fell within the standard value for those electrolyte. Sodium regulates the total amount of water in the body and the transmission of sodium in and out of individual cells plays a critical role in the body functions (Hanukoglu & Hanukoglu, 2016).
Many processes in the body, especially in the brain, nervous system, and muscles, require electrical signals for communication (Ighodaro, 2012). The movement of sodium is critical in generation of these electrical signals. Too much or too little sodium therefore can cause cells to malfunction, and extremes in the blood sodium levels (too much or too little) can be fatal (Ighodaro, 2012). Reduction in serum Na & Cl below the clinically accepted range is indicative of dehydration and shock, while an increase in serum albumin is indicative of stress on the liver organ and a reduction of the same is indicative of chronic liver diseases (Coso et al., 2008), this was however not the case in the present study as the albumin level of all the food ratio groups fell within the normal standard range of 30-51g/L, however there was a significant (p<0.05) difference between the group 5 and the control group. Calcium is a major structural element in bones and teeth. It is necessary to stabilize or allow for optimal activity of a number of proteins and enzymes. The binding of calcium to the protein, troponin-c, initiates a series of steps that lead to muscle contraction. The binding of calcium to the protein, calmodulin activates enzymes that breakdown muscle glycogen, which provides energy for muscle contraction (Hargreaves, M., & Spriet, L. L. 2020). Also the binding of calcium ions is required for the activation of the seven "vitamin K-dependent" clotting factors in the coagulation cascade, with the exception of group 5 (1.87mmol/l), which showed a significant (p<0.05) difference with the control, rats fed on the other experimental diets has no significant (p> 0.05) difference with the control, indicating that the feed may be a rich source of calcium, (Hargreaves, M., & Spriet, L. L. 2020 The magnesium for all the other groups had no significant difference with the control hence the feed had no effect on the magnesium level, though all the values fell within the clinical standard for infants. The phosphorus, glucose and total protein, though had significant difference but fell within the clinical standard values for infants, this shows that the feeds supports the electrolytes levels in the right proportion in infants of weaning age.

Conclusion
Based on the findings of this study, it is concluded that local resources have great potentials in the formulation and preparation of 1106 infant weaning foods since most Nigerian children are already accustomed to weaning preparations from rice, or other cereals, the greatest task was to improve the protein contents of this weaning food. This is achieved by the addition of beniseed to rice.
This work also shows that the ratio of sesame in the feed actually has the ability to increase or decrease the protein and other nutritional contents of the formula, so depending on the values and levels of performance needed, the ratio of rice to sesame will then be decided on. It was thus concluded that, the food ratio of Rice-Beniseed 70:30 diet was potentially viable in the formulation of an infant weaning diet; though all the other ratio did well too. The implication of this finding is far reaching since all the components used in the formulation were obtained from local sources. This would indicate that the product would be cheaper and more accessible. When produced on a commercial scale, it will go a long way in ameliorating the usual symptoms of Protein-Energy Malnutrition (PEM) commonly prevalent in the developing country like Nigeria.

Recommendation
Quantitative phytochemical analysis should be performed on the individual meal ratios. This would give a clearer view of the percentage composition of each phytochemical contained in each group ratio, hence buttress studies on its potential properties for weaning meal preparation.
This research should be carried beyond this limit to see the effects of these group ratios each on the outcome of serving as the chosen ration for weaning meal preparation.
Toxicity study on the meal should be studied too.

Ethical approval
The ethical consideration was obtained from the ethical committee of college of veterinary medicine teaching hospital, Joseph Sarwuan Tarka University Makurdi, Nigeria.

Funding
This piece of work did not have any grant. It was carried out through the shared financial efforts of the contributors.

Author's contribution
The authors confirm contributions to the paper as follows: the first author wrote the paper, the second author contributed data and performed the analysis. the third author conceptualized, designed and proof-read as well as serve as the corresponding author.