Oocyte cryopreservation represents one of the most recent developments in the field of reproductive technologies. However, despite of significant progress, the efficiency of oocyte cryopreservation is still very low. Cryopreservation of mature metaphase II (MIl) oocytes has been reported to induce disorganization of the meiotic spindle and chromosome damage. However, cryopreservation of immature oocyte at germinal vesicle (GV) stage may provide an alternative which avoids these problems. Slow freezing protocols have more recently been replaced by vitrification approaches. In this thesis, recovery, viability and subsequent developmental potential following in vitro fertilisation (IVF), parthenogenetic activation or somatic cell nuclear transfer (SCNT) of ovine oocytes vitrified at GV stage and matured in vitro were studied. Solid surface vitrification (SSV) and cryoloop technologies share the advantages of using a containerless system and small volumes of solution (less than t J.ll) which favours rapid cooling. Maturation, fertilisation, cleavage and blastocyst development were significantly decreased in SSV vitrified oocytes as compared to controls. Following cryoloop vitrification, frequencies of in vitro maturation (43.4 vs 63.2%), oocytes with normal spindle and chromosome configuration (50.0 vs 70.4%) and fertilisation (54.0 vs 74. t %) did not differ significantly between vitrified and control oocytes. Numbers of cleaved embryos that developed to the blastocyst stage following IVM/IVF/IVC did not differ significantly between vitrified and control groups (29.4 vs 45.1 %). In vitro matured ovine oocytes vitrified at GV stage using cryoloop were activated by two different protocols (I) a combination of calcium ionophore (A 23187), cycloheximide and cytochalasin 13 (CA+CHX/CI3), (2) strontium and CB (Sr/Cll). No blastocysts developed in vitrified oocytes activated by CA+CHX/CB; however, 3.8% were obtained following Sr/CI3 activation. Developmental competence of ovinc oocytes vitrified at GV stage and used as cytoplast recipients for SCNT was evaluated. Although the frequencies of cleaved embryos were significantly decreased in vitrified oocytes as compared to control, development to morula and blastocyst stage embryos was not significantly different. No significant differences were observed in total cell numbers, number of apoptotic nuclei as detected by Hoechst and TUNEL assay and proportions of diploid embryos in day 7 blastocysts produced following IVF or seNT of vitrified oocytes as compared to control. Pre-treatment of ovine GV-oocytes with cytochalasin 13 (7.5 J.lglml for 60 min) or demecolcine (0.1 flg/ml for 20 min) prior to vitrification improved frequencies of maturation, fertilisation and subsequent development following IVF or parthenogenetic activation. Caffeine treatment during IVM (10 mM for 6 h) increased the frequencies of blastocyst development in vitrified/thawed GV ovine oocytes. Taken together, these studies suggest that, ovine oocytes vitrified at GV stage can be matured, fertilised and develop in vitro to blastocyst stage embryos. Cryoloop vitrification resulted in higher maturation, fertilisation and subsequent development as compared to SSV. Strontium can be used effectively for parthenogenetic activation of vitrified/thawed ovine GV oocytes. Ovine oocytes vitrified at GV stage can be used effectively as cytoplast recipients for SCNT.

Salmonella enterica subspecies enterica (S. enterica) infection remains a global problem in a wide range of animals and in man. Poultry-derived food is a common source of human infection with the non-host-adapted Salmonella strains while fowl typhoid and pullorum disease are serious diseases in poultry. Development of novel immune-based control strategies against Salmonella infection necessitates a better understanding of the host-pathogen interactions at the cellular level. This study characterizes, in vitro and in vivo, the immune responses that develop following infection of avian species with typhoid and non-typhoid Salmonella serotypes. Salmonella serovars Typhimurium, Enteritidis, Hadar and Infantis showed a greater level of invasion and/or uptake characters to both chicken macrophages (HD11) and chicken kidney epithelial cells (CKC), when compared with S. Pullorum or S. Gallinarum. Nitrate and reactive oxygen species were greater in Salmonella-infected HD11 cells compared with the non-infected controls. HD11 cells revealed higher mRNA gene expression for CXCLi2 (IL-8), IL-6 and iNOS genes in response to S. Enteritidis infection when compared to S. Pullorum-infected cells. S. Typhimurium- and S. Hadar-infected HD11 showed higher gene expression for CXCLi2 versus S. Pullorum-infected cells. Higher mRNA gene expression levels of pro-inflammatory cytokine IL-6, chemokines CXCLi1 (K60) and CXCLi2 and iNOS genes were detected in S. Typhimurium- and S. Enteritidis-infected CKC followed by S. Hadar and S. Infantis while no significant changes were observed in S. Pullorum or S. Gallinarum-infected CKC. Epithelial cell response and production of pro-inflammatory cytokines and chemokines were greatly influenced by Salmonella virulence markers, including Salmonella pathogenicity island type-1 (SPI-1), SPI-2 and bacterial flagella. In chicken infections, S. Enteritidis and S. Infantis colonized the caeca more efficiently than S. Gallinarum and S. Pullorum. High numbers of B-lymphocytes and macrophages were observed in the caecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds elicited the expression of CXCLi1 and CXCLi2 chemokines in the caecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the caecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes. In conclusion, data from the present study provide further insights on the interaction of Salmonella with poultry, and while both S. Typhimurium and S. Enteritidis are strong inflammatory serotypes, S. Pullorum and S. Gallinarum are not.

Chicken meat is an important source of high quality protein in the diet of most people in the world. Consequently, the increasing demand for this meat has made chicken meat production the most important growth sector among other meat species. This has been achieved by a half century of intensive genetic selection for growth traits; however, this was coupled with poor reproductive performance of broiler breeders. Ross 708 represents a broiler breeder strain that has been developed for breast-meat yield, and has been reported to exhibit poor reproductive performance in comparison to Ross 308, a typical broiler breeder strain. Accordingly, the current study investigated key points involved in the reproductive process that might influence variation in reproductive performance. Ovarian follicles number was the first point to investigate, as they are the main material of the egg. Liver fatty acid profiles were also investigated in order to identify lipid metabolism and the efficiency of dietary fat utilisation, as the liver is the main site that supplies different body tissues with fatty acids. Carcass fat content was also examined as its negative relationship with reproduction is well documented. Finally, the content of calcium in the tibia bone was examined to identify whether variation in egg production was associated with differences in the metabolism of this element. Accordingly, broiler breeder females from Ross 308 and 708 strains, reared under the standard production system on two different commercial farms of PD Hook, were collected throughout the reproductive cycle; starting at 25-week-old and in five weeks interval until 55-weeks of age. Findings showed no difference in the number of both the large yellow follicles (P=0.332), and the small yellow follicles (P=0.134); whereas the number of large white follicles was higher in the 708 ovaries (P=0.005). Differences in lipid metabolism were identified with a strong tendency for the 708s towards having lower content of linoleic acid (P=0.056) in addition to significantly lower a-linolenic acid (P=0.005). Of particular importance is the latter fatty acid as it is the precursor to (n-3) fatty acids, some of which were found to be less (P<0.001) in the 708s including docosapentaenoic (DPA) and docosahexaenoic (DHA). The importance of these fatty acids in follicular maturation is well documented in addition to the importance of the linoleic acid; these findings indicate that the 708s were not receiving adequate levels of the essential fatty acids which might have contributed to their poor reproductive performance. The 708s also laid down significantly more fat (P<0.001) in comparison to the 308s which could be another factor that has impaired their reproduction performance. This could indicate different levels of metabolic hormones which, in turn, have been found to act in concert with the reproductive hormones. 708s also exhibited a trend towards lower content of calcium in their tibiae, with an age by strain interaction and thus suggesting a difference in the metabolism of this element at some ages. The current study has addressed changes of the investigated parameters with age, but the effect of genetic selection on reproductive performance was difficult to address. Rather, some physiological differences have been identified; 708s were found to be receiving inadequate amounts of essential fatty acids, calcium content was found to be less at some ages and they exhibited a higher content of carcass fat. All these factors have the potential to contribute to poor reproductive performance, and once they are taken into consideration better assessment for the effect of the continued genetic selection for more growth traits can investigated.

Bambara groundnut is an indigenous African legume grown mainly in sub-Saharan Africa; it is an important source of protein to the rural majority. There are no established varieties and subsistence farmers grow locally adapted landraces which are generally low yielding. Bambara groundnut is a predominantly self-pollinating crop and is expected to exist as non-identical inbred lines, although the previous lack of co-dominant markers has prevented a formal assessment of heterozygosity within bambara groundnut genotypes. A total set of 75 microsatellites that were characterised in this study were used to investigate the genetic diversity of a set of 24 bambara groundnut landraces, to provide an evaluation of the markers for polymorphism and provide a link with DArT marker data that were previously analysed. Sixty eight microsatellites were identified that were found to be consistent and reproducible, from which a set of markers were selected and used for genetic variability studies of bambara groundnut, to compare the use of molecular markers with morphological markers, and to investigate using SSR markers in pure line selection. The genetic diversity of bambara groundnut was assessed based on morphological characters for two seasons; in a glasshouse experiment at the University of Nottingham, Sutton Bonington Campus, UK and in a field experiment that was conducted at the Botswana College of Agriculture (Notwane farm), Gaborone in a randomised block design with three replicates. The landraces were characterised for 24 quantitative and 13 qualitative characters. The results indicated considerable variation for quantitative characters, while significant morphological differences were also recorded for most characters. Multivariate data analysis was conducted using principal component analysis, cluster analysis and heritability estimates were developed. The low cost, simplicity and agricultural relevance of morphological characterisation makes it an important tool in germplasm genetic variation studies. Thirty four lines from field experiments were investigated for genetic diversity based on 20 microsatellites. The expected heterozygosity (He) had an average of 1 in agreement with the fact that bambara groundnut is predominantly self-pollinating. Both cluster analysis and principle component analysis (PCoA) grouped landraces based mainly on their areas of origin. A thorough molecular analysis of genetic and morphological variation in bambara groundnut was conducted to investigate the relationship between the two assessment techniques. This comparison will assist in breeders making informed decisions as to which approach is best to use in germplasm characterisation and plant breeding and how best to apply such knowledge in practical situations. DNA markers could then aid with the selection of germplasm for breeding, quality control within breeding programmes and, potentially, direct selection via Marker Assisted Selection (MAS). Euclidean distance estimates for morphological data and (Nei’s 1972) genetic distance estimates for SSR data were strongly correlated (r = 0.7; P < 0.001) in the agronomy bay and (r = 0.6; P< 0.001) in the controlled growth room. These results suggest the two approaches are generating the same pattern of genetic diversity, and as such can be used as a surrogate for each other.

As a major food source potato delivers significant levels of minerals to the human diet. The aim of this study was to understand the control over the mineral concentrations found in tubers. The three-dimensional patterns of mineral distribution in tubers give clues to the processes leading to storage in the tuber. Within the tuber flesh, calcium and phosphorus content decreased towards the centre of the tuber (on FW basis). The elements iron, magnesium, zinc, manganese, sulphur and chlorine were higher at the stem end, while potassium was higher at the bud end. Remobilisation of minerals within the tuber was evident after six months of cold storage. Mineral variation was explored in potato germplasm. Three diverse germplasm collections, the Commonwealth Potato Collection, the Phureja and Tuberosum Core Collection and the Neotuberosum Population demonstrated wide variation for tuber mineral concentrations, an interaction with tuber yield and, on multivariate analysis, consistent parallels between some minerals suggesting unsuspected shared processes affecting their concentrations. The 12601ab1 x Stirling tetraploid mapping population was used to identify QTls for tuber mineral concentration using REML analysis to account for local field variation. Transgressive segregation for tuber mineral concentrations was detected. The genetic map for this population was extended using DArT markers and QTLs were identified on all 12 linkage groups for all minerals studied. Two bulk segregant analyses were performed to add precision to the QTL analysis. One approach identified candidate genes on the potato genome sequence and used nearby SSRs to seek association in the tetraploid mapping population. A second approach used the variation present in the highly diverse Neotuberosum Population to identify DArT markers which were associated with the tails of the distribution of minerals. Using the latter approach, single superscaffolds containing candidate loci and trait-associated DArT markers could be aligned with a small part of mapping population QTLs, providing additional resolution.

Date palm (Phoenix dactylifera L.) is a diploid with 18 pairs of chromosomes and an estimated genome size of 658 Mb. It is a dioecious perennial monocot, with a long generation time (a period of 4-5 years until first flowering). Date palm is one of the major fruit crops grown in the Gulf countries and particularly in the Sultanate of Oman. Approximately 250 varieties of date palm are recorded throughout the country with evaluation and characterization based on morphological and reproductive traits (e.g. fruit color, fruit shape and fruit weight). Limited molecular characterization work has been undertaken for date palm germplasm in general and Omani date palm germplasm, in particular. The principal focus of this study was to: investigate the genetic diversity of Omani date palm germplasm and compare it with 'exotic' germplasm, to differentiate between female and male plants at the molecular level and to construct an initial genetic map for date palm. Samples were taken from eight parents of the available Omani date palm controlled crosses (Khalas 4, Khalas 13 male, Um-Alsela, Khori male, Bami, Naghal, Bahlani male, and Khasab) with 90 date palms from the BC1 and F1 populations, from 194 Omani date palm accessions (151 female cultivars and 43 male trees), together with samples from Italy (Sanremo and Bordighera), USDA-ARS, France, Iraq, Libya, Sudan and Iran. The F-statistics analysis showed that the genetic variation between female and male accessions based on random markers was only 2.1 %, while within the broader group of Omani female and male accessions the molecular variation was 97%, suggesting that the Omani female and male accessions have little consistent divergence, compared to the large-scale divergence within Omani germplasm, so male palm have been derived from most genetic origins in Oman. Additionally, the Principal Coordinates Analysis (PCA) and bootstrap consensus phenetic tree showed that the Omani accessions were closely related to each other and there was no clear genetic differentiation between female and male cultivars. A high degree of genetic variation was observed between germplasm from Oman, Italy, USDA-ARS, France, Iraq, Libya, Sudan and Iran as measured by Fst (19.7 %). The PCA showed that the Europe-Africa (Italy, France, Libya and Sudan) accessions are distinguished from West-Asia (Oman, Iraq and Iran) accessions and have their own autochthonous origin, a finding which was strongly validated by bootstrap consensus tree test. A medium density genetic map in date palm was constructed using 53 individuals from BC1 and 30 individuals from F1 populations. The BC1 map consisted of 270 markers (28 SSR and 242 SNP) distributed into 29 linkage groups with total genetic length of 1.486.7 cM, while the F1 map consisted of 591 markers (21 SSR and 570 SNP) distributed into 30 linkage groups with total genetic length of 2,385.6 cM. A total of 25 combined linkage groups were possible by combining both BC1 and F1 maps through common markers. A sex-link marker locus was developed and found to predict a high level of discrimination between male and female date palms among multiple varieties distributed across the wide range of cultivation, with an accuracy of 100% in the Omani crosses, 96% in the broad Omani material and 86% in the broadest date palm germplasm. This marker was also mapped in both BC1 and F1 at 42.8 cM and 4.9 cM in linkage groups 18 and 29, respectively and on combined group 19 at 42.8cM.

This thesis presents laboratory investigations on the competitive interactions which take place within and between bethylid parasitoids. Part one investigates the compatibility of three bethylids (Cephalonomia hyalinipennis, Cephalononlia stephanoderis and Prorops nasuta) for biocontrol releases against the principal pest of coffee, the coffee berry borer (CBB), Hypothenemus hampei. Cephalonomia hyalinipennis is able to hyperparasitise and consume pupae of C stephanoderis and P. nasuta. Cephalonomia stephanoderis also engages in intra-guild predation, consuming pupae of C hyalinipennis. In contests for CBB hosts, fatal fighting occurs in 69% of inter-specific replicates but never occurs in intra-specific replicates. This suggests that interspecific competition is stronger than intraspecific competition and that species coexistence may be compromised. Cephalonomia tephanoderis is the superior interspecific contestant while P. nasuta is the least successful and never kills an opponent. Where CBB infested coffee berries are provided to the three bethylids, coexistence between species is possible, but rare, within a single coffee berry. Prorops nasuta is the most successful species in interspecific replicates and replicates containing C. hyalinipennis generally have low production, regardless of the species combination added. Part two investigates contest interactions, the variables that influence contest outcome between Goniozus nephantidis females and chemical release. Prior ownership and difference in contestant weight have positive influences on contest outcome. Host weight positively influences the outcome of contests between two 'owners' and 'intruder' take-over success increases when intruders are older than owners. Seven bethylid species are found to release volatile chemicals when stressed. A pilot study identifies the volatile chemical in G. nephantidis and employs Atmospheric Pressure Chemical Ionisation-Mass Spectrometry for real-time analysis of chemical release during contest interactions. The appendix contains an advanced investigation using this technique. Bethylids are useful model organisms for the study of competitive interactions but appear to be generally ineffective as biological control agents.

Human epidemiological studies have indentified obesity as an independent risk factor for renal disease. In addition, maternal nutrient restriction (MNR) during gestation results in a series of adaptations that may predispose those offspring to obesity and hypertension. Recent reports demonstrated that obese sheep exposed to MNR during early to mid gestation have a predisposition to ectopic lipid deposition in the heart and a rise in necrotic adipocytes, which are markers of severe metabolic dysfunction. Surprisingly, in this model of MNR, the renal tissue of those offspring showed an apparent reduction in cell apoptosis. However, renal diseases associated with obesity have a slow progression and their mechanisms are not completely understood. In the light of these results, the main hypothesis of my thesis is that the renal amelioration observed in those nutrient restricted (NR) obese offspring is a product of post-injury responses, inducing scarring and others adaptations to obesity. Therefore, some of the main regulatory factors in renal and perirenal adipose tissue (PAT) development were analysed in seven-day-old and one-year-old obese sheep offspring exposed to MNR (3.5 KJ/days) from 30 to 80 gestational days (term ≈ 145 days). At one week of age, the renal composition and gene expression showed small changes between NR offspring and those born to control mothers. However, in PAT of NR offspring, an increased in expression of the methyltransferase DNMT-1 and a decrease in mRNA abundance of IGF-2 were observed. At six months of age, obesity onset was accompanied by raised plasma cortisol and leptin concentration in NR offspring compared to control. By one year of age, whilst plasma leptin concentration was similar between the obese groups, in the PAT of the NR offspring there was an increase in gene expression of pro-inflammatory factors and DNMT-1, suggesting advanced adipose tissue remodelling. In kidney, regardless of in utero diet, obesity induced similar amounts of oxidative stress, activation of cellular proliferative factors and collagen deposition. Although, both obese groups had equal activation of pro-apoptotic factors (e.g p-53 and Bax), renal iron and mRNA abundance of the death receptor, Fas only increased in obese offspring born to control fed mothers. A major finding in the NR kidney was increased ectopic triglyceride deposition, indicating early onset post-injury in response to sympathetic activation and lipotoxity. The main conclusion of my thesis is that functional changes observed in the adipose tissue lead the kidney to an initial cycle of cell proliferation, apoptosis and arrest, followed by tissue remodelling, characterised by the presence of collagen. However, this adaptation to obesity is accompanied by an increase in lipid deposition in the kidney of the NR group that may be a sign of an advanced state of metabolic dysfunction.

Wheat (Triticum aestivum L.) is the most widely grown of any crop and provides one-fifth of the total calories of the world's population. Since the 1960s, increases in productivity have been achieved as a result of wide-scale adoption of Green Revolution technologies. However, in spite of growing demand, the challenges of increasing production to feed an estimated world population of 9 billion in 2050 are still considerable. Due to the increased demand, it is estimated that food production must be increased by about 50% by the year of 2050. Improving wheat productivity through developing cultivars with high yield potential and with high adaptability to specific environments is the key objective in the wheat breeding programs worldwide to fill the gap between the production and the demand. The overall aims of the present study were to: (i) investigate the physiological basis of yield potential progress from 1966 to 2009 in spring bread wheats released at the International Center for Maize and Wheat Improvement (CIMMYT) in the irrigated high potential environment of NW Mexico, (ii) investigate the physiological basis of effects of the tiller inhibition Tin1A gene on ear-fertility traits and yield potential and interactions with plant density in NW Mexico and UK environments in lines of a doubled-haploid (DH) population segregating for Tin1A/non-Tin1A alleles and (iii) identify breeding targets for new cultivars with higher yield potential. Four experiments were conducted in NW Mexico at the CIMMYT research station at Ciudad Obregon. Two of these experiments studied a set of 12 historic CIMMYT spring wheat cultivars released from 1966 to 2009 in 2008/9 and 2009/10. The other two experiments examined selected lines from a doubled-haploid (DH) population derived from a cross between CIMMYT spring wheat L14 and UK winter wheat Rialto contrasting for the presence/absence of the TinlA allele for tiller inhibition and their interaction with seed rate in 2008/9 and 2009/10. In addition, two other field experiments were conducted in the UK, one in 2008/09 at KWS UK Ltd in Thriplow, Hertfordshire and one in 2009110 at the University of Nottingham Farm, Sutton Bonington campus, Leicestershire. The plant material for both of these experiments was selected lines from the CIMMYT spring wheat advanced line Ll4 (+Tin1A allele) x UK winter wheat Rialto (-TinlA allele) DH population and the Rialto parent. In the experiment at Thriplow in 2008/09 the DH lines were examined at one seed rate and in the experiment at Sutton Bonington in 2009/10 at two seed rates. At the CIMMYT site in 2008/9 and 2009/10, a randomized complete block design was implemented with four replications for the experiments examining the CIMMYT wheat historic releases and a split-plot randomised complete block design with three replications was implemented for the experiments examining the +/- Tin1A DH lines, with three seed densities (50, 150 and 450 seeds per square metre); seed rates were randomized on main plots and eight genotypes randomized on sub-plots. At the UK site, in the KWS experiment, 24 DH lines (12+Tin1A allele) and (12-TinlA allele) from the L14 x Rialto population were used. There was only one seed rate (300 seeds m-2) and a completely randomised design in three replicates was implemented. The same 24 DH lines were examined in the experiment at the SB site, at two seed rates (40 and 320 seeds m-2) in a split plot randomised complete block design in three replicates. Seed rate was randomized on main plots and DH lines were randomized on sub-plots. In all experiments examining the DH lines of the Ll4 x Rialto population, lines were selected in pairs so that the two groups of +Tin1A and -Tin lA lines were approximately balanced for flowering time and plant height, i.e. every +TinlA line has a non-Tin1A pair with similar height and flowering date. Plots were sampled for destructive measurements of dry weight and DM partitioning and ear-fertility traits at four stages in the historic experiments at (GS3l, GS39, GS61+7d and at maturity) and at two stages in the DH population experiments (GS61+7d and at maturity). The water soluble carbohydrate (WSC) content of the stems plus attached leaf sheaths was also measured at GS61+7d and at maturity. In the historic experiments, at GS 61+14 days, a degraining treatment was implemented by removing all spikelets from one side of the ear (i.e. ca. 50% of the spikletes) in the histories experiment. Non-destructive measurements were taken for stomatal conductance, canopy temperature, fractional photosynthetically active radiation (PAR) interception and normalized difference vegetative index at various dates both pre- and post-anthesis in the historic experiments. In the experiments examining the set of 12 historic CIMMYT spring wheat releases, results showed that from 1966 to 2009 the linear rate of genetic gain in yield potential was 32 ha-1 yr-1 (0.59 % yr-1) (r = 0.76. P = 0.01). Yield progress was primarily associated with harvest index (percentage above-ground DM as grain DM) in the period from 1966 until about 1990 increasing from 43% to 49%, but deceased with year of release thereafter. A non-linear genetic gain in AGDM was evident over the 43-yr period with AGDM increasing from about 1990 from which point it increased rapidly to 2009. There was no association between genetic progress in grain yield and grain number per m2 in this set of 1 cultivars; a small increase in ears per m2 was counteracted by a decrease in grains per ear. However, grain weight tracked the improvement in yield potential over the 43-year period with a linear increase of 0.23 mg yr-1. No change was found in rachis length with plant breeding; however, number of fertile spikelets per ear decreased since about 1990 and was associated with the decrease in grains per ear. There were statistically significant differences in above-ground DM production at all growth stages and a tendency to produce more biomass during the GS31 to GS61+7d phase with year of release. No differences amongst cultivars were found in the amount of radiation intercepted by the whole canopy from GS3l to GS6l+7 days. Although not conclusive, since Bacanora was an exception to the trend and radiation-use efficiency (above-ground biomass per unit PAR interception; RUE), there was a tendency for RUE to increase with year of release which was consistent with a positive association with crop growth rate (above-ground DM per m2 per day; CGR) and the trend for an increase in biomass accumulation during the stem-elongation phase with plant breeding. Although there was a trend for an increase in biomass accumulation from GS31 GS61+7d this was counteracted by a decrease in ear DM partitioning so that ear DM per m2 at GS61+7d and grains per m2 did not change with plant breeding. Results showed that the improvement in the individual grain weight from 1966 to 2009 in this set of cultivars was associated with improvements in the grain filling rate from 1966 to ca. 1990 and in the duration of grain filling from ca. 1992 to 2009. Averaging across years, there was a significant positive association between post-flowering canopy-temperature depression and grain yield. Fractional PAR interception by the canopy layers of the ear, flag leaf and the penultimate leaf was increased with year of release since about 1990. This increase in the fractional interception of PAR correlated significantly with the grain weight and grain yield amongst the 12 cultivars. Grain growth of the cultivars in this historic set was generally sink limited rather than source limited. There was no change in source-sink balance as indicted by grain growth responses to the degraining treatment with year of release.

BACKGROUND Chronic respiratory inflammation is a major cause of recurrent airway obstruction (RAO) in mature horses. RAO has aetiological and clinical similarities to human asthma. Remodelling of airway tissue after bronchial inflammation is evident in RAO and human asthma. Severe asthma in humans is associated with defective lipoxin A4 (LXA4) synthesis and abnormal expression of specific lipoxin receptor (ALX). Arachidonic acid metabolite LXA4 modulates acute inflammation in a number of species and models of acute inflammation. Dysfunctional LXA4 synthesis and/or expression of the ALX receptor may contribute to the chronic inflammatory response seen in asthma. Abnormal LXA4 and/or ALX expression may also be present in horses with RAO thereby promoting airway remodelling. HYPOTHESIS Equine inflammation and its resolution can be modelled in vitro using respiratory epithelial and smooth muscle models. AIMS To develop an in vitro equine respiratory model of respiratory epithelium and airway smooth muscle. Characterise the response of this model to exogenous lipolysaccharide (LPS) and lipoxin A4 on selected molecules associated with inflammation and inflammatory resolution. METHODS Primary equine tracheal epithelial (ETE) cells were obtained from trypsindissociation of tracheal epithelial tissue derived from healthy horses with no sign of inflammation at post-mortem. Primary airway smooth muscle (ASM) cells were cultured from explants of equine trachealis muscle. Near confluent (70-75%) ETE and ASM cells were stimulated with 0.1, 10 and 100µg/ml LPS at 4 and 24 hrs (ETE cells) or 12, 24 and 72hrs (ASM cells). Expression of COX-2 mANA in these cells was used to determine a suitable time point and LPS concentration to induce inflammation. Inflammatory resolution was then examined by comparing the selected time points and LPS concentrations with the response of ETE and ASM cells to 15 minutes incubation with 100µM LXA4 and LXA4/LPS treatment. Finally, the inflammatory relationship between the epithelium and smooth muscle layer was examined by a co-culture model of ETE and ASM cells. Conditioned media from ETE cells treated with 0.1µg/ml LPS, 100µM LXA4 and LXA4/LPS treatment for 24hrs was used to culture ASM cells for a further 24hrs. To examine inflammation and its resolution, selected genes, namely ALX, toll-like receptor 4 (TLA-4), tumour-necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were amplified and quantified by real time PCR. ALX and COX-2 proteins were monitored by Western blot and nitric oxide (NO) levels measured by fluorometric analysis. Values for statistical analysis of ETE and ASM cells were obtained with a two and one-way ANOVA and Tukey's multiple comparisons test. RESULTS Primary ETE and ASM cells were identified by positive immunocytochemical staining with pan cytokeratin-26 and alpha-smooth muscle actin respectively. Treatment of ETE cells with 0.1µg/ml LPS for 24hrs increased iNOS and COX-2 mRNA levels, and significantly increased mRNA expression for ALX, TLR-4 TNF-α and IL-1 j β mRNA (p values <0.05). ASM cells incubated over 72hrs incubation with 0.1119/ml LPS showed increased expression of selected genes but only significant increases in COX-2 mRNA were observed (p values <0.05). Incubation of ETE and ASM cells with LXA4 did not significantly increase or inhibit inflammation as measured by real time PCR, Western blot and fluorometric anlaysis. Western blotting showed some inhibition of COX-2 protein in ASM cells but not ETE cells at 72hrs after LXA4 treatment. Fluorometric analysis of NO levels in ETE and ASM cells showed no significant difference after treatment in either cell type. No noticeable evidence of inflammation or inhibition of inflammation was observed in the co-culture model of ETE and ASM cells. CONCLUSION It was concluded that an in vitro equine model of respiratory epithelium and smooth muscle was successfully established. It was possible to induce partial inflammation in ETE and ASM cells but inflammatory resolution could not be definitively shown in either cell type.