Climate change is one of the biggest challenges facing agriculture today. Transient or prolonged heat stress can be detrimental to plant reproductive development. The male gametophyte, pollen, is particularly sensitive to heat stress, resulting in sterile pollen (pre-anthesis) or deformed/stunted pollen tubes (post-anthesis). Quinoa (Chenopodium quinoa Willd.) has recently seen a rise in global interest due to its nutritional qualities, but global expansion of quinoa is partially hindered by its susceptibility to heat. It has been hypothesized that introgression of heat tolerance traits from wild relatives that occupy warmer environments can increase thermotolerance in quinoa. The goal of this research was to investigate the effect of heat stress on mature pollen grains from quinoa and its wild relatives, C. berlandieri and C. hircinum. To answer this question, several experiments were performed: 1. The nuclear number of the pollen of wild relatives was assessed to determine whether mature pollen grains were released at the trinucleate stage. All pollen was found to be trinucleate. 2. Pollen germination medium was optimized for four accessions: C. quinoa (QQ74; PI 614886), C. berlandieri var. zschackei (CB; BYU14118), and C. hircinum (CHA; Hircinum-069 and CHC; BYU17105). Optimal sucrose and PEG concentrations were determined to be: 5% sucrose/20% PEG for QQ74 and CB; 20% sucrose/0% PEG for CHA; and 10% sucrose/20% PEG for CHC. 3. Temperature optima for pollen germination for QQ74 was 32°C-36°C; CB was 30°C-34°C; CHA was 36°C; and CHC was 32°C -34°C. Overall, pollen from wild relatives was not found to be more heat-tolerant than pollen from domesticated quinoa. 4. Pollen tube elongation over time was observed for all four accessions at 34°C and 38°C, with CHA and QQ74 having the lowest decrease in rate at 38°C (35 and 45%, respectively). This study provides a new method for pollen collection for quinoa and its wild relatives, further optimizes the pollen germination media for QQ74, introduces pollen germination media for three wild accessions, and investigates the effect of heat stress on mature pollen grains. These observations can be employed in future studies investigating heat stress response of pollen in quinoa and its wild relatives.

Bumblebees are experiencing declines and range contractions globally that are, in some cases, independent of anthropogenic pesticide- or land-use change, leaving rising global temperatures as the primary driver of such loses. With ambient temperature (Ta) and thermal limitations being a crucial component in these observed declines, I sought to determine the physiological limitations that high Ta imposes on both individuals and colonies of a temperate bumblebee species, Bombus impatiens. Through Chapter 2, I first established the upper thermal tolerance (CTmax) of the species, testing both adults and larvae to determine which of these colony castes are most thermally sensitive to heat. Collective thermoregulation at the colony-level is then important to ensure that the most heat sensitive individuals are protected from changes in optimal nest temperature (Tn). I thus identified the energetic costs associated with colonial thermoregulation and whether large colonies could successfully achieve thermal homeostasis under various Ta. Chronic bouts of heat stress are also of concern as colonies invest time and energy into thermoregulation, especially given that heatwave events are becoming more frequent. In Chapter 3, I examined whether there exists a trade-off between thermoregulation and foraging effort for colonies under chronic heat stress and how various measures of colony success are impacted. Finally, foraging requires individuals to employ flight for the procurement of resources. In Chapter 4, I investigated if the temperate adaptation of an insulative pile layer would hinder flight performance under high Ta by assessing the metabolic rates of adult castes during flight. I found that larvae were more thermally sensitive compared to bumblebee adults, which emphasizes the importance of colonial thermoregulation – a task successful at low Ta. Under heat stress, however, Tn could not be maintained despite elevated energetic investments (Chapter 2). These findings suggest that Ta which exceeds optimal Tn may pose significant challenges to colonies; not only energetically but also to the health of thermally sensitive larvae within. A trade-off between thermoregulation and foraging effort did not emerge for colonies experiencing chronic exposure to high Ta. Instead, only high incidences of thermoregulation were observed which failed to prevent increases in Tn. Furthermore, a greater number of individuals were found to abandon the colony at high Ta, and fewer offspring were produced (Chapter 3). Here, findings suggest that chronic high Ta may pose the greatest risk to the production of thermally sensitive offspring by way of reduced worker population and failed thermoregulation. Finally, the metabolic output during flight at high Ta was not found to be affected by an insulative layer of pile (Chapter 4), indicating that either pile may play a role in limiting other measures of flight performance at high Ta, or that alternate physiological mechanisms may be responsible instead. Together the findings from this thesis broaden the understanding of how a temperate species of bumblebee responds physiologically to high Ta both at the individual and colonial level, providing further evidence on thermal limitations in a changing climate.

Lactating dairy cows heavily rely on mammary gland functionality to maximize milk production. The number and activity of secretory mammary epithelial cells (MEC) plays a pivotal role in defining the synthesis potential of the gland. This dissertation aimed to investigate the effects of increased milking frequency (IMF), heat stress (HS), and cell heterogeneity as key contributors to the regulation of mammary gland milk synthesis capacity in lactating Holstein cows. The first study evaluated the implementation of IMF with 2x and 4x udder halves at early and mid-lactation for 21 and 20 d on milk yield (MY) and its association with changes in cistern and alveolar capacity. Results showed that udder halves milked 4x produced 2.27 kg more MY. Additionally, cows milked during early and mid-lactation had increased cistern capacity, while alveolar capacity remained unaffected. This suggests that increased cistern capacity may support MY enhancement through possible systemic responses caused by IMF. The second study examined the effects of 4 days of HS on mammary gland tissue structure, MEC number, and activity using a pair feeding model. Heat stress reduced MY of 4.3 kg/d. At the tissue level, HS decreased alveolar area and increased alveoli number and nucleated MEC per area. Gene expression analysis revealed unaffected activity-related targets but showed reduced phosphorylation of protein synthesis (pSTAT5) and cell survival (pS6K1) markers, as well as upregulation of an autophagosome-related protein (LC3 II). These findings indicate impaired pathways that could explain the reduction in MY after acute HS. The final study utilized single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of epithelial and immune cell subpopulations in milk. Analysis revealed multiple subpopulations with distinct gene expression profiles, including different subtypes of mammary epithelial cells expressing representative marker genes (CSN3, CSN2, CSN1S1, CSN1S2, and LALBA) and immune cell types such as T cells, granulocytes (including neutrophils), macrophages, and B cells. Understanding the populations of hematopoietic cells in milk provides valuable insights into mammary gland function during lactation. The investigation of factors influencing cell number and activity in MEC is crucial for optimizing milk production and maintaining udder health. By identifying and addressing these factors, dairy farmers and researchers can implement strategies to enhance mammary gland function, improve milk production efficiency, and ensure the overall well-being of dairy cows. / Doctor of Philosophy / Milk production capacity in dairy cows relies on specialized cells in the mammary gland called secretory mammary epithelial cells (MEC). This study investigated how management practices, environmental factors, and individual cow factors affect the regulation of milk synthesis in Holstein cows. In the first study, we compared milking frequency in udder halves milked two times or four times per day during early and mid-lactation. The cows that were milked four times produced 2.27 kg/d of additional milk. This perhaps happened because the mammary gland's storage capacity increased with more frequent milking. Next, we studied the effects of short-term heat stress on the structure of the mammary gland tissue and the number and activity of MEC. Heat stress lowered milk production by 4.3 kg/d. We observed changes in the size and number of certain cells in the mammary gland, which likely affected the observed milk production findings. We also noticed differences in the activity of proteins related to protein production, cell survival, and the recycling of cell materials. In the final part of the study, we used single-cell characterization techniques to examine the different types of MEC and immune cells in milk. We found that there are various subgroups of MEC, as well as different types of immune cells such as T cells, neutrophils, macrophages, and B cells. Understanding the variety and abundance of these cell populations helps us learn more about how the mammary gland works during milk production. Studying the factors that influence the number and activity of MEC is essential for optimizing milk production in dairy cows. By identifying and addressing these factors, dairy farmers and researchers can develop strategies to enhance mammary gland function, improve milk production efficiency, and ensure the overall well-being of dairy cows.

No description available.

In response to increasing interest in greenhouse production and difficulties imposed by adverse environmental conditions in the southeastern United States, two studies were conducted with green and red-leaf lettuce cultivars. The first study analyzed effects on yield and quality of green and red-leaf lettuce subjected to increasing nutrient solution electroconductivity (EC) across three growing seasons. Results indicated that the interaction between season and cultivar had the greatest effect on growth, flavonoids and phenolics, and leaf mineral content. The second study analyzed effects on yield and quality of increasing solution potassium (K) in red Lollo lettuce at two temperatures. The results suggested that temperature is a stronger regulatory factor than increasing K in the determination of lettuce yield and quality. However, increasing K concentrations to 234.6 mg·L-1 results in higher concentrations of leaf mineral concentrations without compromising lettuce yield and quality.

No description available.

Most proteins must fold into native three-dimensional structures to be functional. But, newly synthesized proteins are at high risk of misfolding and aggregating in the cell. Stress, disease or mutations can also cause protein aggregation. A cyanobacterial small heat shock protein, Hsp16.6, can act as a chaperone to prevent irreversible protein aggregation during heat stress. This thesis is focused on the properties of proteins that were associated with Hsp16.6 during heat stress, and which therefore may be “substrates” of Hsp16.6. Bioinformatics were used to determine if Hsp16.6 preferentially binds to proteins with certain properties, and biochemical studies were performed to investigate how the substrates actually behave with Hsp16.6 during heat stress. It was found that Hsp16.6 preferentially binds to proteins with higher molecular weight, higher acidity, higher percentage of charged residues (especially negatively charged residues), and a lower percentage of hydrophobic residues compared to all proteins encoded by the Synechocystis genome. Proteins bound to Hsp16.6 were also slightly enriched in VQL motifs. The potential substrate fructose bisphosphate aldolase class II (FBA) was expressed in E.coli and purified. FBA could be protected by Hsp16.6 from aggregation through forming a complex with Hsp16.6 during heat stress in vitro, consistent with it being a substrate of Hsp16.6. Another potential substrate, elongation factor G1 (EF-G1) was also expressed in E.coli and purified. EF-G1 did not form insoluble aggregates even at 47°C, but circular dichroism spectroscopy revealed the secondary structure has melted at this temperature, and the protein eluted earlier than unheated protein on size exclusion chromatography. Thus, EF-G1 appears heat sensitive, and may also be an in vivo substrate of Hsp16.6. Lastly, in vivo study studies were performed to determine the amount of FBA and EF-G1 in Synechocystis cells. Both proteins are abundant, with FBA levels (around 2% of total cell protein) being about twice that of EF-G1. Further in vivo experiments will be needed to confirm that FBA and EF-G1 are substrates of Hsp16.6.

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.

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

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