Silvopasture is the intentional integration of trees with forages and livestock. In Blackstone, Virginia a silvopasture management plan was created in a thinned, timber stand seeded with a cool-season forage mixture. Treatment pastures for this study included an open pasture, a thinned pine silvopasture, a thinned hardwood silvopasture, and a cleared and replanted new pine silvopasture. Cattle were introduced in 2017 and rotationally stocked within each pasture according to forage availability. Objectives were to determine the forage availability, forage nutritive value, and the performance of heifers in silvopasture and open pasture systems. Additionally, the new pine silvopasture was grazed to determine the effect of cattle on tree seedlings without protection. Forage availability was affected by date and year and was significantly lower in 2018 (3560 kg ha-1) versus 2017 (5350 kg ha-1). Pre-grazing forage availability was lowest in the pine and hardwood silvopastures in both years (4500 kg ha-1) compared to the open pastures (4920 kg ha-1; p < 0.0001). Date significantly influenced nutritive value, but only had date by treatment interaction in the 2017 grass crude protein and neutral detergent fiber sample. In 2017, the new silvopasture (61% TDN) had greater total digestible nutrients as compared to the open pasture and thinned hardwood silvopasture (57% TDN; p = 0.0292); there was no significant difference (p=0.3733) in total digestible nutrients in 2018 between pastures (58% TDN). In 2017, average daily gains of the heifers were greatest in the silvopastures in June (p = 0.0346). In 2018, average daily gain was lowest among silvopastures later in the summer compared to open pastures and new silvopastures (pJuly = 0.0051; pAugust = 0.0008). Remote temperature loggers were used to collect vaginal temperatures of the heifers over eight days in 2018. Silvopasture heifers had an average core temperature of 39.4 °C from 2-5 PM while heifers in the open pastures had an average temperature of 40.0 °C. A drone with a thermal camera was used to collect external hide temperatures in the morning and afternoon. Heifers in the silvopastures had lower heat loads in the afternoon while animals without shade experienced a 65% greater temperature increase between morning and afternoon when compared to the shaded animals. Silvopastures provide an opportunity to improve the welfare of grazing livestock in the summer, while improving the overall productivity and efficiency of land. Tree seedlings that were planted into pasture to create a new silvopasture experienced a 16% mortality rate while over 75% of tree seedlings had less than 50% damage after two years and would continue to produce trees with future economic and shade value. Future research should focus on how to implement silvopasture as part of a holistic grazing and management plan while continuing to evaluate cattle, forage, and tree response to silvopastures over multiple years. / Master of Science / Silvopasture is the intentional integration of trees with forages and livestock. In Blackstone, Virginia a silvopasture management plan was created in a thinned, timber stand seeded with a cool-season forage mixture. Treatment pastures for this study included an open pasture, a thinned pine silvopasture, a thinned hardwood silvopasture, and a cleared and replanted new pine silvopasture. Cattle were introduced in 2017 and rotationally stocked within each pasture according to forage availability. Objectives were to determine the forage availability, forage nutritive value, and the performance of heifers in silvopasture and open pasture systems. Additionally, the new pine silvopasture was grazed to determine the effect of cattle on tree seedlings without protection. Forage availability was lowest in the pine and hardwood silvopastures in both years compared to the open pastures. Nutritive value of forages was most greatly influenced by time rather than tree presence. In 2017, average daily gains of the heifers were greatest in the silvopastures in June. In 2018, average daily gain was lowest among silvopastures later in the summer compared to open pastures and new silvopastures. Internal and external body temperatures of heifers during the afternoon decreased with increasing shade availability with the greatest internal body temperatures occurring in unshaded animals. Silvopastures provide an opportunity to improve the welfare of grazing livestock in the summer, while improving the overall productivity and efficiency of land. Tree seedlings experienced minimal damage by cattle and would continue to produce trees with future economic and shade value. Future research should focus on how to implement silvopasture as part of a holistic grazing and management plan while continuing to evaluate cattle, forage, and tree response to silvopastures over multiple years.

Heat stress and immune challenge are costly issues to the swine industry causing significant loss in production and health including reduced efficiency in muscle accretion and energy utilization. Alterations to metabolism and immune response may participate in these shortcomings. The study objectives were to examine the metabolic profiles and immune status of swine subjected to a dual challenge of thermal stress and porcine reproductive and respiratory syndrome virus (PRRSV). To determine this, pigs were subjected to four treatments: thermo-neutral (22° C; TN), thermo-neutral PRRSV infected (TP), heat stress (HS), and heat stress PRRSV infected (HP), during two experimental phases. The first phase consisted of infecting half the experimental group with PRRSV while the rest remained infection free in thermo-neutral conditions. A second phase further divided infected and non-infected into heated conditions for three days of constant heat (35° C) or TN conditions. Venous blood was collected prior to each phase and before sacrifice to analyze for metabolites. At sacrifice liver and longissimus dorsi skeletal muscle samples were collected for gene expression analysis. Pigs in challenged conditions had increased body temperatures, reduced feed intake, and lighter body weights compared to controls, with greatest detriment to dual challenged pigs. In addition, challenged pigs had increased markers of muscle degradation. In challenged pigs, differences (p<0.05) were observed in the metabolic and cytokine gene expression profiles suggesting heat stress blunts the immune response of viral infection in muscle and liver. In conclusion, heat stress and immune challenge directly and indirectly affect metabolism and cytokine expression and both variables may contribute to decreased growth parameters. / Master of Science

Heat stress is detrimental across many, if not most aspects of animal agriculture. Heat stress can have direct effects on the animal, effects on their environment and effects on the feedstuffs they consume. The work presented here will address direct effects on the animal (using dairy cattle) and effects on their feed (using pigs). Dairy cattle were chosen to study direct effects on the animal because they exhibit unique metabolic adaptions during heat stress. One adaptation of interest is a heat-stress induced state of hyperinsulinemia and hypoglycemia while self-limiting feed intake. In regards to effects on feedstuffs, high ambient temperatures are associated with elevated concentrations of mycotoxins in cereal grains. Mycotoxins are problematic because, at sufficient concentrations, they induce specific, toxic effects. Zearalenone is one such mycotoxin that is particularly problematic for swine, as it is a potent phytoestrogen. As such, it can reduce productivity by disrupting reproductive processes. Although these topics are disparate, the overarching goal of this work is to improve agricultural animal productivity during periods of heat stress. Overall, the three objectives of this thesis were to: 1) isolate the production-related effects of hyperinsulinemia with hypoglycemia from heat stress, 2) determine whether glucose supplementation during heat stress could improve or rescue milk production during heat stress, and finally, 3) investigate reproductive tract morphology following different durations of zearalenone consumption in peri-pubertal gilts. The first experiment compared the effects of thermoneutral, thermoneutral + hyperinsulinemic-hypoglycemic clamp (HHC), environmental heat stress, and heat stress + euglycemic clamp (EC) on milk production, milk components, and circulating glucose concentrations. Milk production, as expected, did decrease under heat stress when compared to thermoneutral conditions. Milk production during the HHC was intermediate, which was likely the result of the induced hypoglycemia. Interestingly, the glucose infused during the EC did not improve milk production over heat stress. Baseline blood glucose concentrations declined during heat stress, and those concentrations measured during the thermoneutral period were correlated with the change in blood glucose (from thermoneutral to heat stress). In the second experiment, zearalenone (6 mg) was fed to peri-pubertal gilts for 0, 7 or 21 days. Gilts were monitored daily for observable signs of zearalenone toxicity, and tissues were harvested after 21 days of treatment. No differences in the lengths or weights of any portions of the reproductive tract were observed. Likewise, there were no differences in body weight, carcass weight, dressing percent or liver weight. In summary, heat stress has varied effects on livestock production and the ultimate outcomes are not always predictable. Future work will be aimed at defining the factors that contribute to this variability. / Master of Science / Heat stress can have direct and indirect consequences that affect the efficiency of livestock production. The overall objective of this work is to improve agricultural animal productivity during periods of heat stress. The first study compared milk production, milk characteristics and blood glucose concentrations in the presence or absence of heat stress. Aspects of productivity were also measured when insulin and/or glucose were administered in a manner that mimicked either heat stress or thermoneutral conditions. Heat stress did cause a decline in circulating blood glucose, feed intake, and milk production. Infusion of insulin during thermoneutral conditions caused a decline in milk production but not in feed intake. As a way to compensate for the naturally low circulating glucose during heat stress, glucose was infused during heat stress in the last portion of the experiment. Simply infusing glucose to return the animal to pre-heat stress concentrations did not improve milk production. Further work is needed to determine factors (other than feed intake) that limit milk production during heat stress. The second study investigated whether the mycotoxin, zearalenone, affects the size or weight of the pig's reproductive tract. If differences existed, they would be indicative of potential problems with the function of those tissues. Young female pigs consumed zearalenone for 0, 7 or 21 days and tissues were evaluated 21 days after the experiment began. No changes in tissue weights or lengths were observed. Future experiments should be aimed at determining the concentration and duration of exposure at which zearalenone becomes problematic for young, female pigs.

Heat stress (HS), a stress response in homeotherms mainly due to elevated ambient temperature and failure of effective heat dissipation, causes a substantial negative economic impact to livestock industry worldwide. Reduced feed intake, a typical phenomenon observed during HS, was thought to be the primary driver for the milk production loss. However, accumulating evidence indicates that HS influences animal metabolism and endocrine profiles independent of reduced feed intake. Previous studies comparing heat-stressed lactating cows with control group pair-fed (PF) to the intake of HS group but housed in thermoneutral conditions, in order to eliminate the confounding factors result from differentiated feed intakes, showed that HS increased circulating insulin and decreased plasma non-esterified fatty acid (NEFA) in lactating cow, the opposite responses typical of PF cohorts. Therefore, the present studies were performed in order to elucidate the mechanism(s) underlying these counterintuitive changes. In response to a glucose tolerance test (GTT), both HS and PF decreased whole body glucose disposal rate, a sign of insulin resistance. Only PF decreased skeletal muscle insulin sensitivity in terms of reduced protein kinase B (PKB/AKT) phosphorylation, a downstream protein of insulin receptor (IR), while HS group maintained similar intact insulin responsiveness in the liver and skeletal muscle as thermoneutral conditions. There was a global reduction in gene expression of the enzymes related to lipid metabolism in adipose tissue of heat-stressed cows. Similarly, β-adrenergic signaling, a major stimulator of lipid mobilization, was suppressed in terms of NEFA release response during a chronic epinephrine challenge in HS group. After the challenge, phosphorylations of cAMP-response element binding protein (CREB) and hormone sensitive lipase, both located downstream of β-adrenergic receptor, were decreased in HS, but not in thermoneutral conditions, another indicator of impaired adrenergic signaling. In contrast, IR and AKT phosphorylation were increased in HS conditions indicating insulin signaling may be elevated during HS in adipose. Collectively, HS reduces lipid mobilization and appears to favor glucose utilization via alterations of lipid metabolism and hormones signaling pathways. These unique alterations in HS might shed some light on developing counter-HS approaches in the future. / Ph. D.

The Virginia-Carolina (VC) region including Virginia, North Carolina, and South Carolina, is the most important peanut production region for the large seeded, virginia-type peanut in the United States.  In recent years, an increased frequency of heat and drought episodes with significant effects on peanut yield was observed in the VC region.  Because limited information is available on the mechanisms virginia-type peanut develops in response to heat and water stress, the present study evaluated several physiological and metabolic characteristics and their relationship with yield for eight cultivars and breeding lines.  Experiments were conducted under rainfed and irrigated field trials in 2011 and 2012, and in a growth chamber under optimum (30/25 "C) and high temperature (40/35 "C) conditions.  The long term goal of this study is to help development of more tolerant peanut cultivars to heat and drought in the VC region. Visible symptoms of water-deficit stress were observed in peanut during the field experiments in both years.  Significant (p d 0.05) variations for yield, membrane injury, chlorophyll fluorescence (Fv/Fm ratio), specific leaf area, SPAD chlorophyll content, and relative levels of polar and non-polar metabolites were observed in response to water regime, growth stage, and genotype in both years during the field studies.  Similarly each year, the Fv/Fm ratio, organic acids, and saturated fatty acids decreased in rainfed vs. irrigated plants, while the sugar and sugar alcohol relative levels increased.  Regardless the water regime, lower levels of saturated fatty acids and sugars, and higher levels of unsaturated fatty acids and sugar alcohols were associated (p < 0.05) with higher pod yield in field conditions.  Genotypes Phillips, SPT06-07, and N05006 showed potential tolerance and N04074FCT, CHAMPS, and Bailey susceptibility to water deficit in field studies.  Significant physiological and metabolic changes were also observed in response to heat stress under controlled conditions in peanut seedlings.  A general decrease in organic acid and saturated fatty acid levels and an increase in membrane injury, sugar, and unsaturated fatty acid levels were observed under both water deficit and heat stress conditions.  Overall, results from both experiments were suggestive of natural stress responses rather than adaptive mechanisms to water deficit and heat stress of the virginia-type genotypes used in this study. Among all genotypes, SPT 06-07 showed improved tolerance to both stresses.  Our results suggest that monitoring chlorophyll fluorescence and changes in the levels of selected metabolites can be used to screen new peanut lines for drought and heat stress tolerance. / Master of Science in Life Sciences

Despite an increase in domestic milk production, the dairy industry in the Dominican Republic (D. R.) has not been able to adequately meet the demand of the ever-growing Dominican population, prompting the government and milk processing plants to sponsor programs which will increase the national production of milk and eventually lead to milk self-sufficiency. One obstacle to this goal is a very low reproductive rate in cows and heifers due in part to the tropical setting. Year-round heat stress may result in abnormal follicular dynamics and decreased oocyte and sperm quality causing reproductive efficiency to decline drastically. The specific purpose of this project was to examine and characterize the reproductive practices and outcomes of the Dominican dairy industry by region and to attempt to identify factors that influence dairy reproductive efficiency. During the course of the study, 43 farms were visited over a 10-week period and evaluated. Farms were chosen throughout 4 of the 5 regions of the country and were selected based on their size (preferably 40 adult cows or more), availability of data, and demonstrated motivation by the owner in improving the herd. Individual herd evaluation was broken into four major components: owner interview, farm evaluation, collection of individual cow reproductive data, and adult cow evaluation. Once observations had been made and catalogued, all data were summarized on the herd level and analyzed descriptively. In addition to descriptive analysis, multiple regression techniques were used to select independent variables which explain most of the variance for each of four reproductive outcomes: days to first service, services per pregnancy, projected calving interval, and service rate. In general, reproductive management practices varied depending on region and farm size. Average lameness within the herd was the most important factor in explaining the variability within services per pregnancy and projected calving interval. As average herd lameness increases by 1 point (based on a 1 to 4 scale), services per pregnancy and projected calving interval increase by 0.65 services per pregnancy and 61.1 days respectively according to the model formulated. Increase in the number of employees involved in estrus detection resulted in higher days to first service. The Santo Domingo region had lower days to first service possibly due to widespread reproductive hormone use within the region. The percentage of Holsteins within the herd was associated with increased services per pregnancy and projected calving interval. Increases in service rate were most closely associated with the type of record category used, indicating that a larger sample population with thorough insemination records may be needed to adequately assess this outcome. In part, reproductive efficiency in the Dominican Republic can be potentially improved by enhancing methods for estrus detection. Mechanical aids to estrus detection (tailhead chalk, K-mar® patches, etc.) may help increase estrus detection efficiency in herds currently only relying on visual observation. Assigning 1-2 people primarily to estrus detection and increasing the frequency of hormone usage may also improve estrus detection efficiency. The main emphasis for Dominican dairy producers, however, should be on preventing new lameness and culling chronically lame cows once it is economically feasible to do so. Reducing the incidence of lameness could, in itself, dramatically improve reproductive efficiency in the Dominican Republic. / Master of Science

The two objectives for this thesis were to 1) validate a bovine luteinizing hormone (LH) ELISA for use in the laboratory and 2) validate interstitial glucose sensors designed for humans for use in lactating dairy cattle. The first experiment required validation of a bovine LH ELISA in order to measure the circulating concentration of LH in the blood of cows in altered thermal and/or metabolic states. Assays from two separate companies were tested. Half of one plate was run at a time resulting in a total of 6 separate analyses (3 plates total). Despite early, promising results, neither LH ELISA could be successfully validated. For many analyses, the standard curves did not even meet the minimal criteria to allow calculation of a formula for determining the concentrations of the unknowns. In analyses where the standard curves were acceptable, the coefficients of variation (CV%) for the unknowns were unacceptable. The second experiment attempted to validate the use of commercially available human interstitial glucose sensors (FreeStyle Libre and Dexcom G6) in lactating dairy cows. Blood glucose concentrations correlated well with sensors secured behind the cow's ear for both FreeStyle Libre (r=0.82) and Dexcom (r=0.88). Unfortunately, however, the absolute relative error's highest value was 47% for the FreeStyle Libre ear. In summary, neither the LH ELISAs nor the interstitial glucose sensors could be validated. / Master of Science / Two separate studies were performed with the purpose of one, measuring a hormone significant to reproduction, Luteinizing Hormone (LH), and two, measuring glucose in interstitial fluid in lactating cows. The first experiment required the use of a commercially available ELISA test to measure LH concentrations. While using radioimmunoassay (RIA) is the preferred method for measuring LH in dairy cattle, an ELISA is an alternative method that is generally more accessible. There was also little published research available illustrating the accuracy of the LH ELISAs. Therefore, we attempted to validate the assays for bovine samples. Despite several attempts, the LH ELISA was unsuccessfully validated. The second study attempted to validate commercially available continuous interstitial glucose sensors designed for humans for use with lactating Holstein cows. Validation of interstitial sensors for use in dairy cattle would decrease the amount of stress and handling when measuring circulating glucose for research, clinical diagnostics and application in industry. While early data suggested that sensors behind a cow's ear may work, further analysis illustrated the sensors were not accurate enough when used on cows. In conclusion, the lab was unsuccessful in proving that continuous interstitial glucose monitors and LH ELISAs could be utilized in the lab.

Dairy cattle characteristically exhibit decreased milk yield and reproductive performance, as well as increased uterine infection rates during periods of high stress. Chromium supplementation has demonstrated efficacy as a feed additive capable of reducing the detrimental effects of stress. As such, its application in dairy production may help to alleviate economic losses associated with seasonal heat stress and the stress experienced during the early postpartum period. Therefore, the objective of the work described in this thesis was to evaluate the potential benefits of short-duration, high-dose chromium (Cr) supplementation in early postpartum dairy cows during the summer months. Multiparous, early postpartum cows (20.95 ± 0.21 DIM, 658.29 ± 13.61 kg) were assigned to one of two treatment groups: 1) normal TMR (Con; n=10) and 2) normal TMR + Cr propionate supplementation (CrPro; 12 mg/h/d Cr; n=12). Body weight (BW), milk yield, and feed intake were measured each day of the experiment. Ambient temperature and humidity were monitored, and the temperature-humidity index (THI) was calculated for the duration of the study as an indicator of the severity of the heat stress experienced by the cows. Transrectal ultrasonography was performed every three days to assess ovarian follicular and luteal dynamics. Respiration rates (RR), rectal temperatures (RT), and blood glucose were recorded concurrently with ovarian ultrasonography. Plasma was collected and used for analysis of progesterone concentrations. Every six days in conjunction with ultrasonography, endometrial cytology samples were collected via cytobrush from each cow to determine the incidences of subclinical endometritis (SCE), as determined by polymorphonuclear leukocyte (PMNL) %. There were no treatment-based differences in RR, RT, blood glucose, feed intake, milk yield, or BW. However, the supplementation strategy did improve reproductive parameters. Within the Con group, there was an increase in PMNL % between samples five and six. Furthermore, at cytology sample six, the Con group had a greater percentage of PMNL than the CrPro group (P=0.01). Chromium consumption did not affect the counts or sizes of most follicles, with the exception being the 6-9 mm category where the CrPro group had a greater average diameter and tended to have more follicles in this category. While CL numbers or size did not differ between treatments, the ratio of progesterone (P4) to corpus luteum (CL) volume was greater in the CrPro group compared to the Con group (P=0.03). The results from this study indicate that the proposed supplementation strategy does not influence DMI or milk yield in cows experiencing stress. Nonetheless, short-duration, high-dose Cr supplementation strategy could benefit reproductive performance and thereby limit economic losses experienced by dairy producers during periods of stress. / Master of Science / Dairy producers continue to experience monetary losses due to the decrease in production performance by dairy cows resulting from physiological changes in response to stress. Elevated ambient temperature and humidity conditions can lead to heat stress, which has been found to decrease both milk and reproductive performance on dairy operations. The period after calving, known as the postpartum period, can also lead to metabolic changes in a cow due to the stress of giving birth and beginning to produce milk. Chromium is an essential trace mineral that can be supplemented with cattle feed to improve the impacts of such stressful periods on production parameters. This study evaluated the effect of a short-duration, high-dose chromium supplementation strategy on reproduction and lactation of early postpartum dairy cattle during summer months. Twenty-two cows were assigned to two treatment groups: control (standard feed only) and chromium supplement (standard feed plus chromium propionate supplementation). Cows receiving the chromium supplement did not experience any changes in respiration rate, rectal temperature, blood glucose levels, feed intake, milk yield, or body weight. Reproductive analyses determined that cows receiving the chromium supplementation had a lower number of immune cells present in the uterus by the end of the experiment, which may be indicative of a healthier uterine environment. Chromium supplemented cows also had a greater level of progesterone concentration to corpus luteum volume compared to cows in the control group. These results indicate that the short-duration, high-dose chromium supplementation strategy could benefit reproductive performance and limit losses experienced by dairy cows under stress conditions.

Commercial laying hens are heavily selected for increased egg production, but little selection has occurred for resistance to extreme temperatures. The objective of the experiment was to determine the effects of a daily cyclic heat stress (HS) on performance, body temperature, egg quality, nitrogen corrected apparent metabolizable energy, and blood chemistry in laying hens at acute and chronic time points. In total, 407 pure line laying hen pullets (18 wk) were sourced from Hy-Line International and housed individually for the duration of the experiment. Feed intake, egg production, and body weights were significantly decreased while egg weight and feed efficiency increased due to HS exposure. All egg quality parameters except yolk weight were decreased by HS exposure, including Haugh unit, eggshell and albumen weights. Egg yolk weights were increased over the first 2 wk of HS before falling. Blood chemistry was affected by HS resulting in respiratory alkalosis likely caused by increased respiration and evaporative cooling. There was a decrease in the PCO2, an increase in pH and a reduction in iCa in the blood within 4 to 6 hours of HS when compared to pre-HS levels. The data indicate that the hens were negatively affected by HS and the response was different for acute and chronic time points. At the acute time point the changes to the hen's physiology were caused by altered blood chemistry, but after chronic exposure, the low feed intake seemed to influence the responses. / Master of Science / High environmental temperatures can cause laying hens to become heat stressed, including reduced egg production, egg quality, and body weight, and in some cases increased mortality. Two mechanisms to reduce heat production and increase heat dissipation are reduced feed intake and increased respiration rate during heat stress. The reduced feed intake can result in insufficient nutrients to maintain egg production and increased respiration rate can change the blood chemistry, reducing ionic calcium and ultimately causing thinner eggshells. The most efficient way to reduce the negative effects of heat stress is by avoidance through controlling the environmental temperature, however, above certain temperatures cooling methods fail. If heat stress conditions are reached, it is important to understand the genetic ability of the hen to minimize the negative consequences of elevated temperature. Therefore, the effects of a four-week heat stress were determined with a focus on hen performance, egg quality, and blood chemistry parameters in laying hens. Some parameters such as blood pH and ionized calcium, shell weight, and body temperature were negatively affected within four to six hours while others, such as shell thickness, Haugh unit, and egg production took longer to be affected. By the end of the experiment blood pH returned to pre-heat stress levels but feed intake was significantly reduced over the 4 wk HS period. This could indicate that the laying hens began to acclimate to the high environmental temperatures but were not able to completely overcome the negative effects.

The increasing globalization of elite soccer match-play means that soccer players are likely to compete in hot, hypoxic and hot-hypoxic environments over a season. Soccer match-play studies have identified a marked decline in soccer-specific physical performance in the heat and hypoxia due to increasing body temperatures and a reduction in partial pressure of oxygen (PO2), respectively. As hot environments are more prevalent in elite soccer match-play, cooling strategies have been assessed within the literature in an attempt to alleviate these heat-induced- decrements. However, utilising a soccer match-play design makes environmental and interventional inferences difficult to ascertain, as a plethora of match factors and adaptive pacing strategies cause high variability in key physical performance measures within soccer match-play. Therefore, the three experiments within thesis aimed to assess the reliability and validity of a non-motorised treadmill (NMT) based soccer-specific simulation [intermittent Soccer Performance Test - (iSPT)], to enable the reliable investigation of environmental stress on soccer performance and the efficacy of pre- and half-time-cooling to attenuate any heat- induced-decrements. The purpose of experiment 1 was to investigate the reliability and validity of iSPT which utilised a novel speed component called a ‘variable run’. This speed component quantified the distance covered at a self-selected speed above the second ventilatory threshold (VT2speed), which attempted to delimit a ‘high-intensity’ threshold. Twenty male University soccer players completed one maximal oxygen (O2) uptake (V̇ O2max ) test, three familiarisation (FAM) sessions and one peak speed assessment (PSA) on the NMT, before completing the iSPT twice (iSPT1 and iSPT2). The total distance, sprint distance and high-speed distance covered were 8,952 ± 476 m, 1,000 ± 74 m and 2156 ± 140 m, respectively. No significant difference (p>0.05) was found between repeated trials of the iSPT for all physical performance measures and physiological responses. Reliability measures between iSPT1 and iSPT2 showed good agreement [Coefficient of variation: <4.6%; Intraclass correlation: >0.80] compared with statistical guidelines. Furthermore, the variable run phase showed high speed running capacity was significantly decreased (p<0.05) in the last 15 min compared to the first 15 min, showing parity with previous match-play data. Experiment 1 validated the iSPT as a NMT based soccer- specific simulation compared to previous match-play data, and is a reliable tool for assessing and monitoring the physical performance and physiological responses in soccer players. Successfully completing the aim of experiment 1 facilitated the quantification of hot (HOT), hypoxia (HYP) and hot-hypoxia (HH) mediated decrements on maximal soccer-specific performance in experiment 2. Twelve male University soccer players completed three FAM sessions, one PSA and four randomised crossover experimental trials of the intermittent Soccer Performance Test (iSPT) in normoxic-temperate (CON: 18oC 50% rH), HOT (30oC; 50% rH), HYP (1,000m; 18oC 50% rH) and HH (1,000m; 30oC; 50% rH). Physical performance and its performance decrements, body temperatures [rectal (Tre), skin (Tsk) and estimated muscle temperature (Tmu)], heart rate (HR), arterial blood oxygen saturation (SaO2), perceived exertion, thermal sensation (TS), body mass changes, blood lactate (Bla) and plasma volume were all measured. Performance decrements were similar in HOT and HYP [total distance (-4%), high- speed distance (~-8%) and variable run distance (~-12%) covered] and exacerbated in HH [total distance (-9%), high-speed distance (-15%) and variable run distance (-15%)] compared to CON. A 4% increase in peak sprint speed was present in HOT compared with CON and HYP and 7% greater in HH. The sprint distance covered was unchanged (p > 0.05) in HOT and HYP and only decreased in HH (-8%) compared with CON. Body mass (-2%), temperatures (+2- 5%) and TS (+18%) were altered in HOT. Furthermore, SaO2 (-8%) and HR (+3%) were changed in HYP. Similar changes in body mass and temperatures, HR, TS and SaO2 were evident in HH compared to HOT and HYP, however, Bla (p < 0.001) and plasma volume (p <0.001) were only significantly altered in HH. Perceived exertion was elevated (p < 0.05) by 7% in all conditions compared with CON. Regression analysis identified that absolute TS and absolute rise in Tsk and estimated Tmu (r = 0.82, r = 0.84 r = 0.82, respectively; p <0.05) predicted the hot-mediated-decrements in HOT. The hot, hypoxic and hot-hypoxic environments impaired physical performance during iSPT. Future interventions should address the increases in TS and body temperatures, to attenuate these decrements in physical performance. Experiment 3 of this thesis aimed to identify three pre- and half-time-cooling strategies to attenuate the heat-induced-decrements previously seen in experiment 2. Eight male University soccer players completed four randomised experimental trials of iSPT, three with cooling and one control (i.e. No pre- or half-time cooling: CON). The pre- and half-time-cooling interventions involved were 30-min or 15 min in duration, respectively. Ice slurry ingestion (SLURRY), ice packs (PACKS) covering the upper legs and mixed-methods (MM: PACKS and SLURRY) were utilised as the three cooling interventions. Physical performance and its performance decrements, body temperatures (Tre, Tsk and estimated Tmu), HR, perceived exertion, TS, body mass changes and Bla were all measured. Compared with CON, both PACKS and SLURRY pre-cooling significantly reduced (p > 0.05) central (Tre) and peripheral (Tsk and estimated Tmu) body temperatures prior to iSPT, respectively. However, body temperature and physical performance were unchanged during the first half of PACKS and SLURRY compared with CON. The MM pre-cooling significantly reduced all body temperatures and TS both prior to and during the first half which coincided with an improvement in total distance (+3%), high-speed distance (+4%) and variable run distance (+5%) covered. Half-time-cooling via PACKS, SLURRY and MM had no ergogenic effect (p> 0.05) upon physical performance in the second half, compared with CON. The 30 min of mixed-method pre-cooling, via ice packs placed upon the upper legs and ice slurry ingestion, significantly improved simulated soccer performance during the first half, however, future research should identify a valid half-time-cooling strategy to offer further improvements to physical performance in the second half. The main findings within this thesis revealed that the iSPT showed validity with previous soccer match-play data and strong reproducibility between two tests (iSPT1 and iSPT2). Furthermore, the variable run component showed efficacy as sensitive measure of the decrements in high-speed running capability. As the iSPT demonstrated low test-retest error compared with the statistical guidelines and previous NMT based soccer-specific simulations, any changes to physical performance can be attributed to an intervention and not the variability of the measure, unlike in soccer match-play situations. No difference was seen for all physical performance measures in both HOT and HYP, however, the heat and hypoxic-induced- decrements stem from increasing body temperatures and changes to both SaO2 and HR, respectively. Such decrements may have a detrimental effect upon the match outcome. These heat-induced-decrements were attenuated in the first half after 30 min of mixed-methods pre- cooling, however, the 15 min of mixed-methods half-time-cooling did not significantly improve any physical performance measure in the second half. The mixed-method pre-cooling strategy tested within this thesis could go some way in maintaining physical performance during the first half of soccer match-play in hot environments (~30oC). However, future laboratory based research within a controlled environment should look to assess different combinations, times and strategies of cooling which may be applicable to the time constraints associated with elite soccer.