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Effect of Feed Supplementation with Riboflavin and Protease Enzyme on Two Physical Heat Stress Parameters of Finishing Turkeys

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28 April 2025

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28 April 2025

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Abstract
This study was carried out to investigate the effect of turkey feed supplementation with riboflavin and protease enzyme on rectal temperature and pulse rate as a measure of heat stress during finishing stages. One hundred one-day old British United broiler turkey poults were randomly distributed into four dietary treatments T1, T2, T3, and T4 (T1 as control, T2 containing riboflavin at 6mg/kg, T3 containing protease enzyme at 1000mg/kg, and T4 containing combination of riboflavin and protease at 6mg/kg and 1000mg/kg. During the finishing stages of the birds, the rectal temperature and pulse rate of two birds from each replicate were recorded. They were recorded twice every week during the experimental period. Also, ambient temperature and relative humidity were recorded at the time of taking records on the physical parameters. The data collected were analyzed using SYSTAT. Results of turkey fed supplementation with riboflavin and protease enzyme showed no effect on rectal temperature and pulse rate of finishing turkeys. It was concluded that the addition of riboflavin and protease enzyme at the specified levels did not influence the rectal temperature and pulse rate of finishing turkeys and therefore heat stress.
Keywords: 
riboflavin
;  
protease enzyme
;  
heat stress
;  
finishing turkeys
Subject: 
Biology and Life Sciences  -   Animal Science, Veterinary Science and Zoology

1. Introduction

According to Selye (1976), “stress is the nonspecific response of the body to any demand”, whereas stressor can be defined as “an agent that produces stress at any time”. Therefore, stress represents the reaction of the organism (i.e., a biological response) to stimuli that disturb its normal physiological equilibrium or homeostasis.
Heat stress results from a negative balance between the net amount of energy flowing from the animal’s body to its surrounding environment and the amount of heat energy produced by the animal. This imbalance may be caused by variations of a combination of environmental factors (e.g., sunlight, thermal irradiation, and air temperature, humidity and movement), and characteristics of the animal (e.g., species, metabolism rate, and thermoregulatory mechanisms). Environmental stressors, such as heat stress, are particularly detrimental to animal agriculture (Nienaber and Hahn, 2007; Nardone et al., 2010; Renaudeau et al., 2012; Rostango and Lara, 2013).
Extreme heat stress during the summer is of great interest in the poultry industry (Park et al., 2013). Heat stress is largely a problem in birds selected for high rates of growth or egg production under temperate conditions. A high rate of production usually carries the penalty of higher metabolic heat production and the necessity to dissipate this heat to a warm or hot environment (MacLeod et al., 2004).
For many years, researchers have been investigating the effect of high environmental temperature on the performance of different poultry species, including turkeys (Kohne and Jones, 1976; McKee and Sams, 1997), young chickens (Henken et al., 1983), broilers (Cooper and Washburn, 1998), broiler breeders (McDaniel et al., 1995), and laying hens (Emery et al., 1984; Muiruri and
Harrison, 1991; Whitehead et al., 1998), and have found that high environmental temperatures have deleterious effects on productive performance (Mashaly et al., 2004). Ambient temperatures exceeding the thermal comfort zone increase birds’ heat load, resulting in a decrease in feed consumption, feed efficiency, body weight gain and livability (Borges et al., 2003; Daghir et al., 2008). In addition, meat quality, intestinal microflora and immune system were adversely affected by high ambient temperatures (Borges et al., 2007).
Riboflavin (Vitamin B2) (7, 8-dimethyl-10-ribityl-isoalloxazine) is a water soluble vitamin present in a wide variety of foods. It was initially isolated, although not purified, from milk whey in 1879 and given the name lactochrome. It can be crystallized as orange-yellow crystals and in its pure form is poorly soluble in water. Its most important biologically active forms, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), participate in a range of redox reactions, some of which are absolutely key to the function of aerobic cells (Powers et al., 2003) . Early classic studies identified a riboflavin binding protein in chicken egg white that is induced by estrogen and is essential to fetal survival (White and Merrill, 1988). Also, negative effects caused by heat stress in males have been shown in different studies. Semen volume, sperm concentration, number od live sperm cells and motility decreased when males were subjected to heat stress (Joshi et al., 1980; McDaniel et al., 1995; McDaniel et al., 2004; Lara and Rostango, 2013).
Recent years have witnessed a phenomenal increase in the use of enzymes as industrial catalysts. Proteases (synonymous as peptidase or proteinase) constitute a very large and complex group of enzymes, widely utilized in a host of industries. They differ in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature optima, and stability profiles. Studies relating to such properties are imperative for the successful application of these enzymes in their respective industry (Sumantha et al., 2005). The main sources of the enzymes were from
animals (e.g., calf stomach), plants (e.g., pineapple, fig, and papaya), microbes (e.g., Bacillus spp., Pseudomonas spp.) etc. but the production of enzymes from plant and animal sources is limited due to climatic reasons and ethical issues, respectively (Rao et al., 1998; Shafee et al., 2005).
Proteolytic enzymes are capable of hydrolyzing peptide bonds and are also referred to as peptidases, proteases or proteinases (Barrett and McDonald, 1986). The physiological function of proteases is necessary for all living organisms, from viruses to humans, and proteolytic enzymes can be classified based on their origin: microbial (bacterial, fungal and viral), plant, animal and human enzymes can be distinguished (Mótyán et al., 2013).
In hot climates, periods of high temperatures have a negative effect on the health and performance of domestic animals. The rectum is the final straight portion of the large intestine in some mammals, and the gut in others. Measures of rectal temperature (RT), pulse-rate (PR) and respiratory rate (RR) are some of the most important determinants of the adaptation of poultry to the tropical environment. They also, to a large extent, determine the profitability of the poultry enterprise (Ilori et al., 2012).
It has been established that heat stress is evaluated by measuring the rectal temperature (RT), which is a true reflection of internal body temperature and a reliable index of thermal balance (Bianca, 1976; Mittal and Ghosh, 1979; Ayo et al., 1998).
Data on the variation in rectal temperature and pulse rate of turkeys in response feed supplementation with riboflavin and protease enzyme during the hot-dry season are currently lacking in available literature.

1.1. Justification

Since no single control method has proven most effective in the control of heat stress in poultry production, different studies have been investigating integrated management practices in poultry heat stress. Therefore, this study seeks to investigate the effect of turkey feed supplementation with riboflavin and protease enzyme on rectal temperature and pulse rate which are among the physical parameters of heat stress.

1.2. Objectives

1.2.1. Broad Objective

To determine the effect of turkey feed supplementation with riboflavin and protease enzyme on two physical heat stress parameters during finishing stages of turkey production.

1.2.2. Specific Objectives

1)
To determine the rectal temperature of finishing turkeys fed diets supplemented with riboflavin (vitamin B2) and/or protease enzyme.
2)
To determine the pulse rate of finishing turkeys fed diets supplemented with riboflavin (vitamin B2) and/or protease enzyme.

2. Literature Review

2.1. Poultry Production

Poultry production has been reported to be the fastest growing in the livestock industry, and more particularly in tropical and sub-tropical regions of the world (Daghir et al., 2009; Holik et al., 2009). Today a large percentage of the world’s poultry population is located in regions where heat stress is a major management problem at some particular moments of the bird’s productive lives (Ajakaiye et al., 2011).
The establishment of poultry farms in new areas means that live birds of all ages have to be transported by road, across different ecological zones throughout the year (Ajakaiye et al., 2010). Many elements of the transport process can be harmful to the birds (Knowles and Brown, 1990; Nicol and Saville-Weeks, 1993). These includes handling by humans, air temperature changes, high ambient temperature (AT) and relative humidity (RH), removal of food and water, novelty, confinement, noise, motion, micro-thermal core within the vehicle and the use of inappropriate vehicles (Mitchell and Kettlewell, 1998; Bedonova et al., 2006; Vecerek et al., 2006). The adverse effects of these factors and their combinations may range from mild discomfort and aversion to death (Ajakaiye et al., 2010).
During the early summer season, the turkey industry reports substantial losses in yield due to formed turkey breast products with poor water-holding capacity, poor texture, and pale color. These meat characteristics are consistent with those observed in pale, soft, and exudative (PSE) pork. According to Ogah (2011), Turkey is not common among poultry growers in Nigeria: a number of farms are beginning to breed the bird at commercial level owing to increasing interest as a provider of meat complementing chicken. They are mostly located in urban areas and are gradually spreading even to village farms. The fast growth in the industry requires and intensive research approach to boast its production especially considering the potentials associated with it. The first approach in livestock characterization apart from evaluation of its production performance is the evaluation of body size and conformation (Ibe et al., 1989). Assessment of body weight and linear body measurements have been found useful in quantifying body size and shape (Ibe and Ezekwe, 1994). Linear body measurements have also been used to predict live weight in poultry (Chhabra et al., 1972; Monsi et al., 1992; Gueye et al., 1998). The multitude of different body measurements available has lead several researchers to use multivariate techniques to simultaneously examine the relationship among body measurements and production traits (Brown et al., 1973). Use of principal component analysis to examine the relationship between measurement of size and shape in poultry have been reported in chicken (Ibe et al., 1989; Yakubu et al., 2009) and duck (Shahin et al., 1996; McCracken et al., 2000; Ogah et al., 2009). This multivariate procedure describes the total variation in a large system of body measurements in terms of a few artificial varieties (Ogah et al., 2011).

2.2. Heat Stress

Heat stress results from a negative balance between the net amount of energy flowing from the animal’s body to its surrounding environment and the amount of heat energy produced by the animal. This imbalance may be caused by variations of a combination of environmental factors (e.g., sunlight, thermal irradiation, and air temperature, humidity and movement), and characteristics of the animal (e.g., species, metabolism rate, and thermoregulatory mechanisms). Environmental stressors, such as heat stress, are particularly detrimental to animal agriculture (Nienaber and Hahn, 2007; Nardone et al., 2010; Renaudeau et al., 2012).
High ambient temperature is a problem in many parts of the world. Heat stress has been associated with decreases in broiler weight gain, feed intake, feed efficiency, nitrogen retention, protein digestibility and total mineral retention (Austic et al., 1985; Sahin and Kucuk, 2003).
The high humidity will aggravate the bad influence of high temperature on the broiler (Yahav et al., 1995; Gu et al., 1999; Lin et al., 2005a, b) reported that humidity could affect the thermoregulation of broiler chickens by redistributing heat within the body at high, low and even thermoneutral temperatures, high humidity above 60% impaired the heat transmission from body core to the surrounding at high temperature. At hot environment, the chemical composition of chicken is changed (Leenstra and Cahaner, 1991; Geraert et al., 1996) and meat sensory quality is decreased by heat stress (Osman et al., 1989; Northcutt et al., 1994; Li et al., 1999). The oxidative damage of tissues induced by heat stress is one of the possible reasons (Sandercock et al., 2001; Lin et al., 2006b).
It has been reported that chronic heat exposure negatively affects fat deposition and meat quality in broilers, in a breed-dependent manner (Lu et al., 2007). In fact, recent studies demonstrated that heat stress is associated with depression of meat chemical composition and quality in broilers (Dai et al., 2012; Imik et al., 2012). Another recent study (Zhang et al., 2012) demonstrated that chronic heat stress decreased the proportion of breast muscle, while increasing the proportion of thigh muscle in broilers. Moreover, the study also showed that protein content was lower and fat deposition higher in birds subjected to heat stress.
Environmental stress causes oxidative stress and impairs antioxidant status in vivo (Halliwell and Gutteridge, 1989; Sahin et al., 2001). Mujahid et al. (2005) shown that superoxide production by the skeletal muscle mitochondria of meat type chickens is significantly enhanced by heat stress.
This in turn was associated with a heat-induced increase in rectal and muscle temperatures, leading to a significant body weight loss.
When the protein level increases under the heat stress, even more metabolic heat is produced, thus it is helpful to increase essential amino acids like methionine and lysine. As well, stimulating feed intake by providing high preference ingredients such as soy oil or molasses, and providing vitamin C are helpful to minimize heat stress (Leeson and Summers, 1991; Park et al., 2013). The benefits of vitamin C, vitamin E, potassium chloride, ammonium chloride, potassium sulphate and sodium bicarbonate in drinking water or feed during the hot period of the day have been reported (Sahin and Kucuk, 2001; Ciftci et al., 2005). For this purpose, vitamin C and vitamin E are used in the poultry diet because of their anti-oxidant properties in the neutralization of the free radicals generated during heat stress (Ramnath et al., 2008). Turkey poults are sensitive to environmental temperature with a lower critical temperature of about 29 °C (Scott et al., 1983). As a consequence of increased environmental temperature, the internal body core and skin temperature of the bird increases. The bird begins to breathe more rapidly, while its heart rate and oxygen consumption increase. The increase in oxygen consumption is a direct reflection of the increased energy demand for maintenance due to panting (Miles et al., 1999). Turkeys cannot tolerate a concurrent high temperature and high humidity. When the surrounding air is moist, it cannot absorb as much moisture from the lungs; consequently the bird must pant faster. Similarly, when the outside temperature is high, respiratory rate is increased. With both high temperature and high humidity the bird may not be able to pant fast enough to remove the heat from its body (El Boushy & Van Marle, 1978). The increase in the breathing rate is accompanied by an increase in the loss of moisture from the body. To compensate for this loss, the bird drinks more water to avoid dehydration. Eventually, the bird drinks more water than it can exhale, and the surplus is excreted through the droppings. The amount of moisture in the ambient air (humidity) also affects the panting rate; the higher the humidity the more rapid the respiration (North et al., 1984). Consequently, either at low or high inside air temperature, the rate of productive performance of turkey poults decreases. Therefore, inside air temperature when brooding turkey should be kept at an optimal level (Konca et al., 2001) by reducing heat loss and using supplementary heating when needed (Anonymous et al., 1987 and Gencoglan et al., 2009).
It has been established that heat stress is evaluated by measuring the rectal temperature (RT), which is a true reflection of internal body temperature and a reliable index of thermal balance (Bianca et al., 1976; Mittal and Ghosh, 1979; Ayo et al., 1998). Changes in reproductive hormone secretion represent the final sequence in the neuroendocrine pathway leading to the diminished reproductive performance associated with stress (Rozenboim et al., 2007).

2.3. Riboflavin (Vitamin B2)

Almost all riboflavin in tissues is enzyme bound, such as FAD covalently bound to succinic dehydrogenase (EC 1.3.5.1) (Singer and Kenney, 1974). Unbound flavins are relatively labile and are rapidly hydrolyzed to free riboflavin, which diffuses from cells and is excreted. The intracellular phosphorylation of riboflavin is therefore a form of metabolic trapping key to riboflavin homeostasis (Gastaldi et al., 2000; Powers et al., 2003).
Riboflavin deficiency is associated with demyelination of peripheral nerves and consequent locomotion difficulty. The classical disease associated with this deficiency is known as curled toe paralysis. It is reported that a condition similar to curled-toe paralysis, which occurred in turkeys fed with 3-Nitrogen, 4-hydroxyphenylarsonic acid. It was found that dietary levels of this substance greater that 50ppm caused demyelization of peripheral nerves (Das et al., 2011).
Milk and dairy products make the greatest contribution to riboflavin intake in Western diets, making riboflavin exceptional among the water-soluble vitamins. National dietary surveys in the United Kingdom report that, on average, milk and dairy products contribute 51% of intake in preschool children, 35% in schoolchildren, 27% in adults, and 36% in the elderly. Cereals, meats (especially offal), and fatty fish are also good sources of riboflavin, and certain fruit and vegetables, especially dark-green vegetables, contain reasonably high concentrations (Powers et al., 2003). A small amount of riboflavin is present in foods as free riboflavin, which is an isoalloxazine ring bound to a ribitol side chain; most is present as the derivative FAD, and a smaller amount occurs as the monophosphorylated form, FMN. FAD and FMN occur predominantly in a non-covalently-bound form to enzymes; flavins that are covalently bound do not appear to be available for absorption (McCormick et al., 1972). In contrast with most foodstuffs, milk and eggs contain appreciable quantities of free riboflavin bound to specific binding proteins (Zanette et al., 1984). The flavocoenzymes such as FMN and FAD comprise the major part of riboflavin in blood plasma (Ohkawa et al., 1982). The bioactive forms of riboflavin are hydrolyzed to riboflavin before they enter into the cells, but riboflavin is accumulated in tisues by resynthesis of flavocoenzymes (A.W et al., 1983; McCormick and Zhang, 1993).

2.4. Protease Enzymes

The use of exogenous enzymes is not a new concept and has been extensively studied and reported (Campbell and Bedford, 1992; Leeson et al., 1996; Seskevicience et al., 1999; Smits and Annison, 1996).
According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified under the subgroup 4 of Group 3 (hydrolases). However, proteases do not comply easily with the general system of enzyme nomenclature due to their huge diversity of action and structure. On the basis of their site of action on protein substrates, proteases are broadly classified as endo- or exo-enzymes (Rao et al., 1998). They are further caegorized as serine proteases, aspartic proteases, cysteine proteases or metallo proteases— depending on their catalytic mechanism. Proteases are also classified into different clans and families depending on their amino acid sequences and evolutionary relationships. Based on the pH optima, they are referred to as acidic, neutral, or alkaline proteases (Rao et al., 1998; and V. N. Jisha et al., 2013).
Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require the use of them, including the production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, use of proteases in cell culture experiments and in tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure- function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing, and proteolytic digestion of proteins in proteomics (Mótyán et al., 2013).
Proteolytic enzymes are capable of hydrolyzing peptide bonds and are also referred to as peptidases, proteases or proteinases (Barrett and McDonald, 1986).
The physiological function of proteases is necessary for all living organisms, from viruses to humans, and proteolytic enzymes can be classified based on their origin: microbial (bacterial, fungal and viral), plant, animal and human enzymes can be distinguished.
Proteolytic enzymes belong to the hydrolase class of enzymes (EC 3) and are grouped into the subclass of the peptide hydrolases or peptidases (EC 3.4). Depending on the site of enzyme action the proteases can also be subdivided into exopeptidases or endopeptidases. Exopeptidases catalyze the hydrolysis of the peptide bonds near the N- or C-terminal ends of the substrate. Aminopeptidases can liberate single amino acids (EC 3.4.11), dipeptides (dipeptidyl peptidases, EC 3.4.14) or tripeptides (tripeptidyl peptidases EC 3.4.14) from the N-terminal end of their substrates. Single amino acids can be released from dipeptide substrates by dipeptidases (EC 3.4.13) or from polypeptides by carboxypeptidases (EC 3.4.16-3.4.18), while peptidyldipeptidases (EC 3.4.15) liberate dipeptides from the C-terminal end of a polypeptide chain. Endopeptidases cleave peptide bonds within and distant from the ends of a polypeptide chain (Rao et al., 1998). Based on the catalytic mechanism and the presence of amino acid residue(s) at the active site the proteases can be grouped as aspartic proteases, cysteine proteases, glutamic proteases, metalloproteases, asparagine proteases, serine proteases, threonine proteases, and proteases with mixed or unknown catalytic mechanism (Rawlings et al., 2012).
The current classification system further classifies the proteases into families based on sequence similarities, furthermore, homologous families are grouped into clans using a structure-based classification (Rawling et al., 2012; Rawlings and Barrett, 1993). Classification and nomenclature of proteolytic enzymes as well as a detailed description of individual proteases is available in the MEROPS database (Rawlings et al., 2012 and Mótyán et al., 2013). Action of the proteolytic enzymes is essential in several physiological processes, e.g., in digestion of food proteins, protein turnover, cell division, blood-clotting cascade, signal transduction, processing of polypeptide hormones, apoptosis and the life-cycle of several disease-causing organisms including the replication of retroviruses (Neurath and Walsh, 1976; Devlin et al., 2002). Due to their key role in the life-cycle of many hosts and pathogens they have great medical, pharmaceutical, and academic importance (Li et al., 2013; Craik et al., 2011; Antonelli and Turriziani, 2012). They are intensively studied to explore their structure-function relationships, to investigate their interactions with the substrates and inhibitors, to develop therapeutic agents for antiviral therapies (Antonelli and Turriziani, 2012) or to improve their thermostability, efficiency and to change their specificity by protein engineering for industrial or therapeutic purposes (Li et al., 2013). Studying proteolytic enzymes is highly justified by their key role in several fields of industry (Rao et al., 1998; Kirk et al., 2002; Rani et al., 2012; Ray et al., 2012), as well.

2.6. Physiological Parameters

According to Altan et al. (2000), exposure of broilers to 39 °C significantly increased rectal temperatures, heterophil and basophil proportions and Heterophil to Lymphocyte (H/L) ratios, and decreased monocyte and lymphocyte proportions.
Rectal temperature (RT), respiratory rate (RR) and heart rate (HR) are important physiological parameters most relevant for on-the-spot evaluation of the health status and adaptability of animals, including poultry species (Bianca, 1976; Ayo et al., 1998). The parameters are easily measured and are of value in the determination of state of stress in birds, especially during the process of transportation in rural areas where laboratory facilities may be lacking.

3. Materials and Methods

3.1. Experimental Location

The experiment was carried out at the turkey unit, College of Animal Science and Livestock Production (COLANIM) Farms, Federal University of Agriculture Abeokuta, Ogun State, Nigeria. The farm is located on latitude 70 10’N and longitude 30 2’E and lies in the southwestern part of Nigeria with prevailing tropical climate and a mean annual rainfall of about 1037mm.The mean monthly ambient temperate ranges from 280C in December to 360C in February with a yearly average relative humidity of about 82%.

3.2. Experimental Birds, Materials and Management

A total of 100 one-day old British United broiler turkey poults were used for the experiment. The poults were bought from a commercial hatchery: O. H. L., Ibadan, Oyo State, Nigeria. The brooding house was prepared and disinfected before the arrival of the poults. The poults were brooded intensively for four weeks at a controlled temperature of about 34.50C for the first two days and reduced gradually every week at 20C to a final ambient temperature of about 270C at the fourth week of brooding. The brooding was carried out on a deep litter system, using wood shavings as the litter materials. Management routine practices were carried out, feeders and drinkers were cleaned daily. The vaccination and medication schedule was strictly adhered to. After the brooding period, the poults were further reared for four weeks (28 days), prior to the commencement of the experiment. Birds were fed with pre-starter diet (0-4 weeks),starter (4-8 weeks),grower (8-12 weeks) and finisher (12-18 weeks). The experiment was commenced at the end of the starter phase.

3.3. Source of Riboflavin and Protease Enzyme

There was an inclusion of riboflavin and protease enzyme in the bird’s diet. Riboflavin (vitamin B2) was procured from a trusted pharmaceutical company in Abeokuta, Ogun State. Also, protease enzyme was procured from Norogen Nigeria Limited, Lagos, Nigeria.

3.4. Experimental Design

Eighty broiler turkeys of similar weight range were selected and allotted on weight equalization basis to four dietary treatment at 56 days. There were four experimental diets formulated for feeding growing and finishing phases of the study. The diets were supplemented with 1000mg/kg of protease enzyme, 6mg/kg of riboflavin (vitamin B2), (1000mg+6mg)/kg of both protease enzyme and riboflavin and a standard basal diet containing no supplement.

3.5. Experimental Diet

Table 1. Experimental Diets.
Table 1. Experimental Diets.
Four experimental diets were used and they are:
Diet 1 basal diet without riboflavin and protease enzyme.
Diet 2 basal diet with riboflavin 6mg/kg.
Diet 3 basal diet with protease enzyme 1000mg/kg.
Diet 4 basal diet with riboflavin and protease enzyme (6mg+1000mg)/kg.

3.6. Data Collection

3.6.1. Rectal Temperature

The rectal temperature of the birds was taken by inserting clinical digital thermometer (0.10C accuracy) into the rectum via the cloaca until an alarm sound beeps, indicating the end of the reading. It was done twice every week during the experimental period.

3.6.2. Pulse Rate

The pulse rate of the birds was taken with the aid of a stethoscope. It was read by the number of heart beats per minutes. This also was taken twice every week during the course of the experiment.

3.6.3. Environmental Indicators in the Poultry House

Ambient temperature and relative humidity were taken with the aid of the hygrometer. They were taken twice in a week at the time of taking records on physical parameters of the birds during the experimental period.

2.7. Statistical Design and Analysis

The data collected was analyzed using factorial experiment of SYSTAT. Results were reported at least square means (LS Means).
Table 2. Gross composition of pre-starter (0-4 weeks) and starter (4-8 weeks) diets.
Table 2. Gross composition of pre-starter (0-4 weeks) and starter (4-8 weeks) diets.
Gross composition of diets (%)
Ingredient Pre-starter (0-4 weeks) Starter (4-8 weeks)

Maize
42.50
50.00
Soybean Meal 40.70 36.00
Fish Meal (72%) 9.00 8.90
Bone Meal 4.50 3.00
Limestone 2.00 1.00
*Vitamin/trace mineral Premix 0.50 0.15
Lysine 0.10 0.20
DL Methionine 0.40 0.50
Salt 0.30 0.25
Total 100.00 100.00
Calculated analyses
Metabolizable Energy (Kcal/kg) 2,840 2878.7
Crude Protein (%) 28.19 26.62
Crude Fibre (%) 3.00 3.97
Ether Extract (%) 3.78 2.89
Calcium (%) 2.16 1.39
Phosphorus (%) 0.86 0.67
Lysine (%) 1.91 1.89
Methionine (%) 0.88 0.68
Arginine (%) 1.82 1.71
Vitamin/Mineral Premix composition per Kg diet: vit A: 40, 000IU, vit D3: 4000IU, vit E: 40.0 mg, vit K3: 8mg, vit B1: 1.0mg, vit B2: 8mg, vit B6: 5mg, vit B12: 0.025mg, Niacin: 60mg, Panthothenic acid: 20mg, Folic acid: 2000mg, Biotin: 150mg, , Iron: 32mg,Manganese: 64mg, Zinc: 40mg, Copper: 8mg, Cobalt: 80mg, Iodine:0.15mg, Selenium: 0.2mg, Choline: 300mg.
Table 3. Gross composition of experimental grower diet (8-12 weeks).
Table 3. Gross composition of experimental grower diet (8-12 weeks).
T1
(Control
) 		T2
(Riboflavin
) 		T3
(Protease) 		T4
(Riboflavin and Protease)
Supplemental levels of
feed additives 		0 6mg/kg 1000mg/k
g		 6mg+1000mg/kg
Ingredient composition
Maize 555 555 555 555
Soya Bean Meal 260 260 260 260
Fish meal (72% CP) 65 65 65 65
Wheat offal 50 50 50 50
Palm oil 17 17 17 17
Bone Meal 20 20 20 20
Limestone 15 15 15 15
*Vitamin/mineral Premix 5 5 5 5
Methaonine 2 2 2 2
Lysine 1.5 1.5 1.5 1.5
Salt (NaCl) 2.5 2.5 2.5 2.5
Total 1000.00 1000.00 1000.00 1000.00
Calculated analysis
Metabolisable Energy (Kcal/kg) 2952.92 2952.92 2952.92 2952.92
Crude Protein (%) 21.90 21.90 21.90 21.90
Crude Fibre (%) 3.70 3.70 3.70 3.70
Ether Extract (%) 3.39 3.39 3.39 3.39
Phosphorus (%) 0.90 0.90 0.90 0.90
Calcium (%) 2.38 2.38 2.38 2.38
Lysine (%) 1.29 1.29 1.29 1.29
Methionine (%) 0.62 0.62 0.62 0.62
Vitamin/Mineral Premix composition per Kg diet: vit A: 40, 000IU, vit D3: 4000IU, vit E: 40.0 mg, vit K3: 8mg, vit B1: 1.0mg, vit B2: 8mg, vit B6: 5mg, vit B12: 0.025mg, Niacin: 60mg, Panthothenic acid: 20mg, Folic acid: 2000mg, Biotin: 150mg, Iron:g, Manganese: 64mg, Zinc: 40mg, Copper: 8mg, Cobalt: 80mg, Iodine: 0.15mg, Selenium: 0.2mg, Choline: 300mg.
Table 4. Gross composition of experimental finisher diet (12-18 weeks).
Table 4. Gross composition of experimental finisher diet (12-18 weeks).
T1
(Control
) 		T2
(Riboflavin
) 		T3
(Protease) 		T4
(Riboflavin and Protease)
Supplemental levels of
feed additives 		0 6mg/kg 1000mg/k
g		 6mg+1000mg/kg
Ingredient composition
Maize 645 645 645 645
Soya Bean Meal 205 205 205 205
Fish meal (72% CP) 45 45 45 45
Wheat offal 37 37 37 37
Palm oil 15 15 15 15
Bone Meal 27 27 27 27
Limestone 15 15 15 15
*Vitamin/mineral Premix 5 5 5 5
Methaonine 2 2 2 2
Lysine 1.5 1.5 1.5 1.5
Salt (NaCl) 2.5 2.5 2.5 2.5
Total 1000.00 1000.00 1000.00 1000.00
Calculated analysis
Metabolisable Energy
(Kcal/kg)		 3030.64 3030.64 3030.64 3030.64
Crude Protein (%) 18.31 18.31 18.31 18.31
Crude Fibre (%) 3.45 3.45 3.45 3.45
Ether Extract (%) 2.84 2.84 2.84 2.84
Phosphorus (%) 0.86 0.86 0.86 0.86
Calcium (%) 2.19 2.19 2.19 2.19
Lysine (%) 1.08 1.08 1.08 1.08
Methionine (%) 0.56 0.56 0.56 0.56
*Vitamin/Mineral Premix composition per Kg diet: vit A: 40, 000IU, vit D3: 4000IU, vit E: 40.0 mg, vit K3: 8mg, vit B1: 1.0mg, vit B2: 8mg, vit B6: 5mg, vit B12: 0.025mg, Niacin: 60mg, Panthothenic acid: 20mg, Folic acid: 2000mg,. Biotin: 150mg, Iron:g, Manganese: 64mg, Zinc: 40mg, Copper: 8mg, Cobalt: 80mg, Iodine: 0.15mg, Selenium: 0.2mg, Choline: 300mg.

4. Results and Discussion

4.1. Results

4.1.1. Effect of Feed Supplementation with Riboflavin and Protease Enzyme on Rectal Temperature of Finishing Turkeys

The effect of feed supplementation with riboflavin and protease enzyme on rectal temperatures of finishing turkeys is presented in Table 5. The analysis of variance showing effect of riboflavin and protease enzyme on rectal temperature of finishing turkeys is presented on Table 6. There was no significant (P<0.05) difference in the rectal temperatures of birds offered the four experimental diets within age. The range of rectal temperature observed was between 40.4500C and 41.1310C. However, age had effect on rectal temperature. Rectal temperature decreased significantly at the age of 16 and 17 weeks compared to earlier ages.

4.1.2. Effect of Feed Supplementation with Riboflavin and Protease Enzyme on Pulse Rate of Finishing Turkeys

The effect of feed supplementation with riboflavin and protease enzyme on pulse rate of finishing turkeys is presented on Table 7. The analysis of variance showing effect of riboflavin and protease enzyme on pulse rate of finishing turkeys is presented on Table 8. There was no significant (P<0.05) difference in pulse rate within age of birds offered the four experimental diets. The range of pulse rate observed was between 150.250 and 171.250. However, age had effect on pulse rate. Pulse rate decreased significantly at the age of 16 and 17 weeks compared to earlier ages.

4.1.3. Result of Ambient Temperature and Relative Humidity of the Poultry House

4.1.3.1. Ambient Temperature

Ambient temperature is shown on Figure 1. The range of ambient temperature observed was between 29.20C and 29.80C. Ambient temperature decreased significantly with the poultry house during week 16 and 17 of the experiment.

4.1.3.2. Relative Humidity

Relative humidity is shown on Figure 2. The range of relative humidity observed was between 72% and 74%. Relative humidity decreased significantly during week 17 compared to earlier periods of the experiment.

4.2. Discussion

In this current study, it was observed that there was no significant difference in the rectal temperatures and pulse rate of birds offered the four experimental diets within age. The addition of riboflavin and protease enzyme at the specified levels did not influence these physiological parameters. This suggest that supplementation of riboflavin and protease enzyme did not effect any changes in metabolic heat generated during the finishing stage of turkey birds. However, the benefits of vitamin C, vitamin E, potassium chloride, ammonium chloride, potassium sulphate and sodium bicarbonate in drinking water or feed during hot period of the day have been reported (Sahin and Kucuk, 2001; Cifti et al., 2005).
However, age had effect on rectal temperature and pulse rate of the birds. The rectal temperature and pulse rate decreased significantly at the age of 16 and 17 weeks compared to earlier ages. In consonance, the ambient temperature and relative humidity decreased significantly at week 16 and 17 compared to earlier periods of the experiment. The simultaneous decrease in body temperature, pulse rate and the environmental parameters suggest interdependence. As the environmental queues, changes in the physiological parameters will be influenced. The rectal temperature and pulse rate of the birds decreased significantly at week 16 and 17 compared to earlier periods of the experiment. This suggest that, high humidity will aggravate the bad influence of high temperature on the turkey. Yahav et al. (1995) and Lin et al. (2005a,b) reported that humidity could affect the thermoregulation of broiler chickens by redistributing heat within the body at high, low and even thermoneutral temperatures. High humidity above 60% will impair the heat transmission from body core to the surrounding at high temperature. The ambient temperature and relative humidity had effect on rectal temperature and pulse rate in reduction of metabolic heat of the birds.

5. Conclusions

It is concluded that the addition of riboflavin and protease enzyme at the specified levels did not influence the rectal temperatures and pulse rate of finishing turkeys and therefore suggest that the two types of supplements at the levels used are not likely to be effective in controlling heat stress in turkey.

5.1. Recommendation

Addition of riboflavin and protease enzyme at 6mg/kg and 1000mg/kg may not be effective on these two physiological parameters. However, further studies are required to ascertain whether other levels of supplementation may be effective.

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Figure 1. Bar chart showing ambient temperature per week.
Figure 1. Bar chart showing ambient temperature per week.
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Figure 2. Bar chart showing relative humidity per week.
Figure 2. Bar chart showing relative humidity per week.
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Table 5. Effect of riboflavin and protease enzyme on rectal temperature of finishing turkeys during hot-dry season.
Table 5. Effect of riboflavin and protease enzyme on rectal temperature of finishing turkeys during hot-dry season.
Age (weeks) Control Basal diet with riboflavin (6mg/kg) Basal diet with protease enzyme (1000mg/kg) Basal diet with riboflavin and protease enzyme (6mg/kg+1000mg
/kg)
11 40.931 ± 0.099 41.081 ± 0.099 40.975 ± 0.099 40.875 ± 0.099
12 41.131 ± 0.099 40.919 ± 0.099 40.794 ± 0.099 40.869 ± 0.099
13 40.931 ± 0.099 40.938 ± 0.099 40.950 ± 0.099 40.788 ± 0.099
14 40.969 ± 0.099 41.113 ± 0.099 40.931 ± 0.099 41.063 ± 0.099
15 40.925 ± 0.099 40.875 ± 0.099 40.919 ± 0.099 41.119 ± 0.099
16 40.525 ± 0.099 40.594 ± 0.099 40.644 ± 0.099 40.588 ± 0.099
17 40.350 ± 0.099 40.431 ± 0.099 40.419 ± 0.099 40.425 ± 0.099
Table 6. Analysis of variance showing effect of riboflavin and protease enzyme on rectal temperature during hot-dry season.
Table 6. Analysis of variance showing effect of riboflavin and protease enzyme on rectal temperature during hot-dry season.
Source Degree of Freedom Mean-square *
Treatment 3 0.854 NS
Week 6 0.000 ***
Treatmeat * Week 18 0.552 NS
Error 420
*** P<0.000, NS: Not Significant.
Table 7. Effect of riboflavin and protease enzyme on pulse rate of turkeys during hot-dry season.
Table 7. Effect of riboflavin and protease enzyme on pulse rate of turkeys during hot-dry season.
Age (week) Control Diet with riboflavin (6mg/kg) Diet with
protease enzyme (1000mg/kg) 		Diet with riboflavin and protease enzyme (6mg/kg+100 0mg/kg)
11 167.50ab±1.708 162.500bc±1.708 164.750b±1.708 171.250defg±1.
708
12 153.25defg±1.708 157.750cd±1.708 157.500cde±1.708 156.000defg±1.
708
13 154.50defg±1.708 151.000fg±1.708 156.000defg±1.708 156.000defg±1.
708
14 156.75def±1.708 157.250de±1.708 155.750defg±1.708 153.500defg±1.
708
15 155.000defg±1.708 156.313def±1.70
8		 155.000efg±1.708 154.500defg±1.
708
16 154.250defg±1.708 152.750defg±1.70
8		 151.750g±1.708 155.500defg±1.
708
17 154.750defg±1.708 154.500defg±1.70
8		 150.250a±1.708 153.500defg±1.
708
Table 8. Analysis of variance showing effect of riboflavin and protease enzyme on pulse rate of turkeys during hot-dry Season.
Table 8. Analysis of variance showing effect of riboflavin and protease enzyme on pulse rate of turkeys during hot-dry Season.
Source Degree of Freedom Standard Error of Mean
Treatment 3 40.437 NS
Week 6 1343.336 ***
Treatment * week 18 85.062 *
Error 420 46.703
*** P<0.001, * P<0.05 and NS: Not Signicant.
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