<p>The first four months of a dairy calf’s life provide the foundation for future production and health. Therefore, it is critical that calves are managed to optimize both health and growth, to maximize their potential. Calf morbidity, which leads to less productive animals and is an economic impact on producers, continue to be areas of opportunity in the dairy industry. In addition, the animal agricultural community is working to reduce the use of antibiotics, due to a growing concern regarding antibiotic resistant bacteria. This provides researchers with the challenge of identifying strategies to reduce calf morbidity and mortality, while also decreasing reliance on antibiotics. The objective of this dissertation was to evaluate nutritional strategies supplemented to calves as well as maternal factors that impact colostrum. The studies focused on strategies to improve the innate and adaptive immune responses and growth of the calf, ultimately reducing reliance on antibiotics. Chapters 2 and 3 discuss feeding dairy calves <i>Saccharomyces cerevisiae</i> fermentation products in milk replacer and solid feeds until 4 months of age. This study concluded that feeding <i>Saccharomyces cerevisiae</i> fermentation products to calves improves average daily gain and feed efficiency post-weaning and reduces antibiotic treatment incidence for respiratory disease. It also increases the evenness of the fecal microbiome and the acute innate immune response, as determined by increased TNF-α, glucose, and respiration rate during a lipopolysaccharide challenge. Chapter 4 evaluated the effects of feeding dairy calves medium chain fatty acids (C8:0 and C10:0) in milk replacer until 60 days of age. Feeding C8:0 and C10:0 to calves reduced plasma NEFA concentrations around weaning, suggesting the mobilization of less adipose tissue to meet the energy demands of the calf. This trial also determined that vaccinating calves at 3 weeks of age with ovalbumin combined with an aluminum hydroxide adjuvant, is an effective way to evaluate their adaptive immune responses. </p> <p>Supplementing calves directly is not the only way to impact calf growth and health, maternal factors also impact calf nutrition through colostrum consumption. Last, chapter 5 explored maternal factors that influence the lipidome of colostrum and therefore the lipid intake of the newborn calf. This study concluded that the metabolic status of the cow affects circulating lipids and the lipid content of colostrum. Also, the lipidome of colostrum is distinct from the circulating lipidome of the calf, which is similar to the circulating lipidome of the cow, except for phosphatidylglycerol, where it appears that colostrum serves as the source for the phosphatidylglycerol present in the circulation of the calf. There are many different nutritional strategies that can impact the health and productivity of calves. Calves can be directly supplemented with nutraceuticals like <i>Saccharomyces cerevisiae</i> fermentation products or medium chain fatty acids, or calf nutrition can be influenced by the maternal factors through the consumption of colostrum. </p>

<p>  </p> <p>In modern society, continuous light exposure and obesity are increasingly prevalent, especially in women of childbearing age. Circadian, metabolic, and reproductive systems have a complex, inter-regulated relationship. Thus, the disruption of one system likely impedes another. Excessive adiposity and circadian disruption alter normal behavior and physiology and disrupt the endocrine milieu. The overall goal of the studies described in this thesis was to develop and test a model system that could tease apart the influence of prepregnancy obesity and circadian disruption, as well as study the combined effects on female reproductive competence. </p> <p>The first study focuses on the prepregnancy period and aims to determine the effect of high fat diet feeding on diurnal eating pattern, body weight over the four-week period, the body composition at the end of the four-week period, hair corticosterone levels, and circadian fecal corticosterone patterns on female ICR mice. Five-week-old female ICR mice were randomly assigned to control (CON; 10% fat) or high fat (HF; 60%) diets and fed for four weeks to achieve adequate adiposity. During this four-week time period, mice had routine light exposure of 12h light and 12h dark. Feed was weighed at 0600 and 1745 Monday-Friday to determine diurnal feed intake. The mice were weighed on a weekly basis. After four weeks on respective diets, mice were anesthetized with isoflurane to measure crown-rump length to calculate BMI and hair was shaved for corticosterone extraction. Once mice recovered from anesthesia, body composition was measured with EchoMRI. After 1 week on diets, HF mice consumed more (P<0.05) during the day than CON mice, which is typically when mice are inactive. After two weeks on diets, HF mice weighed more (P<0.05) than CON, as well as had higher BMI and percent body fat (P<0.05) than CON after four weeks on diets. After four weeks on diets, HF mice had high hair corticosterone (P<0.05). Sampling feces over a 48h period at the end of the four weeks demonstrated that HF mice had elevated basal corticosterone, attenuated circadian rhythms, and a shift in corticosterone amplitude. The prepregnancy period demonstrated that high fat diets alone alter circadian eating pattern and corticosterone rhythms.</p> <p>The remainder of the study continued the dietary treatments assigned during the prepregnancy period, as well as implemented light conditions to create a 2Χ3 factorial study design. There were three light conditions: 12h light and 12h dark (LD), 24h dim light (L5), or 24h bright light (L100). Mice were moved into experimental light conditions after the observation of a vaginal plug or after 5 days with males. This portion of the study aimed to determine the effect of diet (CON or HF) and light exposure (LD, L5, or L100) on gestation length, number of pups born, milk composition, litter weight on postnatal day 12, as well as dam feed intake, hair corticosterone levels, and plasma prolactin. Continuous light exposure increased gestation length, with L5 (19.1 d ± 0.23) and L100 (18.9 d ± 0.21) having longer gestation lengths (P<0.05) than LD (18.1 d ± 0.25). Diet affected the number of pups born (P<0.05), with HF dams having fewer pups (9.99 ± 0.4) than CON (11.4 ± 0.4). Despite no difference in birth weight of standardized litters (n=8 pus/litter), litters of HF dams weighed more than CON by day 4 postnatal. The greater litter weight of HF dams continued until the end of the study on day 12 of lactation (P<0.05). Light had a tendency to increase litter weight (P=0.07). Diet, light, and stage of reproduction influenced dam feed intake (P<0.05). L100 dams had higher plasma prolactin, as well as final dam and mammary wet weights (P<0.05). Constant light exposure decreased ATP content in the mammary gland (P<0.05) and decreased milk lactose concentration (P<0.05). Pearson’s correlation analysis showed a positive relationship between mammary weight, fat pad weight, BMI, kcal of feed intake, and gestation length (P<0.05). In CON mice, hair corticosterone was negatively correlated with litter weight on lactation day 12. Liver weight was positively correlated with d 12 litter weight in HF mice. Together, these studies demonstrate that feeding high fat diets and continuous light alter maternal behavior and physiology, which may impact offspring health and development, however continuous light may not be the best approach to studying circadian disruption. Elevated maternal plasma prolactin and increased dam weight suggests a long day photoperiod was likely induced, thus potentially mitigating the circadian disruptive effects from constant light. Other model systems should be considered, such as using a chronic jet lag model that changes the light exposure every 3 d. </p>

<p dir="ltr">Boars in the swine industry are one of the select animals subject to limit feeding. Limit feeding boars occurs once boars are selected to enter the boar stud. Before selection boars are <i>ad libitum</i> fed like a nursery or grow finishing pig. Research involving boar nutrition and the impact it has on growth, development, and semen characteristics are limited compared to the rest of the swine industry. The objective of these studies conducted at Purdue University were to determine how varying nutritional feeding strategies impact semen quality and quantity as well as growth and development of artificial insemination boars.</p><p dir="ltr">Feeding fiber is a commonly used strategy in gestating sow nutrition to mitigate some of the negative side effects of limit feeding animals. A variety of fiber sources are available to the swine industry, however most of the common sources used are wheat bran, alfalfa meal, sugar beet pulp, and soybean hulls. Soyhulls are one of the more easily accessible sources of fiber in Indiana and the Midwest U.S. due to large amounts of soybean production and processing which will continue to grow with bi-energy demands. Boars fed the fiber diet (14.3% soyhulls) had increased bodyweight and body condition changes over the twelve-week period. Additionally, fiber inclusion in the diet had no impact on semen characteristics but semen was impacted by the age and breed of the boar. Salivary cortisol levels were reduced in boars fed fiber as well as some changes in behavior were observed.</p><p dir="ltr">The addition of fiber had impacts on growth and development but no impacts on semen. Since boars are limit-fed for so long from selection after the grow-finish period until they are culled this drew up the question if feeding boars different levels after selection (1.8 kg/d or 2.7 kg/d) impacted trainability, first time semen collection characteristics, growth and development of young boars. No differences were observed for feeding level impacting trainability with 92% of all boars regardless of treatment being successfully trained. Semen volume was increased in boars fed 2.72 kg/d by approximately 50% but no other semen characteristics were impacted. Boars fed 2.72 kg/d also had increased bodyweight and body condition score measures.</p><p dir="ltr">The boars subject to the training study unknowingly arrived at the farm naturally infected with porcine circovirus three (PCV3). Research has been conducted on the ability of viruses (PRRSv, PEDv) to be detected and transmitted through semen. Porcine circovirus three is a relatively new variant of circovirus so the opportunity to evaluate the question of if this virus was able to be detected in seminal plasma and the impacts it has on semen characteristics was presented. Serum and semen samples were collected for polymerase chain reaction analysis of detection of PCV3 conducted at Purdue Animal Disease Diagnostic Laboratory. Porcine circovirus three was able to be detected in the serum of positive boars however no positive detection occurred for seminal plasma samples. Morphological abnormalities such as distal midpiece reflex, distal droplets, and head and tail differed between infected and non-infected boars. Young boars sero-positive for PCV3 had decreased total sperm, total normal sperm motility, and head and tail abnormalities but had decreased distal midpiece reflex abnormalities when compared to PCV3 negative young boars. Young boars were also able to maintain the virus for 98+ days, while some old boars were able to clear the virus withing 14 weeks of becoming positive.</p><p dir="ltr">The next chapter evaluated maintenance energy equation listed in the swine NRC 2012 and if it was still accurate for sexually active boars and if weight management through the use of this equation impacted boar performance. Boars were subject to three treatments (150%, 100%, 80%) of maintenance energy for the first nine-week period and 150% and 80% diets were switched for second nine-week period while 100% stayed the same the entire time in a cross-over design. Semen, bodyweight, and body condition parameters were analyzed throughout the two nine-week periods. Boars fed 80% of maintenance had the largest reduction in semen parameters compared to 150% and 100% maintenance fed boars. Additionally, switching 80% feed intake boars to 150% maintenance feed intake, boars were able to recover some of the negative impacts, however they were not able to fully recover in the second nine-week period. Boars fed 100% and 80% maintenance energy lost weight regardless of age. The loss of body weight of the 100% maintenance fed boars indicates the swine NRC 2012 equation is underestimating the modern AI boar maintenance needs. Old boars were able to handle changes in weight and body condition better than young boars as there was reduced variation in serum metabolites analyzed with old boars compared to young boars.</p><p dir="ltr">The last chapter of this thesis focused on a big data project from commercial sow farms. The objective of this study was to determine if breeding gilts at first signs of estrus or after has an impact on sow performance and economic value to parity three. A regression model was developed from on farm data to determine the difference in total born piglets to parity 3 between gilts bred on their first estrus or after. Gilts bred on their first estrus had six fewer total piglets to parity three compared to gilts who were bred after the first estrus. Also, gilts bred after their first estrus had a larger percentage of sows being profitable compared to gilts bred on first estrus. This model data was compared to actual data for comparison between the two. The model with the limited variables inputted and compared to the actual data was fairly accurate with economic profitability and with more refinement the accuracy can be increased.</p><p dir="ltr">Overall, nutrition of the boar needs to be an area of interest in the swine industry to continue improving efficiency of production. Feeding fiber to boars has minimal impact on semen characteristics but can be used to reduce stress in boars that are limit-fed for weight and body condition management. Limit feeding boars beginning at selection reduces growth and development and had negative impacts on first time semen collection volume. Porcine circovirus 3 was not found in the seminal plasma of naturally infected boars when serum analysis was positive. Boars fed below maintenance levels listed in the swine NRC 2012 have negative impacts on semen, bodyweight, and body condition parameters. Once diets were changed boars were able to recover from the feed restriction but not meet baseline measures prior to feed restriction. Young boars are more sensitive to feeding changes than old boars, this could be due to the young animal still growing while the older animal is closer to mature size. Big data projects can aid producers in making production changes decisions to estimate the economic impact it can have to an individual farm or the entire production system.</p>

<p>Postmortem aging has been extensively practiced as value-adding process due to the beneficial impacts on meat palatability. Meat tenderization occurred through proteolytic fragmentation of myofibrillar structural proteins via endogenous protease systems, which is considered as the primary drive to enhance major palatability attributes including tenderness, juiciness, and flavor. Recent theoretical framework proposes apoptosis, or programmed cell death, as the preceding step that initiates postmortem proteolysis. Whereas small heat shock proteins have been consistently recognized as meat quality biomarkers, probably due to their protective activities against proteolysis through anti-stress, anti-apoptotic, and chaperoning functionalities. To shed light on detailed mechanisms controlling postmortem proteolysis and consequential impacts on the development of fresh meat quality characteristics, postmortem proteolytic changes of small heat shock proteins, apoptotic factors, and myofibrillar structural proteins were profiled in postmortem skeletal muscles under different metabolic backgrounds and across species. </p> <p>In beef, three muscles, <i>longissimus lumborum</i> (LL), <i>semimembranosus</i> (SM), and <i>psoas major</i> (PM), have been selected to represent glycolytic, intermediate, and oxidative muscle types. Tenderness and water - holding capacity were determined, and proteolysis, apoptotic features, and small heat shock proteins were measured in 8 beef carcasses at 1, 2, 9, 16, and 23 days of aging. PM exhibited limited aging potential in quality developments shown by lower extents of shear force, water-holding capacity, and proteolytic changes, including calpain 1 autolysis, troponin T, and HSP27 compared to LL and SM. Conversely, LL had an increase in tenderization and water-holding capacity, which was accompanied with more extended calpain 1 autolysis, proteolysis and HSP27 degradation, compared with other muscles. The results of this study suggest that postmortem proteolytic changes of myofibrillar proteins, small HSPs and apoptotic factors occur in a muscle-specific manner, which is likely attributed to different rate and extent of meat quality developments of each muscle during aging. </p> <p>Callipyge lambs are a unique genetic background showing calpastatin over-expression, muscle hypertrophy in loin and hindquarter area, substantially compromised meat tenderization potential, and a shift of muscle fiber composition towards fast-glycolytic directions. Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < 0.05), including metabolic co-factors, polyphenols, and AA/short peptides.</p> <p>Pig production is facing increased public pressure regarding antibiotic usage restriction. Recently, dietary L-glutamine at cost effective level (0.2%) was identified as an effective antibiotic alternative in post-transport nursery pig diets. To evaluate carcass and meat quality characteristics in market-ready pigs when 0.2% dietary L-glutamine was applied as for early-life post-weaning and transport recovery, pigs (N=480) were weaned and transported in two replication trials in SPRING (April of 2017) vs. SUMMER (July of 2016), fed 3 different diets (Non: no antibiotic, Anti: 441 ppm chlortetracycline and + 38.6 ppm tiamulin, Gln: 0.20% L-glutamine) for 14 days after transport, and fed basal diet until reaching market weight. Pairs of <i>longissimus dorsi</i> (LD) and <i>psoas major</i> (PM) muscles from each carcass (n=10/diet/trial) were separated at 1 d and 7 d postmortem, respectively. Carcass yield and meat physical and quality attributes were evaluated. Overall impacts of Gln on physical attributes of carcasses and porcine muscles were minimal. No dietary effects were found in carcass, proximate composition, water-holding capacity, or shear force. Significant difference between trials were found in terms of productivity and pork/carcass qualities, where SPRING replicates showed increased body weight, faster pH decline, paler surface color, higher intra-muscular fat deposition, and improved tenderness and water-holding capacity as indicated by lower shear force values, thaw-purge loss, and cooking loss (P < 0.05).</p> <p>The pork and carcass quality results give rise to a postulation that different metabolism and animal growth might have been occured between the two production trials, consequentially differentiated meat quality development. In this regard, myofibrillar proteolysis, small heat shock proteins, and apoptotic factors were characterized during 7 d postmortem aging in porcine LD and PM muscles from both seasonal trials, combined with metabolomics profiles of 1d samples using the GC-TOF-MS/MS platform. Compared to SUMMER counterparts, SPRING muscles showed concurrence of more extended apoptosis, further calpain 1 autolysis, and increased structural protein degradation (P<0.05). SPRING muscles showed more ATP catabolism compounds and increase in carbohydrates, branched-chain amino acids, and 16-18 carbon fatty acids, which could be chemistry fingerprints of increased cellular oxidative stress, consequentially favoring onset of apoptosis and proteolysis. Meanwhile, SUMMER pigs showed increased stress-defending metabolites, such as ascorbic acid, antioxidant amino acids, and decreased inhibitory neuro-transmitter GABA, which may indicate elevated stress-defending activity in SUMMER pigs that possibly inhibited apoptosis and proteolysis. </p>

<p>(From abstract) </p> <p>In the first study, the urine collection method was effectively applied for evaluation of intestinal permeability using Cr-EDTA, an indigestible oral marker, demonstrating the applicability of the procedure in 1-week-old and 6-week-old neonatal heifer calves (n=15 calves). Calf health observations were recorded during the entire urinary catheterization process and collection period to evaluate any negative health reactions to the procedure, or localized reactions. Proportion of localized reactions were analyzed, and the proportions did not exceed 20% for the calves catheterized at either 1 week or 6 weeks of age. </p> <p>In the second study, the developed catheterization procedure and urine collection method was applied using Cr-EDTA as an oral marker to investigate if L-GLN supplementation would offer improvement to intestinal permeability. In this larger study, 30 Holstein heifer calves [1.5 ± 0.5 days old; 37.1 ± 0.86 kg body weight (<strong>BW</strong>)] were blocked by serum total protein, BW, and age, and randomly assigned to 1 of 2 treatments: <strong>GLN</strong> [24% crude protein (<strong>CP</strong>)], 17% fat milk replacer (<strong>MR</strong>) +10 g L-GLN/kg MR powder) or <strong>NS</strong> (24% CP, 17% fat MR). MR was reconstituted to 12.5% solids with warm water and fed 3.8 L/calf/d until weaning. Calves were weaned at 56.4 ± 0.5 days of age, and had <em>ad libitum</em> grain (17% CP, 2% fat) and water access throughout the experimental period.</p> <p>During the preweaning period, calves were individually housed in hutches and health observations, which included respiratory and fecal scores, were assessed daily. Body weight was measured weekly, and grain and MR intake was assessed daily to calculate average daily gain (<strong>ADG</strong>), average daily feed intake [<strong>ADFI</strong>; grain intake (dry matter (<strong>DM)</strong> basis) + MR intake (DM basis)], and feed efficiency (<strong>G:F</strong>; ADG:ADFI). At weaning, calves were weighed, moved to pens (n = 3 pens/treatment, 4-5 calves/pen), provided free access to grain and grass hay, and then weighed 2 weeks post-weaning. Additionally, urinary catheters were placed at 1 and 6 weeks of age, and calves were orally dosed with 1 L Cr-EDTA in their MR. Urine samples were then collected over a 24-hr period for Cr output analysis as an <em>in vivo</em>biomarker of intestinal permeability. </p> <p>Blood was collected on study days 1, 2, 5, 7, 14, 21, 42, 56, and 70 to measure haptoglobin, serum amyloid A, leukocyte data, neutrophil: lymphocyte (<strong>N:L</strong>), glucose, non-esterified fatty acids, insulin, and cortisol. Two study periods were identified for data analysis representing greater (<strong>P1</strong>; weeks 1-3) and reduced (<strong>P2</strong>; weeks 4-8) enteric disease susceptibility. Data were analyzed using PROC GLIMMIX or PROC MIXED in SAS 9.4 with calf as the experimental unit. There was a decrease in total preweaning Cr output (<em>P</em> < 0.05) for GLN calves, and Cr output in 1 week old calves was decreased (<em>P</em> = 0.04) in GLN versus NS calves. The N:L was decreased overall (<em>P</em> = 0.03) and during P2 (<em>P</em> = 0.01) and P2 neutrophil count tended to be reduced (<em>P</em> = 0.07) in GLN versus NS calves. There were no MR treatment differences for ADFI, ADG, body measurements, post-absorptive metabolic biomarkers, disease scores, and therapeutic treatments (<em>P</em> > 0.10). In summary, L-GLN supplementation improved intestinal integrity and biomarkers of physiological stress in pre-weaned Holstein heifer calves managed under production-relevant conditions.  </p>

<p>  </p> <p>Little is known about pullet stress and welfare and even less is known about pullets in cage-free housing. The studies included in this dissertation evaluated bird responses to stocking density, feeder space, and ACTH stressors in pullets. The project and procedures were reviewed and approved by the Purdue University Animal Care and Use Committee (Protocol#:2001002002 and # 1908001938).  </p> <p>Two studies of external stressors and one study of an internal stressor were applied to Lohmann LB-Lite (Brown) and Lohmann LSL-Lite (White) pullets. For both studies of an external stressor, bird-based welfare parameters included evaluation of bird appearance through feather coverage, foot condition, and keel condition; blood parameters, i.e., cholesterol, corticosterone, cortisol, NDV/IBV IgG/IgY titer, and differential WBC (%) and heterophil to lymphocyte ratio; measurements of the spleen, bursa, thymus, and liver; and production values measured through body weight, body weight uniformity, shank length, feed consumption, and feed conversion ratio (FCR). Birds were followed from 2-16 weeks of age (WOA) for the external stressors and were housed on the floor.</p> <p>The first external stressor was applied through a 2 x 2 factorial randomized complete block design (RCBD) using Brown and White strains and two stocking densities- high (HSD) and low (LSD). Stocking densities were adjusted over time, with the ending density of 670 cm2/bird for HSD and 1352 cm2/bird for LSD at 16 WOA. Density alone affected relative bursal weight (%), with LSD generating heavier bursas. LSD also generated lower, better FCR for both white and brown strains though this difference was not statistically significant. Within a density, opposing strain effects were seen for body weight uniformity and feather coverage. White was improved at HSD while Brown was improved for LSD. </p> <p>The second external stressor study was a 2 x 2 x 3 factorial RCBD with Brown and White, 2 feeder space allocations (3.5 or 7 cm/bird), and 3 stocking densities. The ending stocking densities were 154.45, 518.76, and 1,247.38 cm2 for the high (HSD), medium (MSD), and low (LSD) stocking density treatments, respectively, at 16 WOA. In this study, less space per bird was associated with poorer feather condition but improved FCR. Reduced feeder space was associated with elevated or worse FCR.</p> <p>In a study of internal stressors, ten each of Lohmann LSL-Lite hens received either a saline-mannitol or synthetic human ACTH injection at 6, 17, and 26 weeks of age. Blood was collected prior to injection and 1- and 2-hours post-injection. H:L ratio, WBC differential counts, corticosterone, and cortisol were measured in blood. Albumen samples were collected to measure corticosterone and cortisol at 26 WOA only but were unaffected by treatment. Serum corticosterone and H:L ratio were also unaffected by treatment. Serum cortisol was increased 1 hour after ACTH injection at 17 and 26 WOA and percent heterophils and lymphocytes were affected by ACTH at 26 WOA indicating potential utility as a measure of stress for adult birds only. </p> <p>The results of these studies show that pullets are resilient and resistant to several stressors. Both acute and chronic as well as internal and external stressors were tested in these studies with limited effects on the birds’ physiology, immunity, condition, or production. Feather coverage and bursal size were improved at lower stocking densities and FCR was improved at greater feeder space but the implications of these differences are unclear. Further research is needed to identify why there are reduced stress responses in pullets; it is possible that domestication or preserved evolutionary pathways explain some of the differences between pullet and adult stress responses. And while the majority of parameters were unaffected by the stressors used, the results of this study presented baseline values for Lohmann LB-Lite and Lohmann LSL-Lite pullets that will help guide future research. Additionally, the results of these studies left perhaps more questions than answers but illustrate perfectly that more research will be critical to an understanding of pullet stress and welfare.</p>

<p dir="ltr">Epigenetics involves the study of mechanisms that influence gene expression. These mechanisms are heritable and dynamic, and despite altering gene transcriptional activity, they do not change the underlying DNA sequence. While epigenetic mechanisms govern gene expression throughout the lifetime of an organism, the dynamic nature and precision of the transcriptional control afforded by processes such as histone modifications and chromatin architecture remodeling are exemplified in early mammalian development. Perhaps unsurprisingly, perturbations to the epigenetic status of a cell can alter its function, and widespread epigenome disruptions due to changes in the developmental environment can compromise the growth, and even viability, of an embryo or fetus. By studying epigenetic mechanisms and the patterns they impart, we can better understand not only how developmental progression is regulated during embryonic development and beyond, but also what the consequences of aberrant epigenetic disturbances may be to developing organisms.</p><p><br></p><p dir="ltr">Many gaps remain in our knowledge concerning epigenetic mechanisms in domestic livestock species, particularly regarding early development. Pigs represent a compelling model organism for study in this area, as they are increasingly being used as a biomedical model for human-oriented research due to their physiological similarities to humans, and they remain a staple meat species in many parts of the world. Chapter 2 investigates the presence and transcriptional dynamics of the SWI/SNF chromatin remodeling complex GBAF in porcine trophectoderm (PTr2) and fetal fibroblast (PFF) cells. These cell lines represent two discrete developmental stages during early swine development, with the PTr2 cells originally obtained from the trophectoderm of a gestation day 12 elongating porcine conceptus, and the fetal fibroblast cells were collected from a fetus on day 40 of gestation. Using immunocytochemistry and Western blotting techniques, GBAF was identified in both cell lines. Further, co-immunoprecipitation of GBAF constituent subunits and other BAF family subcomplex subunits revealed a previously undescribed interaction between the GBAF subunit GLTSCR1 and the BAF subunit BAF170, the latter of which has not been shown to be present in human and mouse GBAF. This may suggest a species-specific GBAF composition in swine. Analysis of RNA-seq data from porcine embryos, PTr2 cells, and PFF cells showed that while transcription of GBAF-specific subunits BRD9 and GLTSCR1 was detectable, expression levels were lower compared to other BAF family subunits. Taken together these results suggest that, while GBAF is detectable in swine early development contexts, it may have a comparatively minor contribution as an epigenetic mechanism during the represented developmental timepoints.</p><p><br></p><p dir="ltr">Details in the literature about the epigenetic landscape and the resulting chromatin state during porcine early development are also limited at present. Chapter 3 involves the global epigenetic profiling of the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in PTr2 and PFF cells using CUT&RUN. The enrichment patterns observed for these features were consistent with known patterns described in the literature. H3K4me3 was primarily enriched in gene promoter regions, and H3K27ac showed enrichment in both promoter regions and distal intergenic regions, some of which are likely active enhancers. H3K27me3 showed broad genomic localization and was detected at genes known to be transcriptionally inactive in these cell types, as well as in distal intergenic regions. BRG1 showed some co-enrichment with H3K4me3 and H3K27ac in promoter regions, as well as several instances of H3K27ac co-enrichment at intergenic sites. The sequencing files were used to build a chromatin state prediction model for 10 states in each cell line, ranging from TSS to repressed genomic regions. Additionally, the transcriptomes of PTr2 and PFF cells were compared to those of human cells taken from comparable gestational time points to determine if these swine cell lines could potentially serve as translational <i>in vitro</i> models. PTr2 cells and human trophectoderm (TE) cells were relatively dissimilar in their cell-type specific gene identities (~24% overlap) and corresponding transcriptional levels, but the porcine and human fibroblast cells shared around 50% of the same cell type-specific genes, and expression levels were broadly similar among them. Altogether, these findings provide foundational epigenetic landscape information for PTr2 and PFF cells and potential insights regarding similarities and differences in cell identity between human and pig trophectoderm and fetal fibroblast cells.</p><p><br></p><p dir="ltr">The placenta is a transient organ that provides essential support to the developing fetus in the form of nutrient and gas exchange. Despite its significance in facilitating fetal development, our understanding of how the placenta is affected by its environment is greatly limited, and only a handful of studies exploring the placental epigenome in swine exist to date. To address these gaps, and building upon the epigenetic profiling methods developed in Chapter 3, Chapter 4 investigated whether, and to what extent, the placental epigenome changes in response to fetal endocrine perturbations. Placental tissue was collected at day 86 of gestation from untreated pregnant gilts and pregnant gilts treated for 21 days with methimazole (MMI) to induce fetal hypothyroidism. CUT&RUN was used to evaluate the enrichment of H3K4me3, H3K27ac, and BRG1 in placental tissue derived from n=6 male and female fetuses in each treatment group (n=12 samples per group). Differential enrichment of all three epigenetic features was seen in placental tissues obtained from MMI-treated fetuses, and, notably, existing sex-specific differences in placental epigenetic features were exacerbated by MMI-induced fetal hypothyroidism. This may suggest that the porcine placenta may be impacted by fetal endocrine status during late gestation. Together, these findings show that sex-specific differences in placental chromatin state exist and that a fetal hypothyroid state is sufficient to perturb the placental epigenome, ultimately providing novel insights into the intricate interplay between fetal endocrine status and regulation of the placental epigenome.</p>

This thesis sets out to identify physical and physiological differences between lambs of different birth ranks at birth, and to use this information to identify practical on-farm management strategies which could improve triplet-born lamb survival. Triplet-born lambs, especially the lightest-triplet-born lambs, not only had a greater capacity to lose heat but also had a reduced capacity to produce heat when compared to twin-born lambs. Due to their lighter birth weights, triplet-born lambs had lower plasma thyroxine (T4) concentrations within the first 24 hours of life than twin-born lambs, and within twin- and triplet-born litters, the lightest- and medium-triplet-born lambs had greater plasma lactate concentrations than all twin-born lambs and the heaviest-triplet-born lambs. Independent of lamb birth weight, triplet-born lambs had lower plasma tri-iodothyronine (T3) concentrations within five minutes of birth, and within twin- and triplet-born litters, the lightest- and medium-triplet-born lambs had lower plasma T4 and T3 concentrations within five minutes of birth than all twin-born lambs and the heaviest-triplet-born lambs. It was hypothesised that because triplet-born lambs had a lighter birth weight and lower plasma thyroid hormone concentrations, they would have inadequate thermoregulatory capabilities when compared to twin-born lambs. The lower rectal temperatures of triplet-born lambs within the first hour of life and the lower heat production on a per lamb basis at 24 to 36 hours of age, and the lack of difference in maximum heat production on a per kg of birth weight basis at 24 to 36 hours of age support this hypothesis. Two practical on-farm management strategies trialled in this thesis to improve triplet-born lamb thermoregulation were offering concentrate supplement during late pregnancy to improve lamb birth weights, and maternal iodine supplementation to improve lamb plasma thyroid hormone concentrations. While offering concentrate showed positive effects such as increasing lamb birth weights, colostrum uptake and triplet-born lamb heat production on a per kg of birth weight basis, the results were either inconsistent across experiments or between birth ranks suggesting additional work is required to determine the repeatability and cost effectiveness of these findings. Maternal iodine supplementation offered no iv | P a g e benefits in terms of lamb birth weights, plasma thyroid hormone concentrations or lamb heat production. Further investigations identified that lamb birth weights, thyroid hormone concentrations, glucose and NEFA concentrations are positively associated with maximum heat production at 24 to 36 hours of age. Practical on-farm management strategies which could target these physical and physiological factors may improve triplet-born lamb heat production, and therefore the survival rates of triplet-born lambs.

Studies of humans and domestic animals have shown that there is a linkage between the neonatal and post-natal health of an individual and its uterine environment during gestation. However, very little information exists for sheep and there have been no studies that have directly examined the stage of gestation at which such effects could be introduced to the conceptus. In the present study, pure-breed embryos were transferred within and reciprocally between large (Suffolk: S) and small (Cheviot: C) breeds of sheep to establish different uterine environments; SinS (large control), SinC (restricted environment), CinS (luxurious environment) and CinC (small control) and their effects upon embryonic, fetal, neonatal and post-natal development and glucose metabolism of lambs were examined. By Day 19 of gestation, conceptuses (embryo and trophoblast) developing in a restricted uterine environment (SinC) were smaller (P<0.05) than in control (SinS). The head length of SinC fetuses was smaller (P<0.05) than in SinS fetuses on Day 55 of gestation and SinC lambs were lighter and smaller (P<0.05) than SinS lambs at birth. During subsequent post-natal life, there was no difference (P>0.05) in the growth rate of SinC and SinS lambs. The liveweight and body dimensions of SinC lambs were lower (P<0.05) than SinS lambs until 9 weeks and 12 weeks of age, respectively. Day 19 peri-implantation embryos and trophoblasts that developed in a luxurious environment were bigger than in control (CinC). However, CinS fetal size did not differ (P>0.05) from CinC fetuses by Day 55 of gestation. There was no difference (P>0.05) in the birthweight and body dimensions of lambs born from these two groups. Dimension of the placentas of SinC and SinS or CinS and CinC did not differ (P<0.05) during gestation or at lambing. Concentrations of ovine placental lactogen (oPL), progesterone, insulin-like growth factor-1 (IGF-1), glucose and free fatty acid (FFA) differed between uterine environments. During glucose challenge tests, there were no differences in the concentrations of glucose and insulin, between SinC and SinS female lambs, however, glucose concentrations declined more rapidly (P<0.05) in CinS than CinC female lambs at one year of age. It was concluded that restricted uterine environment affects embryonic, fetal and neonatal development of lambs, and that these effects perpetuates until at least one year of age; but there was no effect upon glucose metabolism. Conversely, a luxurious uterine environment enhances the early development of embryos but had no effects upon subsequent fetal, neonatal and post-natal development; however glucose metabolism of post-natal female lambs was improved. It appears that these effects of uterine environment were mediated through the trophoblast during the early embryonic period and via the placenta during subsequent gestation. oPL, progesterone, IGF-1, glucose and FFA were implicated in feto-maternal dialogue. These results suggest that uterine environment significantly influences the biology of young sheep with possible economic consequences.

<p>Thyroid hormone is essential for regulating adult metabolism and proper fetal development. Under normal conditions, maternal and fetal thyroid hormones are subject to metabolism at the placenta and within fetal tissues through deiodination and sulfation to regulate fetal exposure to the bioactive hormone. Disruptions of the thyroid hormone system can result in non-thyroidal illness syndrome (NTIS), which is classified as the dysregulation of thyroid hormone homeostasis. The exact cause of the alterations in circulating thyroid hormone levels during NTIS is not well- known. In comparison, hypothyroidism results from the absence of thyroid hormone production and presents as low thyroid hormone levels.</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) crosses the late gestation placenta and causes suppression of circulating maternal and fetal thyroid hormone. Chapter 2 investigates the potential role of thyroid hormone metabolism in this disruption. Pregnant gilts were challenged with PRRSV2 (n=22) or sham inoculated (n=5) at gestation day 85. Samples were collected on day 106, and viral load was assessed in fetal serum and thymus. From the entire fetal population, three distinct subsets of fetuses representing biological extremes were identified, including uninfected with no detectable viral load (UNIF), high viral load viable (HV-VIA), or high viral load with severe meconium staining (HV-MEC). In addition, control fetuses from sham inoculated gilts (CON) were used as a reference group. Samples of fetal liver, kidney, and the corresponding fetal placenta and maternal endometrium for n=10 fetuses per group were then used to evaluate gene expression. A total of 11 genes associated with thyroid hormone metabolism including deiodinases (DIO1,2,3), sulfotransferases (SULT1A3,1B1,1C2,1E1,2A1), sulfatase (STS), and solute carriers (SLC16A2,16A10) were quantified using absolute quantification qPCR. Evidence of fetal decompensation was observed within the high viral fetuses in the form of decreased DIO1 expression within the fetal liver and increased DIO3 expression in both components of the placenta. Circulating levels of T4 and inactive thyroid hormone metabolites, reverse-triiodothyronine (rT3) and two diiodothyronines (3,5-T2 and 3,3’-T2), were measured in fetal serum. While T4 was depressed, no change was observed in circulating rT3 levels, and neither T2 metabolite reached the lower detection limit. This may suggest that alterations in thyroid hormone metabolism generate a localized effect on hormone metabolites in the respective tissues.</p> <p>Alternatively, the low levels of available T3 and T4 limit the production of downstream metabolites to be found in serum.</p> <p>The cause-and-effect relationship between PRRSV infection, fetal thyroid disruption, and the effects on fetal thyroid hormone metabolism are unclear. Therefore, Chapter 3 developed a non-pathogenic model using methimazole (MMI) to induce hypothyroidism in the late gestation fetus and evaluate the impact on fetal development and thyroid hormone metabolism. Pregnant gilts were either treated with oral methimazole or equivalent sham from gestation day 85-106 (n=4/group), followed by classification of all fetuses as live, live but meconium stained, or dead. Fetuses exposed to MMI in-utero were notably hypothyroid with significantly suppressed serum T3 and T4 and histological evidence of goiter. Surprisingly, fetuses from MMI-treated dams were substantially larger but appeared to exhibit non-allometric growth with an increase in girth but not length. The liver, kidney, and the corresponding fetal placenta and maternal endometrium were collected from a subset of 16 fetuses per group to evaluate the relative expression of five genes associated with thyroid hormone metabolism, including three deiodinases and two solute carriers known to transport thyroid hormone. Compensatory transcription of DIO3 was observed in all tissues evaluated, suggesting increased vertical transfer of maternal thyroid hormone at the placenta and decreased breakdown of thyroid hormone within fetal organs.</p> <p>The evaluation of thyroid hormone metabolism within the fetus and within the placenta has allowed us to differentiate suppressed thyroid hormone levels of the pig fetus under pathogenic and non-pathogenic conditions. In the context of PRRSV infection, the observed decompensation of thyroid hormone metabolism would further exacerbate the hypothyroid state and is therefore consistent with NTIS. In contrast, fetuses with induced thyroid hormone suppression following maternal exposure to MMI showed compensatory thyroid hormone metabolism in the same tissues. This indicates true hypothyroidism and clearly demonstrates a fetal capacity to respond to such endocrine disruption.</p>