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.

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.

Supplementing ruminants with dietary nitrate (NO₃) is an effective methane mitigation strategy if it can be managed so as to not expose ruminants to any risk of clinical nitrite (NO₂) toxicity. The objective of this thesis was firstly to deepen the understanding for NO₃ metabolism in sheep and secondly to develop practical strategies to reducing risk of NO₂ toxicity in sheep supplemented with dietary NO₃.

It has been previously established, that in the rumen NO₃ is reduced to NO₂ and then to NH₃, and that supplementing with excessive amounts of NO₃ can expose ruminants to NO₂ toxicity due to the absorption of NO₂. This thesis reports a series of five investigations of NO₃ metabolism by sheep and identifies:

Nitrate, like urea, is ‘recycled’ within the ruminant. Transfer of ruminal ¹⁵NO₃^--N into the blood and transfer of blood NO₂-N into the rumen being quantified. Only 20% of rumen NO₃^-and 30% of blood NO₂^- were recovered in urine.

That in hourly fed sheep approximately 90% of dietary NO₃^- was rapidly converted to NH₃ in the rumen, with the remainder leaving the rumen by absorption into the bloodstream or passage to the lower gastro-intestinal tract.

Within the rumen, the conversion of NO₃^-to NH₃ is neither simple nor complete. In vitro and in-vivo studies showed NO₃^-is reduced to gaseous nitrous oxide (N₂O) and N₂O may be further metabolised to N₂ gas by the rumen microbiota. Approximately 0.04% and 3.0% of dosed NO₃^--N was recovered over 10 h from sheep as N₂O and N₂ respectively, and this was not affected by whether sheep had prior adaption to NO₃^- or not, identifying denitrification as a reaction not previously reported from the rumen.

From this understanding and a review of the literature on ruminant NO₃ metabolism, eight critical control points for reducing the risk of nitrite toxicity (methaemoglobinaemia), were identified and the potential for manipulating five of these evaluated.

Reducing the rate at which NO₃ became available to the rumen biota by coating calcium nitrate with paraffin wax significantly reduced blood methaemoglobin level (MetHb; an indicator of NO₂ toxicity) in sheep supplemented with NO₃.

The extent of methaemoglobinaemia could also be reduced by the daily ration being consumed at shorter intervals rather than in a single bout, and this established that feed management is pivotal to safe feeding of NO₃^-containing diets.

Enhancing the rumen’s capacity to reduce potentially toxic NO₂ ^-by supplying Propionibactericum acidicpropionici as a direct fed microbial was ineffective in reducing blood MetHb or NO₂^-concentration of sheep fed NO₃^- supplemented diets.

Attempts to increase the rate of removal of NO₂^-from the rumen by providing a substrate (glycerol) to stimulate NADH availability in the rumen, and accelerate the nitrite reductase enzyme system did not reduce the concentration of NO₂ in incubations of rumen contents supplemented with NO₃^-.

We found no evidence that adapting sheep to dietary NO₃^- protected them against NO₂^- toxicity. Indeed, in vitro more NO₂^- accumulated in incubation when donors where adapted to dietary NO₃^-. Also, no signs of reduced MetHb were noticed after several weeks of NO₃^-supplementation in vivo.

Other critical control points such as regulating microbial uptake of NO₃ and ruminal absorption of NO₃ and NO₂ were unable to be assessed in this thesis.

The studies reported here also confirmed the practical impacts of NO₃ as an effective supplement for reducing enteric methane emissions and increasing wool growth of sheep. As well as providing a better understanding of NO₃^-metabolism, studies also showed that the greenhouse gas (GHG) abatement impact of methane mitigation may be partly offset by an associated production of the potent GHG, N₂O. Discovery of the production of N₂O and N₂ from NO₃^-in the rumen and identification of recycling of blood NO₂^- to the rumen has expanded our understanding of NO₃^-metabolism. Coating NO₃^-to decrease the rapidity of NO₃^- release in the rumen as a strategy to reduce NO₂ toxicity was effective but needs further investigation. The applicability of feed grade NO₃^-as a commercially available feed additive will also depend on the cost of NO₃ and the additional cost of the technology to ensure its safe feeding, compared to the cheaper alternative non-protein nitrogen source, urea.

Detailed findings of the potential use of sorghum grain in diets for broiler chickens are presented in this thesis. A comprehensive literature on the above subject was reviewed, followed by feeding trails.

An initial experiment (Chapter 3) in this thesis evaluated the chemical composition and nutritive value of South African sorghum varieties as feed for broiler chickens. Physical and proximate composition, total phenolic and antioxidant activity, mineral content, amino acid profile and digestibility and true metabolisable energy of four sorghum varieties were studied. The thousand-kernel-weight ranged from 33 to 28 g and kernel texture was somewhat corneous to floury. The condensed tannin sorghum varieties, PAN8625 and NS5511, had higher total phenolic content and antioxidant activity than the non-tannin varieties, PAN8816 and PAN8906. Starch and gross energy contents differed significantly across the sorghum varieties. The sorghum varieties had somewhat similar total and individual mineral contents. Threonine, leucine, phenylalanine, valine, proline and alanine contents of varieties PAN8625 and PAN8906 were higher than those of NS5511 and PAN8816, which had similar contents. The amino acid digestibility and metabolisable energy of the tannin sorghum varieties were generally lower than those of the non-tannin varieties.

In addition to determining the physio-chemical properties of sorghum grains the influence of xylanase inclusion in sorghum-based diets on performance of broiler chickens was determined in another experiment (Chapter 4). Gross performances were assessed and carcass yield and visceral organ weights were measured at day 21. On day 25, birds were euthanized by intravenous injections of sodium pentobarbitone, and digesta contents from the distal ileum were collected and processed to determine the nutrient digestibility. Broiler chickens offered sorghum variety Pan8816 supplemented with xylanase had higher feed intake, weight gain, and better feed conversion ratio (FCR) than those given diets composed of sorghum variety Pan8625 without xylanase addition at 1-7 days of age. Crude protein digestibility of Pan8625, a tannin variety not supplemented with xylanase, was lower than that with xylanase. Enzyme inclusion significantly increased the crude protein digestibility.

In Chapter 5 of this thesis the response of broiler chickens to whole sorghum inclusion and feed form were evaluated. Body weight and feed intake were measured on a pen basis at 10, 25, and 35 days of age and feed conversion ratio (FCR) calculated. Pellet diets affected feed intake, body weight and carcass parts of broiler chickens aged 1-35 days. Heavier relative gizzard weights with lower pH were recorded for broiler chickens offered mash diets at ages 1-35 days. FCR at 1-35 days increased with an increase in WS and levelled off with higher inclusion rates. Relative gizzard weights at 35 days marginally increased with the increase in whole sorghum inclusion levels. Similarly, relative bursa and liver weight at 35 days increased with the increase in WS inclusion. Overall, the results showed that pelleted diets were superior to mash diets. Although higher levels of WS inclusions enhanced the gizzard development, performance parameters of birds offered these levels were not affected.

The influence of age of introducing whole sorghum grain and xylanase supplementation to broiler chicken diets was evaluated in this thesis (Chapter 6). A whole sorghum inclusion level of 50 % with or without xylanase was offered to birds at different ages. Gross performance parameters on a pen basis at 10, 24, and 35 days of age were measured. Age of introduction had improved feed intake and body weight of broiler chickens at ages 1-10 and 1-35 days. Chickens offered whole grain from hatch had higher feed intake and body weight at 1-10 days. The relative visceral organ weights, meat parts yield, meat colour and pH were not influenced by age of WSG introduction or xylanase supplementation. Apparent metabolizable energy (AME) of diets and of the sorghum was improved by age of introduction. Birds to which whole grain was introduced from hatch or day 11 had significantly higher AME than birds not receiving whole grain inclusion. Age of introduction had an effect on gross performance of broiler chickens. Feeding whole grain as early as hatch marginally improved body weight and feed intake of broiler chickens.

The main finding in this thesis is that non-tannin sorghums fed to broiler chickens are superior to the tannin-containing varieties although the relatively high anti-oxidants activities of the latter may have health-promoting benefits. Feed form affected the performance of broiler chickens offered pelleted diets, resulting in better performance than mash diets. Whole sorghum grain inclusion level did not affect feed intake and body weight gain of broiler chickens; however, feed efficiency increased with an increase in whole sorghum. This indicates that WSG can be added to broiler diets at any inclusion rate without negatively affecting performance.