Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T08:58:10Z (GMT). No. of bitstreams: 1 Marinho_ArianeCassiaSalustiano_M.pdf: 2625506 bytes, checksum: 4b9c6de7f98fb06e145d2b86f601d06f (MD5) Previous issue date: 2013 / Resumo: Lipopolisacarídeos (LPS) - endotoxinas são capazes de estimular células a produzirem citocinas pró-inflamatórias envolvidas na destruição tecidual periapical. Os objetivos do presente estudo foram: 1) Verificar e quantificar endotoxinas em canais radiculares infectados de dentes com periodontite apical crônica e sua relação com sinais e sintomas clínicos de origem endodôntica; 2) Avaliar a efetividade do preparo químico-mecânico (PQM) com hipoclorito de sódio (NaOCl) 2,5%; clorexidina gel (CLX) 2% e soro fisiológico (SS-controle) na eliminação de endotoxinas; 3) Avaliar o potencial inflamatório do conteúdo endodôntico, antes (C1) e após a instrumentação do canal radicular (C2) com NaOCl 2,5%, CLX 2% ou soro fisiológico e após uso de EDTA 17% (C3) em cultura de células de macrófagos quanto à produção de citocinas pró-inflamatórias - IL-1?, TNF-?. Amostras foram coletadas de 30 canais radiculares com necrose pulpar e presença de lesão periapical em C1, C2 e C3 utilizando cones de papel estéreis/apirogênicos. Endotoxina foi detectada em 100% dos canais radiculares estudados, representada pela mediana de 18,70 EU/mL. Dentes com presença de dor à percussão e exsudação intracanal foram relacionados com níveis elevados de endotoxina (p<0,05). Após o PQM, significativa redução de endotoxina foi obtida nos canais radiculares: NaOCl 2,5% + EDTA17% (99,75%), CLX gel 2% + EDTA 17% (98,27%), SS+ EDTA 17% (98,71%) (p<0,05). IL1- ? e TNF-? foram produzidos por macrófagos estimulados pelo conteúdo endodôntico (C1>C2>C3). Foi possível concluir que 1) Endotoxinas estavam presentes em todos os casos investigados, apresentando níveis mais elevados nos dentes com dor à percussão e exsudato intracanal. 2) O preparo químico-mecânico foi eficaz na redução do conteúdo de endotoxinas, independente da substância química auxiliar testada; 3) O potencial inflamatório do conteúdo endodôntico foi demonstrado pela produção de IL1- ? e TNF-?, exercendo maior atividade inflamatória contra macrófagos nas amostras iniciais, quando comparado com as obtidas após o preparo químico-mecânico / Abstract: Lipopolysaccharide (LPS)-endotoxin are able to stimulate cells to produce proinflammatory cytokines involved in the periapical tissue destruction. The aims of this study were: 1) To verify and quantify endotoxin in infected root canals of teeth with apical periodontitis and correlate the levels with clinical signs and symptoms of the primary endodontic infections; 2) To assess the effectiveness of chemo-mechanical preparation (CMP) with 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) and saline solution (SS), as negative control, for the elimination of endotoxins; 3) To evaluate the inflammatory potential of endodontic content before (C1) and after the CMP (C2) with 2.5% NaOCl, 2% CHX or SS and after final rinse with 17% EDTA (C3) in macrophage cell culture regarding the production of proinflammatory cytokines - IL-1 ? and TNF-?- via the enzyme-linked immunosorbent assay (ELISA). Samples were collected from 30 canals with pulpal necrosis and apical periodontitis in C1, C2 and C3 using sterile non-pyrogenic/paper points. Endotoxin was detected in 100% of root canals studied, represented by the median of 18.70 EU/mL. Teeth with tenderness to percussion and intracanal exudate were related to high levels of endotoxin (p< 0.05). After CMP, significant reduction of endotoxin was obtained in root canals instrumented with 2.5% NaOCl + 17% EDTA (99.75%), CHX 2% + 17% EDTA (98.27%) and SS + EDTA 17% (98,71%) (p< 0.05). IL1-? and TNF-? were produced by the macrophages in response to the endodontic content (C1>C2>C3). It was possible to conclude that 1) Endotoxin were present in all cases investigated, showing higher levels in the teeth with tenderness to percussion and intracanal exudate; 2) CMP was effective in reducing the endotoxic content, regardless of the auxiliary chemical substance; 3) The inflammatory potential of endodontic content was demonstrated by the production of IL1-? and TNF-? in all cases. The infectious content present in the root canal in the initial samples exerted greater inflammatory activity against macrophages compared to the residual content after CMP / Mestrado / Endodontia / Mestra em Clínica Odontológica

Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T00:41:43Z (GMT). No. of bitstreams: 1 Melo_EstevaoAzevedo_M.pdf: 1050744 bytes, checksum: b28d2d7f1bf1683ed75a9b3e47166ad2 (MD5) Previous issue date: 2015 / Resumo: A ácido graxo sintase (FASN) é a enzima responsável pela biossíntese endógena de ácidos graxos e apontada como uma oncoproteína metabólica, por favorecer a proliferação e sobrevivência das células tumorais nas quais sua expressão é elevada. Vários são os compostos capazes de inibir a atividade de FASN, dentre eles o orlistat (Xenical®), que possui efeitos antiproliferativos previamente mostrados em células de câncer de mama, próstata, boca e melanoma. O sistema imunológico apresenta um importante papel na prevenção e defesa do organismo contra neoplasias malignas. As células do sistema imune que se infiltram nos melanomas são produtoras de uma vasta gama de citocinas, dentre elas interleucina 12 (IL-12) e interferon gama (IFN-?) que favorecem uma resposta imune bem sucedida contra os tumores, porém, as células dos melanomas possuem capacidade de produzir interleucina 10 (IL-10) e fator de crescimento transformante beta (TGF-?), capazes de inibir as células imunocompetentes, favorecendo a progressão tumoral e disseminação metastática. O objetivo deste estudo foi avaliar a secreção das citocinas IL-10, IL-12, IFN-? e TGF-? pelas células de melanoma murino B16-F10 após tratamento com orlistat. Para isto, inicialmente determinou-se a dosagem de orlistat capaz de inibir a proliferação celular em 50% (IC50). Em seguida, as células foram tratadas por 24 e 48 horas, quando realizou-se a quantificação da secreção das citocinas por ELISA. Após 24 horas de tratamento, observou-se aumento da secreção de IL-10 e IL-12, no entanto, após 48 horas de tratamento não foi detectada diferenças estatisticamente significantes na secreção de ambas as citocinas, quando comparadas aos seus controles. IFN-? e TGF-? não foram detectáveis. Assim, os resultados desta pesquisa mostram que o tratamento com orlistat alterou a produção das citocinas IL-10 e IL-12, sugerindo que o tratamento promove um equilíbrio entre estas citocinas pró e anti-inflamatórias nas células estudadas / Abstract: Fatty acid synthase (FASN) is the enzyme responsible for the endogenous biosynthesis of fatty acids suggested as a metabolic oncoprotein by promoting proliferation and survival of cancer cells. Several compounds are known to inhibit FASN activity, including orlistat (Xenical®), which has antiproliferative effects in breast, prostate, and oral cancer as well as melanoma cells. Melanoma is an aggressive malignant tumor of melanocytes with high propensity for metastatic spread and resistant to chemotherapy. The immune system plays an important role in the prevention and defense against malignant neoplams. In fact, immune cells that infiltrate melanomas produce a wide range of cytokines, such as interleukin 12 (IL-12) and interferon gamma (IFN-?), which favor a successful immune response against the tumor. However, melanomas cells are able to produce interleukin 10 (IL-10) and transforming growth factor beta (TGF-?) and in turn inhibit immunocompetent cells, favoring tumor progression and metastatic spread. The aim of this research was to evaluate the effect of the FASN inhibitor orlistat on the secretion of the cytokines IL-10, IL-12, IFN-? and TGF-? by B16-F10 mouse melanoma cells. For this purpose, we first searched for the IC50 Of orlistat in B16-F10 cells. Then, cells were treated for 24 and 48 hours with the drug and the secretion of cytokines quantified by ELISA. After 24 hours of treatment the secretion of IL-10 and IL-12 was increased, however, after 48 hours there were no statistically significant changes in the secretion of both cytokines, compared to their controls. IFN-? and TGF-? were not detectable. Thus, the results of this study show that the treatment with orlistat change the production of IL-10 and IL-12, suggesting a balance between the secretion of pro- and anti-inflammatory cytokines / Mestrado / Estomatopatologia / Mestre em Estomatopatologia

Asma alérgica é uma inflamação pulmonar crônica mediada por células Th2. A Hipótese da Higiene é a teoria aceita para explicar o aumento das alergias nas últimas décadas e preconiza que a menor exposição dos indivíduos a componentes microbianos prejudica a geração mecanismos imunorregulatórios. Nosso estudo abordou a modulação da resposta alérgica pulmonar induzida por ovalbumina, por cepas de bacilo Calmette-Guérin recombinantes (rBCG) que expressam fragmentos de toxinas bacterianas. Observamos que dependendo do antígeno heterólogo expresso, a imunização intranasal com rBCG pode levar tanto a supressão como a exacerbação da resposta alérgica pulmonar. Demonstramos que tanto em um contexto profilático quanto terapêutico, rBCG é capaz de suprimir os parâmetros alérgicos, e a supressão não envolve o recrutamento de células T regulatórias, é um fenômeno local, está associada a maior produção de IFN-g do que IL-4 e é dependente de IL-12. Estes dados sugerem que a infecção pulmonar por rBCG gera um milieu capaz de bloquear a migração de células Th2 inflamatórias. / Allergic asthma is an atopic disorder mediated by Th2 cells. The Hygiene Hypothesis is the accepted theory to explain the increasing in allergy in recent deacades. It states that modern health care and hygiene practices have led to a reduced exposure to microorganisms components which impairs the generation of immunoregulatory mechanisms. The present study analysed how the intranasal infection with recombinant bacillus Calmette-Guérin (rBCG) strains expressing fragments of bacterial toxins could modulate an allergic pulmonary inflammation induced by ovalbumin. We demonstrated that the rBCG strains could supress or exacerbate the allergic inflammation depending on the expressed heterologous antigen. We analysed the effect of the mycobacterial infection in a prophylactic and in a therapeutical contexts, and we have identified that for both situations the of supression allergic features does not involve the recruitment of regulatory T cells to the lungs, is a local phenomena, is associated with an increased production of IFN-g, and is an IL-12 dependent mechanism. Taken togheter, this data suggest that the rBCG pulmonary infection generates a milieu capable to supress the chemotaxis for Th2 cells, which suppress the establishment of the allergic inflammation in the lungs.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the United States. Aldo-keto reductase 1 B10 (AKR1B10) is highly expressed in colon and small intestine of normal humans, but its expression is lost or markedly down-regulated in tissues of patients with ulcerative colitis (UC) and CRC. AKR1B10 is a monomeric cytosolic enzyme with strong enzymatic activity to α, β-unsaturated carbonyl compounds, protecting cells from carbonyl lesions; AKR1B10 also mediates de novo synthesis of long chain fatty acids and membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2). To study the etiopathogenic role of AKR1B10 in UC and CRC, our lab generated AKR1B8 deficient (AKR1B8 -/-) mice. AKR1 B8 is the orthologue in mice of human AKR1B10,

More than 85% of American adults do not consume recommended amounts of fruits or vegetables. Preterm birth and hypertensive disorders of pregnancy are common adverse conditions affecting pregnancy and are leading causes of maternal and fetal morbidity and mortality. Preterm birth affects nearly 10% of all births in the United States and is on the rise, as are hypertensive disorders, which have increased by 25% over the last two decades. Pregnancy is a state of controlled inflammation, and dysregulation has been linked to preterm birth and other adverse gestational outcomes. A healthy diet is recommended in pregnancy, but little is known about the effect fruit and vegetable intake on perinatal outcomes. Omega-3 (n-3) fatty acids are essential dietary components and are known to affect inflammatory state, but little is known about how they affect inflammation in pregnancy. As current evidence is lacking, further research is needed to investigate the relationships between maternal nutrition in pregnancy, inflammation and birth outcomes. The purposes of this dissertation were to: 1) to review and evaluate the current evidence on the relationship between n-3 fatty acids and inflammation in pregnancy; 2) to evaluate the current state of the science on the impact of maternal dietary consumption of fruits and vegetables on preterm birth, gestational diabetes, preeclampsia, small for gestational age, gestational weight gain and measures of inflammation or oxidative stress in pregnancy; and 3) to examine relationships between maternal dietary intake of fruits and vegetables, cytokine expression in early and mid-pregnancy, preterm birth and gestational hypertension. A critical review of literature examining the relationship between inflammation and n-3 intake during pregnancy found that multiple inflammatory cytokines in maternal and fetal tissues were lower in women who received n-3 supplements. A second review of literature review supported an inverse relationship between fruit and vegetables and risk of preeclampsia and suboptimal fetal growth. The available evidence was insufficient to establish relationships between fruit and vegetable intake and gestational diabetes, preterm birth or inflammation. A study evaluating the relationships between maternal fruit and vegetable intake, inflammation and birth outcomes was conducted. This study provided evidence supporting a relationship between first and second trimester cytokine expression and maternal dietary intake of fruits and vegetables. Those who met recommended vegetable intake in the first trimester had higher first trimester serum CRP, IL1-α, IL-6 and TNF-α and lower first trimester cervicovaginal IL-6 levels. Those who met recommendations for first trimester fruit intake had 56% lower risk for preterm birth. Those who met second trimester vegetable intake recommendations had more than twice the risk of developing gestational hypertension. The results of this dissertation provide support for the beneficial effects of omega-3 fatty acids and fruit and vegetable intake in pregnancy. Maternal intake of these dietary components may promote optimal immune status during pregnancy. Supplementation of maternal omega-3 fatty acids may help regulate inflammation via the anti-inflammatory effects their bioactive eicosanoids exert. Fruit and vegetables have antioxidant and anti-inflammatory effects that may also help balance the inflammatory state during pregnancy. These dietary components may help promote favorable immune status during pregnancy and reduce risk of adverse perinatal outcomes such as poor fetal growth, hypertensive disorders of pregnancy and preterm birth.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22327号 / 医博第4568号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 江藤 浩之, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22830号 / 医博第4669号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 武藤 学, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The longevity of high productivity sows in the herd has become a challenge in pig production. Several factors may contribute to increased mortality rates observed over the past few years as well as lower retention rates of young sows. Chronic inflammation and metabolic disorders are conditions that sows had evolved over the years together with the greater productivity. This dissertation underlines the immunomodulatory effects of using yeast fermentation product fed to lactating sows. In the interest of determining patterns of local and systemic immune response, a new methodology to access cytokine profiles in puerperium sows was developed. In Chapter 2, one hundred-forty sows were used to evaluate the effects of two different <i>Saccharomyces cerevisiae</i> fermentation product (SCFP), a liquid source (LIQ) and a dry source (XPC®; Diamond V), on sow and litter performance. Sows were fed a common gestation diet until d 112 of pregnancy and then allotted to one of four treatments: 1) Control diet (CON), 2) CON + 15 mL/d of LIQ from d 112 to weaning (LIQ), 3) CON + 0.20% of XPC from d 112 to weaning (DRY), and 4) DRY + 15 mL/d of LIQ from d 112 to d 7 post-farrowing (D+L). Colostrum immunoglobulin concentrations were estimated using Brix refractometer. Plasma of piglets (2/sow) was collected 24 h after birth for immunocrit ratio analysis and for determination of plasma IgA and IgG concentrations. Lactation water and feed intake (ADFI) were recorded daily. Post-weaning follicle growth was evaluated by transrectal ultrasonography. Sows had the same initial BW (P > 0.13) but those fed any SCFP were heavier at weaning (P = 0.03) while not affecting sow backfat and loin depth (P>0.05). Overall, sows fed SCFP had greater ADFI than CON fed pigs (P < 0.01) while water intake, reproductive performance (total born, stillborn, weaned) did not differ among treatments (P > 0.05). Sows fed LIQ had the greatest ADFI on weeks 1, 2, 3, and overall compared to CON (P < 0.05). Litter ADG from SCFP treatments tended to be greater than CON (P = 0.10) and litter weight variability was lower (P = 0.10). No treatment effects were observed in colostrum Brix values (P > 0.77), in the piglet plasma IgG and IgA, and serum immunocrit ratio (P > 0.21). The average daily post-weaning follicle growth was greater for SCFP treatments than CON (P = 0.05). The wean to estrus interval was shorter for sows fed LIQ than CON and DRY (P < 0.01).<br><div><br></div><div>In Chapter 3 a non-invasive methodology to assess cytokine profiles from post-partum uterine lavage is described. The uteri of fourteen second and third parity sows were flushed with sterile saline solution (0.9%) on days 2, 4, and 14 post-parturition. Uterine fluid collected was immediately centrifuged and the supernatant stored at -20°C. Samples were freeze-dried, re-suspended in sterile saline (2 mL), and stored at -80°C. Cytokine profiles of the uterine fluid were evaluated using a multiplex ELISA panel including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Cytokine concentrations were calculated relative to protein content (pg/mg of protein). IFN-γ and TNF-α were lower than the limit of detection in most samples (5/38 and 1/38, respectively). IL-4 and IL-10 concentrations did not differ among days of collection (P>0.14). IL-8 was greater on day 4 than on days 2 or 14 (P<0.05). IL-1β and IL-6 were greater on days 2 and 4 than on day 14 (P<0.05).<br></div><div><br></div><div>The study presented in Chapter 4 refers to a subsample of sows (n=40) from the entire group of sows used in the study presented in Chapter 2. In this case, the methodology presented in Chapter 3 was used to evaluate SCFP effects on blood and uterine cytokine profiles in sows. A similar set of cytokines from Chapter 3 were evaluated on d 112 of gestation, d 2 and 6 post-farrowing in the plasma, and from uterine fluid collected on d 2, 4, and 6 post-farrowing. Serum C-Reactive protein (CRP) and haptoglobin concentrations were evaluated. No interactions between treatments and day of collection were observed (P>0.13). LIQ and D+L sows had the greatest serum IL-10 concentration (P<0.001) and sows fed CON tended to have lower serum concentration of IL-8 (P<0.06) vs. other treatments. Serum CRP concentrations were greatest on d 2 (P<0.001), serum IL-10 (P<0.04) and IL-4 (P<0.07) linearly decreased while serum haptoglobin (P<0.02) and IFN-γ (P<0.001) linearly increased post-farrowing. In the uterine fluid, LIQ and D+L sows had greater IFN-γ (P=0.04) concentrations and CON tended to have the least concentration of TNF-α (P=0.08). Uterine fluid IL-1 tended to linearly increase (P<0.07) and IL-6 linearly decrease (P<0.01) post-farrowing. LIQ sows had the greatest daily feed intake and CON the least during the first week of lactation (P=0.04).<br></div><div><br></div><div>In conclusion, feeding SCFP to lactating sows improved feed intake and litter growth while not affecting milk yield and colostrum quality. Besides improvements on litter ADG, the uniformity was better for all sources of SCFP. The liquid sources had slightly better results over the other sources and CON, including the greatest feed intake, less body weight mobilization, and a reduction in WEI. The method proposed to evaluate cytokine profiles in the uterine fluids of sows after farrowing, accomplished the objective of being a non-invasive procedure to be applied in puerperium sows. This new procedure was applied to analyze the immunomodulatory effects of SCFP. The correlations observed between the uterine and serum cytokines lead to a refined description of immune response in puerperium sows. Feeding SCFP to lactating sows stimulates the immune system allowing sows to build a desirable immune responses. Thus, the quicker resolution of acute phase reaction as demonstrated by greater daily feed intake in the first week post-farrowing can be attributed to SCFP immunomodulatory effects, ensuring better lactation performance.<br></div>

In vitro produced bovine embryos are critical to the cattle industry. However, these embryos have altered morphology, epigenetics, and metabolism when compared to their in vivo counterparts. The aim of this thesis was to alter maturation and culture media to improve the developmental competence of in vitro bovine embryos. This thesis is comprised of three experiments and one proof of concept study. Each experiment followed the same general layout: oocyte aspiration from Jersey or Holstein ovaries, oocyte maturation for 24 hours, fertilization with bull semen for 24 hours, then embryo culture for 7-8 days in 38.5 °C in 5% O2, 5% CO2 and 90% N2. A proportion of stage 7 grade 1 blastocysts were fixed and stained with Nile Red to evaluate lipid content, Mitotracker Red CMX-Rosamine to measure mitochondrial activity, or Cell Rox Green to assess reactive oxygen species (ROS). Using a confocal microscope, images were taken of each stained embryo to detect and measure fluorescence. Any stage 7 embryos that were not imaged were slow frozen and evaluated for re-expansion when thawed. Experiment 1 was a one-way treatment designed to compare the conventional maturation media (control) containing fetal bovine serum (FBS), to an alternative media replacing FBS with a human platelet lysate serum substitute (SS). Both abattoir and ovum pick up (OPU) oocytes were used. The results suggested that maturing in vitro and OPU oocytes with serum substitute maintained developmental competence, including a similar yield of embryos and re-expansion rate. Resulting in vitro SS embryos had lower lipid content (p<0.05) and ROS levels compared (p<0.05) to the FBS control. Experiment 2 was a 2x2 factorial design testing how the addition of FGF2, LIF, and IGF1 cytokines to maturation and culture media affected in vitro embryo development. The first factor was maturation media (Mcon: industry standard and Mcyt: added cytokines) and the second factor was culture media (Ccon: industry standard and Ccyt: added cytokines). The two maturation media crossed with the two culture media equated to four treatments, including a control. The results suggested that cytokine addition had no effect on blastocyst rate or re-expansion rate. The combination of MCyt x CCyt media produced the lowest lipid levels (p<0.05) while the MCon x CCon treatment led to the highest mitochondrial activity (p<0.05). Experiment 3 was a 2x2 factorial design testing how the addition of melatonin to cytokine supplemented maturation media affected embryo developmental competence. The maturation factor had two levels: no supplementation (NoM) and melatonin with cytokine supplementation (MM). The culture factor had two levels: no supplementation (NoC) and cytokine supplementation (CC). We found no difference in blastocyst or re-expansion rate between any treatments. NoM showed higher mitochondrial activity than MM (p<0.05). NoC showed higher mitochondrial activity than CC (p<0.05). The NoM x NoC treatment showed the highest lipid levels of any treatment (p<0.05). The NoM x NoC treatment showed the highest mitochondrial activity of any treatment (p<0.05). The final part to this thesis focused on the preliminary use of phasor-fluorescence lifetime imaging microscopy (FLIM) and deep imaging via emission recovery (DIVER) technologies to autofluoresce endogenous compounds and predict the viability of an embryo without the use of invasive labels. In conjunction with the University of California Irvine, we tested the technologies on morula and blastocyst stage embryos to see if developmental competence was altered. Results suggested FLIM successfully captured NADH levels and DIVER successfully captured ROS and lipid content. Future studies are planned to fully investigate the effects of the microscopes on development and to accurately predict bovine embryo viability for transfer. Overall, human platelet lysate was a successful replacement for FBS, likely due to its similar content of protein and growth factors. Neither cytokine nor melatonin supplementation had conclusive results, further trials are needed to fully determine effectiveness.