The commercial poultry industry is considered the most rapidly growing of all the agricultural sectors. Feed costs constitute around 70% of the total cost of poultry production. The most important feed ingredients for poultry production are energy and protein sources. The poultry industry mainly relies on a limited number of animal and vegetable protein ingredients, such as oilseed meals, legumes and animal by-products (Broomhead, 2013; FAO, 2013). The commonly used animal protein sources such as blood, meat, meat and bone and fish meals are recognized as high- quality protein, with excellent nutritive value and balanced amino acids. Furthermore, chickens tend to prefer animal by-products to vegetable proteins (Hossain et al., 2013). On the other hand, there are some constraints to the use of animal by-products as feed ingredients for animals; high prices, restricted hygienic conditions and the risk that birds may suffer from zoonotic diseases if the animal by-products are processed under sub-optimal conditions. Therefore, for ethical and/or health reasons, animal proteins are excluded from production systems in some parts of the world such as the European Union (Hamilton, 2002).
There are numerous vegetable protein sources of local importance around the world, such as soybean, canola, cottonseed, sunflower seed, peanut, and sesame meals, but these have less nutritive value than animal protein sources (Hamilton, 2002; Aftab, 2009). Most of the vegetable protein sources contain one or more anti-nutritive factors, which can limit the digestion of their nutrients and eventually affect overall animal health, for instance trypsin inhibitors, glucosinolates, and gossypol in soybean meal, canola and cottonseed meals, respectively (Akande et al., 2010). The average crude protein content of different vegetable protein sources ranges between 235 g/kg in peas and 480 g/kg in soybean meal (SBM). Soybean meal is the primary plant protein source used by the poultry industry around the world. However, canola meal (CM) is increasing in importance (Hamilton, 2002; Nagalakshmi et al., 2007). The price of both soybean and canola meals do fluctuate but are generally high, particularly in importing countries. Besides CM, there are other vegetable protein sources close to SBM in nutritive value, low in prices and locally produced such as cottonseed meal (CSM) and sunflower seed meal (SFM).
Cotton (Gossypium), a genus of the Malvaceae family, covers approximately 2.5 % of the agricultural land around the world. Cotton production worldwide is estimated as 23013 thousand tonnes. The highest cotton producing countries in 2015/2016 were India, China, United States, Pakistan, Brazil, Uzbekistan, Turkey and Australia with 5748, 4790, 2806, 1524, 1285, 827, 577 and 566 thousand tonnes, respectively (USDA, 2017).
Cotton yields a number of by-products which are of great value to humans and domesticated livestock. Cottonseed is one of the most valuable by-products produced after the fine cotton fibres are harvested. Jones (1985) reported that for each kg of fibre produced there is 1.5‐1.7 kg of cottonseed separated out in the ginning process. Cottonseed meal or cake is a by-product of oil extraction from cottonseed. It has been reported that crushing one tonne of cottonseed produces around 200 kg of oil, almost 500 kg of cottonseed cake and 300 kg of cottonseed hulls or exteriors (Campbell et al., 2009). Several factors affect the quality of cottonseed obtained, including genetic differences, environmental conditions and harvesting techniques, which indirectly affect the composition of the resulting cottonseed meal. In addition to the genetic differences and environmental effect, the differences in the produced cottonseed meal arise from the residual oil content due to the method of extraction. For this reason there are different types of cottonseed meals, in terms of their protein, fibre and oil contents. The three main methods used by the oil industry to extract oil from oilseeds are: mechanical, solvent and pre-press solvent extraction. Mechanical extraction is the traditional method; it uses a circular motor and hydraulic press or expeller. In this method the seed may need to be decorticated, dried and/or heated before extraction. Besides the cakes produced by this method being tough and large, another important disadvantage is that around 20% of oil remains inside the meal. This high amount of oil, although it considered as valuable energy source, but it may increase the cost and reduce the palatability and storage period of diets. The difference between the mechanical method and the direct solvent extraction method is that in the latter method the oil is extracted by solvents (hexane or ethanol) alone without mechanical pressing and the meal produced has lower oil content. The third method, the pre-press solvent extraction, was developed from a combination of the preceding two methods. This method is considered an integrated method because screw-pressing is followed by solvent extraction, resulting in the extraction of almost 97 % of the oil content of oilseeds (Morgan, 1989; Ash, 1992; O'Brien et al., 2005).
Cottonseed meal is a palatable and excellent source of protein for ruminants. Although it's nutritive value is less than SBM, but its low cost in some regions makes it the main source of protein for cattle especially in parts of India, Australia and United States. Furthermore, CSM can replace all other oilseed meals in dairy cow feeds without affecting milk production (McGregor, 2000). Using whole cottonseed as a major source of protein has been tested to some extent in large animals, but its use in poultry diets as such results in decreased feed consumption and conversion, reduced nutrient digestibility, and poor growth (Devanaboyina et al., 2007). Furthermore, incidence of lameness and a high mortality rate are also associated with feeding entirely CSM as a source of protein to birds (Kakani et al., 2010). The presence of anti-nutritional factors such as gossypol and cyclopropenoid fatty acid, high fibre content and a deficiency in lysine are the well-known factors that limit the use of CSM in poultry diets (Swiatkiewicz et al., 2016). Cottonseed meal has a high crude protein content that ranges between 220 g kg-1 in the in the non-decorticated and 560.2 g kg^-1 in the completely decorticated seed, with metabolizable energy in the range of 7.4 to 11.99 MJ kg^-1. Furthermore, the fibre content of CSM exceeds that of SBM by 25% in the non- decorticated to 5% in the fully decorticated seed (Nagalakshmi et al., 2007). This promising nutrient profile of CSM, along with the fluctuation in the price of SBM around the world encourages poultry nutritionists and producers to trial CSM as a cost-effective and best nutritional alternative to SBM (Aftab, 2009).
Numerous ways have been reported that help in alleviating the limitations associated with the inclusion of CSM in poultry diets and raise its nutritive value. These include genetic manipulation of Gossypium through conventional breeding approaches and/or modern biotechnology, ingredient processing, using effective feed processing techniques, to decrease and inhibit anti-nutrients, and supplementation with nutrients such as synthetic amino acids, fat and vegetable oils. However, microbial enzymes appear to be the most effective solution to overcoming the limitations of the high-fibre and the non-starch polysaccharide (NSP) contents of alternative vegetable proteins that limit their inclusion at high levels in poultry diets (Scott et al., 1998: Leeson and Summers, 2001). All the above-mentioned techniques have helped to increase the CSM inclusion rate from 5% to around 30% of complete formulated diets for broiler chickens without compromising birds' performance (Watkins et al., 1995). Poultry lack specific enzyme systems to target NSP. For this reason, researchers are concentrating on developing single and composite microbial enzyme products that target NSP and enhance the nutritive value and nutrient digestibility of diets containing fibrous vegetable protein meals (Scott et al., 1998).
The poultry industry has employed microbial enzymes to improve the quality of temperate cereals and oilseed cakes. Therefore, inclusion of appropriate exogenous microbial enzymes in poultry feeds has clearly been demonstrated to increase the bio-availability of poorly digested diets, promote utilization of fibrous diets and improve the feed conversion ratio. These positive effects of the usage of exogenous enzymes have been frequently reported in recent studies as a result of the use of newly developed products for specific ingredients (Creswell, 1994; Slominski et al., 2006; Raza et al., 2009).
Much research and many industry field studies have been conducted to investigate the possibilities of replacing more expensive plant protein sources, like SBM, with alternatives with a similar nutritive value but lower prices such as CM, CSM and SFM. The present study is one of these investigations, and, hence, the main objectives of this study are to:

• Test the response of broiler chickens to CSM-containing diets supplemented with new microbial enzyme products (Avizyme 1502 and Axtra XB).
• Assess the potential of microbial enzymes in improving the nutritive value of CSM in diets for broiler chickens, especialy NSP-targeting enzymes.
• Evaluate CSM as a cost-effective alternative protein ingredient to SBM without compromising broiler performances.
• The study is intended to, among other things, determine the optimum levels of CSM and the test microbial enzymes in diets for broiler chickens and establish CSM as a competitive alternative to SBM.

The major objective of this doctoral research project was to investigate the influence of varying levels of dietary minerals (Ca, NPP, Na, Fe and Zn) on phytase activity and its subsequent impact on broiler chickens. Along with an extensive review of literature (Chapter 2) related to this subject, the key findings of one in vitro study followed by four feeding trials are summarised in this thesis. Diets of all feeding trials were formulated by considering mineral matrix (Ca, P and Na) value of tested phytase and this matrix value appeared to be correct on the basis of the overall results of all trials.

In the in vitro experiment, the effect of different dietary minerals (Ca, Na, Fe and Zn) on phytase activity at different pH was examined (Chapter 3). Calcium (0, 0.6, 0.8 and 1.0 %), Fe (0, 70, 80, 90 mg /diet), Zn (0, 30, 40, 50 mg kg/diet) or Na (0, 0.15, 0.25 and 0.35 %) were incubated (30, 60 and 90 mins) with a Na-phytate (0.27 %) solution, with phytase enzyme (500 FTU kg/ diet) at pH 2.5 or 6.5. There was a reduction (p < 0.05) in phytate hydrolysis by phytase at high concentrations of Ca, Fe and Zn (10 g, 90 and 50 mg respectively), particularly at pH 6.5. Although, increasing Na concentration reduced (p < 0.05) phytate hydrolysis, mostly at pH 2.5, the pattern was indefinite. In the presence of high concentrations of Ca, Zn and Fe, residual phytate content after phytate digestion was higher (p < 0.05) at pH 6.5 than 2.5, while the reverse was the case in the presence of Na. The findings of this in vitro study was further evaluated in four subsequent feeding trial.

The influence of different levels of Ca (0.6, 0.8 and 1.0 %) and NPP (0.3 and 0.4 %) with phytase (500 FTU/kg) or without phytase supplementation was evaluated in first feeding trial (Chapter 4). In general, phytase supplementation improved (p < 0.05) the body weight gain (BWG), feed intake (FI) tibia bone breaking strength (BBS), tibia ash content, ileal digestibility of Ca, P and protein. However, the positive effect of phytase on these variables was reversed (p < 0.05) in diets containing high Ca (1.0 %) and low NPP (0.3 %). This combination of minerals and phytase also reduced (p < 0.001) the activities of alkaline phosphatase (AP), Ca-ATPase and Mg-ATPase activity of jejunum. High Ca diet reduced the carcass yield of bird even with phytase supplementation (Ca × phytase, p < 0.041).

In Experiment 3 (Chapter 5), the effect of dietary Na (0.15, 0.25 and 0.35 %) on phytase (500 FTU/kg) activity and broiler performance was evaluated and presented. Varying levels of dietary Na, phytase and their interaction did not statistically affect the performance and tibia bone development. High dietary Na (0.35 %) reduced (p < 0.001) excreta dry matter (DM). The ammonia excretion was higher (p < 0.007) in phytase supplemented diets than unsupplemented diets. The negative effect of high Na diet on AME (apparent metabolisable energy), ileal digestibility (Ca and P) and the total tract retention (Ca, P, Na and Mg) of nutrients was countered by phytase supplementation. Supplementation with phytase increased (p < 0.05) the activities of Na-K-ATPase in the jejunum.

The activity of phytase in the presence of varying dietary levels of iron (60, 80 and 100 mg/kg) in broiler chickens was investigated in Experiment 4 (Chapter 6). The phytaseinduced improvement in BWG (p < 0.001) and FCR (p < 0.045) at d 35 was significantly reduced by high dietary Fe content (100 mg/kg), indicating significant interaction between Fe and phytase. The combination of high dietary Fe and phytase also reduced (p < 0.001) the ileal digestibility of N, P, Mg and Fe. The high Fe diet reduced the tibia BBS which was counteracted (p = 0.059) by phytase inclusion. High dietary Fe increased (p < 0.001) the deposition of Fe in tibia bone and liver. Phytase improved (Fe × phytase, p < 0.001) the activity of Ca-Mg ATPase, Ca-ATPase and Mg-ATPase in the jejunum when supplemented to diet containing 80 mg Fe/kg.

In the final experiment, the response of birds to different levels of dietary Zn (30, 40 and 50 mg/kg) supplemented with phytase were assessed and presented in Chapter 7. The low Zn (30 mg/kg) diet reduced (p < 0.041) FI but only during 1-10d. Irrespective of Zn level, phytase supplementation improved (p < 0.012) the BWG at 1-24d. Bone development of birds was not affected by Zn, phytase or their interaction. Phytase supplemented to low Zn diet improved (p < 0.001) the ileal digestibility of P but reduced (p < 0.05) the Fe and Zn digestibility. The accumulation of Fe (p < 0.001) and Zn (p < 0.002) in liver was increased in birds on high Zn (50 mg/kg) diet. Phytase supplemented to diet containing 40-50 mg Zn/kg improved (p < 0.008) the net energy for production (NEp) and the fat and protein deposition rate in the tissues of broiler chickens. The activities of AP, Ca-ATPase and Mg-ATPase in jejunal mucosa was high (p < 0.001) in birds on the phytase-supplemented mid-Zn diet.

In general, it can be concluded that high Ca and Fe had significant negative effect on phytase activity and subsequently on broiler performance. The inhibitory effect of high Ca on phytase activity was more pronounced in low NPP diet. Phytase supplemented to high Zn showed better effect on birds’ performance. The negative effect of high Na only observed on utilization of some minerals and N which was countered by phytase supplementation. Finally, a careful consideration of dietary mineral levels in phytase supplemented diets can be a useful way to sustain the activity of phytase, improve productivity and reduce mineral excretion into the environment.