Progress 03/01/14 to 02/28/19
Outputs
Target Audience:Professional animal scientists Animal producers Research scientists in animal science Research scientists in reproductive physiology Veterinarians Undergraduate students Graduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate students learned skills necesary for functioning in an anmal science environment, including animal laboratory study. The post-doctorat learned skills needed to surpervisinga research laboratory including scientific writing and oral presentations. How have the results been disseminated to communities of interest?Reports at scientific meeetings, in scientific publications as well as discussions in regional projects and in seminars locally. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact Mastitis, an endemic disease of dairy cattle, can be acute or chronic. Mastitis reduces milk marketed and causes losses of pregnancy if the infection occurs shortly after insemination. Increased intervals to pregnancy create financial loss and increased culling. Pregnancy loss is due to conception failure, i.e., early embryo loss. Mastitis causes inflammatory changes, but mimicking the disease in cattle is expensive both in materials for research and animal maintenance. We developed a model by injecting 2.5 μg/kg lipopolysaccharide (LPS), an endotoxin, into sheep five days after mating. Experiments used that model to understand effects of inflammation on the maintenance of pregnancy. From that knowledge, prophylactic treatments might be developed to reduce economic losses. For each experiment, ewes were restrained by cradling its head in the crook of the animal holder's arm. Injections and blood sampling were via the jugular vein. Samples were taken frequently and then at 9 and 16-18 days after injection when animals were restrained for ultrasonographic visualization of the reproductive tract for observation for pregnancy. Animals show variable degrees of normal inflammatory acute responses to LPS: increased body temperature (1-2 C), nasal discharge, coughing, watery eyes, vaginal discharge, depressed appetite, lethargy, and restlessness. Animals recover within 12 hours. Body temperatures were recorded at blood sampling, and other symptoms were monitored throughout. The expectation was that animals challenged with LPS, or with the mediator released by immune cells in the animal, TNF, would show the most changes and exogenous anti-inflammatory agents, e.g., dexamethasone or NSAID would lessen these changes. Outcomes We developed a sheep model using endotoxin that allows replicating of causal factors of mastitis in dairy cows. Scientists can use this model to investigate factors associated with early reactions (APR) to illnesses. In dairy cattle, body condition was not a predictor of reproductive success, but the prevalence of health disorders in early postpartum decreased pregnancy success. Changes in biomarkers in milk, blood, and uterine secretions occurred with no relationship among tissues. Neither hematocrit nor plasma haptoglobin in the late transition period was indicative of disease status or a useful predictor of fertility Although pretreatment with dexamethasone, which blocks Toll-like receptor 4 actions, decreased clinical signs and some APR, neither it nor flunixin meglumine, which blocks synthesis of prostaglandins, prevented LPS-induced pregnancy loss . Acute infections may cause embryonic loss by shifting the animal's immune system to be pro-inflammatory. Enhancing the dietary intake of ω-3 PUFAs could reduce the inflammatory response to gram-negative bacteria. LPS challenge during early pregnancy stimulates expression of pro-inflammatory genes in both the uterus and WBC. The LPS-immune reaction has a dimorphic expression; females were more responsive, and jugular catheterization exacerbated the APR in both sexes. Different gene regulation in response to LPS explains the different APR in breeds, which could impact management practices of animal producers. Objective 1 Aim 1. Effects of LPS-induced acute phase response (APR) with or without blocking prostaglandin synthesis. In Exp. 1, mated (d 0), mixed breed ewes received (d 5) either 2.5 µg LPS or LPS and 2.2 mg/kg BW (at -6 and 0 h) flunixin meglumine (Banamine). LPS increased BT for six h. Inhibition of prostaglandin with flunixin meglumine attenuated fever but did not affect TNF, which increased dramatically, but transiently. More ewes (p = 0.16) were pregnant after Banamine (6/9) than LPS alone (3/9). In Exp 2, mated, mixed breed ewes received (iv) saline, LPS, 1 μg/kg TNF at 0 and 30 min, or LPS after 0.14 mg/kg BW dexamethasone (DEX) at -12 and 0 h. More control and TNF ewes (13/19) lambed than ewes (6/19) treated with LPS or LPS and DEX. DEX blunted LPS-induced fever and increases in TNF and blocked mucosal and lethargy responses. Exogenous TNF slightly increased BT and half showed a mucosal reaction, whereas none were lethargic. All three treatments increase SAA, but only LPS affected Hp. Aim 2. Method of bleeding and sexually dimorphic responses to LPS. Dorset and Suffolk wethers and ewes were assigned to venipuncture + LPS venipuncture + PBS, jugular catheter + LPS, and jugular catheter + PBS. WBC was measured at collection (jugular catheter or venipuncture). LPS increased cortisol, fever, lethargy and mucosal discharge, and decreased WBC and appetite. Indwelling catheter increased WBC but not cortisol. However, LPS+catheters had higher cortisol. Wethers had higher fevers after LPS, but ewes showed more leucopenia. Aim 3: Breed and gene expression of pro- and anti-inflammatory genes in LPS ewes. Suffolks had higher fevers and behavioral/physical responses and an enhanced APR (Hp and cortisol) than Dorsets. After LPS, CXCL8, TLR4, SOD2, and C3 increased, while PPARG and MRC1 decreased. Dorsets had higher TLR4, IL10, PPARG, FOXP3, and SOD2; whereas, Suffolks had higher IL6, IFNG, PTGS2, and C3. Suffolks had higher IL6 and C3 in response to LPS. Gene expression may predispose Suffolks to be more responsive to infection than Dorsets. Aim 4: Metabolic stress could exacerbate immune responses to bacterial challenges. At 45 d prepartum 72 Holsteins were categorized: lean (< 3.25), moderate, and over-conditioned (> 3.75) BCS. We related changes in BCS with yield and health status at d 0- 10 and d 20-26 on conception to first service (CR). BCS changed most in over-conditioned cows but did not impact CR. Disease status d 0 to d 26 postpartum affected CR (0 vs. 42.5%). WBC at d 20-26 but not hematocrit was negatively related to pregnancy. Hp at d 20-26 was not associated with disease status. In exp 2, changes in WBCs and Hp were not correlated significantly between milk or blood and the uterus. CR was negatively related to calving difficulty, reproductive diseases, and incidence of health disorders during 0-26 DIM. Plasma Hp at 20-26 DIM was not associated with illness but was a marker at subsequent periods. Illness status between 0-26 DIM was the most predictive variable for CR with positive uterine culture as the second most predictive. Plasma Hp 54-60 DIM, calving difficulty, clinical illness, and uterine culture produced the model most predictive of fertility. Objective 2 Aim 1 Feeding ω-3 polyunsaturated fatty acids could alter APR genes Ewes fed flaxseed in Spring exhibited less fever, and WBC recovered sooner after LPS than in Fall. Also, Spring ewes fed flaxseed had lower CXCL8 and TLR4 and higher IL-10 and decreased cortisol and TNF and increased Hp than Fall when they had higher fever and cortisol. After LPS, CXCL8, TLR4, C3, and SOD2 increased, while PPARG, FOXP3, and MRC1 decreased. LPS- flaxseed-ewes had less CXCL8 and TLR4 in Spring than Fall when C3 increased. Aim 2 Uterine genes that potentially mediate LPS-embryonic loss. Mated, mature Suffolks received either PBS or LPS. LPS-ewes had fever and lower WBC. After 4-6 hours, uteri were placed on ice, and 0.5 mm uterine body biopsies were placed into RNAlater. RNA was extracted and sequenced (RNA Seq). Quality of sequencing was determined with FastQC, with no trimming. Reads were mapped to Ensembl version 3.1 of the sheep genome using STAR, and genes were enumerated using featureCounts and Ensembl annotation. Fold changes in gene expression were determined using DESeq2 in R. LPS ewes had 1,444 genes downregulated, and 1425 upregulated: for TLR-2, TLR-3, and TLR-4 and MyD88, STAT4, NFκB, MAPK, STAT3, NOD1, NOD2, NLRP, and PPARγ, downstream transcriptional regulators, and IL1β, ΙL-6, IL-18, and IL-33, pro-inflammatory cytokines. PGDH, was 6-fold higher in control than LPS, which would increase uterine PGs in LPS ewes.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Nikpour, Hoda. "Influence of Sex and Blood Collection Method on Innate Immune Response Following Lipopolysaccharide Administration in Sheep." Order No. 10275071 West Virginia University, 2017. Ann Arbor: ProQuest.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Hadfield, Jessalyn Marie. "Characterization of the Immune Response to Lipopolysaccharide in Early Pregnant Ewes as a Model to Study Bacterial Infection Induced Embryonic Loss." Order No. 10275836 West Virginia University, 2017. Ann Arbor: ProQuest.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Bowdridge, E. C., Dailey, R. A. (2017). Systemic LPS Challenge during Early Pregnancy in Sheep Stimulates Expression of Pro-Inflammatory Genes in the Uterus. 99th Annual meeting of The Endocrine Society. April 14 Orlando Fl. Endocrinology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Nikpour, H., Bowdridge, E. C., Dailey, R. A. (2017). Effect of Sex and Catheter on Cortisol Levels and Leukocytes Following LPS Challenge in Sheep. Biology of Reproduction. Society for Study of Reproduction 50th Annual Meeting.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Graham, M. R. (Graduate Student), Bowdridge, E. C., Bowdridge, S. A., Holaskova, I., Elsasser, T. H., Dailey, R. (2018). Effect of lipopolysaccharide-induced immune responses on pregnancy loss in ewes. (vol. 08, pp. 421431). Wuhan: Open Journal of Animal Science. https://file.scirp.org/Html/5-1400704_87905.htm
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Hadfield, J. M. (Graduate Student), Bowdridge, E. C., Holaskova, I., Elsasser, T. H., Dailey, R. (2018). Breed specific differences in the immune response to lipopolysaccharide in ewes. Journal of Animal Science (4th ed., vol. 81, pp. 4220-4228). Champaign, Illinois: Journal of Animal Sciences. https://doi.org/10.2527/2003.814912x
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Research scientists in animal science Research scientists in reproductive physiology Veterinarians Undergraduate and graduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Three studies were conducted in a refinement of the objectives of1 and 2. Objective 1:A breed study was initiatedto determine if the response to LPS differed by the breed of ewe. Results indicated quantifiable differences after LPS challengebetween Suffolk and Dorset ewes with Suffolk ewes displaying more signs of sickness and more prolonged cortisol response. Suffolk ewes hadgreater gene expression of pro-inflammatory elements (IL-6, IFNG, PTGS2, and C3), whileDorset ewes hadgreater gene expression of TLR4 and anti-inflammatoryelements (IL-10, PPARG, FOXP3, and SOD2 (superoxide dismutase 2, which neutralizes toxic peroxide radicals). In a separate study,wethers and ewes were used to study the effect of sampling through an indwelling jugularcatheter or repeated venipuncture and their interaction with sex on circulating cortisol, leukocyte concentrations and physical behaviors following administration of LPS. During the LPS challenge, the catheter exacerbated the innate immune response and cortisol levels with males attaining higher rectal temperatureand females showed greater leukopenia. Thus, catheter placement created an additional stressor that augmented the LPS-induced immune response. Objective 2:A studyexaminedif omega-3 PUFAs in the form of supplementary whole flaxseed could reduce the inflammatory response to an LPS challenge. The response was affected by season. The flaxseed-fed ewes expressed less CXCL8 and TLR4 in the Spring but not in the Fall and had increased expression of C3in the Fall.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Graham, M. R. (Graduate Student), Bowdridge, E. C., Bowdridge, S. A., Holaskova, I., Elsasser, T. H., Dailey, R. (2018). Effect of lipopolysaccharide-induced immune responses on pregnancy loss in ewes. (vol. 08, pp. 421431). Wuhan: Open Journal of Animal Science. https://file.scirp.org/Html/5-1400704_87905.htm
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Hadfield, J. M. (Graduate Student), Bowdridge, E. C., Holaskova, I., Elsasser, T. H., Dailey, R. (2018). Breedspecific differences in the immune response to lipopolysaccharide in ewes. Journal of Animal Science (4th ed., vol. 81, pp. 4220-4228). Champaign, Illinois: Journal of Animal Sciences. https://doi.org/10.2527/2003.814912x
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
"Influence of Sex and Blood Collection Method on Innate Immune Response following Lipopolysaccharide Administration in Sheep"
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Nikpour, H., Bowdridge, E. C., Dailey, R. A. (2017). Effect of Sex and Catheter on Cortisol Levels and Leukocytes Following LPS Challenge in Sheep. Biology of Reproduction. Society for Study of Reproduction 50th Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Bowdridge, E. C., Dailey, R. A. (2017). Systemic LPS Challenge during Early Pregnancy in Sheep Stimulates Expression of Pro-Inflammatory Genes in the Uterus. 99th Annual meeting of The Endocrine Society. April 14 Orlando Fl. Endocrinology.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
adfield, Jessalyn Marie. "Characterization of the Immune Response to Lipopolysaccharide in Early Pregnant Ewes as a Model to Study Bacterial Infection Induced Embryonic Loss." Order No. 10275836 West Virginia University, 2017. Ann Arbor: ProQuest. Web. 30 Jan. 2019.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Nikpour, Hoda. "Influence of Sex and Blood Collection Method on Innate Immune Response Following Lipopolysaccharide Administration in Sheep." Order No. 10275071 West Virginia University, 2017. Ann Arbor: ProQuest. Web. 30 Jan. 2019.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Scientific: The results of the investigation reached other investigators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student training towards MS thesis. How have the results been disseminated to communities of interest?Results presented to animal and dairy scientists What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Mastitis has major economic impacts to the dairy industry in terms of lost profitability due to milk losses and early culling of cattle. We developed an alternative animal model (ewe) to dairy cattle to examine the factors associated with acute responses to endotoxin challenge. This model allows replicating the causal factors of diseases in an animal that shows comparable responses but is less expensive to use in experiments. Other scientists can use this model to investigate factors associated with early responses to disease. Objective 1: In the initial objective, we compared the impact of tumor necrosis factor alpha (TNF), which is assumed to be a key determinant in inducing inflammatory responses to endotoxin, to that of lipopolysaccharide (LPS). Bred ewes were challenged with LPS or TNF and monitored for inflammatory reponses on day 5 after mating. Challenge with LPS induced acute phase responses (fever, mucosal responses, lethargy and increased serum TNF, haptoglobin and serum amyloid A) and decreased pregnancy rates. Exogenous TNF alone induced mucosal and serum amyloid A responses but did not affect pregnancy status. In a related objective, we investigated whether inhibiting the increase in prostaglandin with flunixin meglumine after an endotoxin challenge would affect responses to the endotoxin. Predictably, flunixin meglumine attenuated fever but did not increase pregnancy rate in LPS-treated ewes. Objective 2: In the second objective, we examined whether the Toll-like receptor 4 initiates the inflammatory cascade by prior treatment with dexamethasone. Pre-treatment with dexamethasone in ewes on day 5 after breeding blocked fever and mucosal and lethargic responses that occur in response to LPS challenge and attenuated increases in TNF and haptoglobin that also occur in response to LPS but did not ameliorate LPS-induced pregnancy loss. These results are compatabile with assumption that activation of the Toll-like receptor 4 by injection of LPS initiate the acute phase responses. Outcome: We learned that acute challenge with LPS in early pregnant ewes mimics bacterial-induced pregnancy losses in dairy cattle. Hence, the ewe can be used as a model for the dairy cow. Although pretreatment with dexamethasone, which blocks Toll-like receptor 4 action, decreased clinical signs and some innate immune responses, neither it nor flunixin meglumine, which prevents synthesis of prostaglandins, prevented LPS-induced pregnancy loss. Because responses to LPS were not mimiced by treatment with TNF alone or by inhibiting prostaglandin synthesis lead to the conclusion that factors in addition to TNF and prostaglandin define the full acute response to an endoxin challenge.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Wise, M. J., Akers, K., Bowdridge, E. C., Holaskova, I., Dailey, R. (2016). 042 Changes in Body Condition from Prepartum to Postpartum on Reproduction in Dairy Cattle. Journal of Animal Science, 94(supplement1), 21⿿21.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Professional animal scientists, graduate students, animal producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate studentshave been trained further in conducting research; the post-doctoral student has gained further experience in maintaining a laboratory and in instructing graduate and undergraduate students in research. How have the results been disseminated to communities of interest?The results have been presented at professionalresearch society meetings as well as annual research symposiumat the College What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, experimentswill address theeffect of breed on the responsivenesss to the immune challenge with LPS. Further, the response of castrated male sheep will be contrastedto the response in same age ewes.
Impacts
What was accomplished under these goals?
An immunological balance has to be established during pregnancy that protects the mother yet tolerates the semi-allogenic fetus. To understand the innate immune response during bacterial infections, such as mastitis, that may cause early embryonic loss, a gram negative bacteria cell wall component lipopolysaccharide (LPS) sheep model was used. The objectives were to characterize the immune response to an acute LPS challenge and to determine if a differential response exists between breeds. Estrus was synchronized by CIDR insertion for 5 days, followed by 20 mg of PGF2? at CIDR withdraw. Early pregnant ewes, 5/6 days post coitus (dpc), received, via the jugular vein, either phosphate buffered saline (PBS; 3 ml) (Dorset n = 10; Suffolk n = 16) or LPS, (2.5 μg/kg) (Dorset n = 11; Suffolk n = 16). Blood was collected via jugular venipuncture at hour: 0, .25, .5, .75, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24. Whole blood samples were used to determine total white blood cell counts (WBCs) before centrifugation collection of white blood cells for RNA extraction. Rectal temperature and change in behavior/physical (lethargy, coughing, nasal discharge, absence of eating) appearance were recorded hourly. After LPS, total WBCs decreased and returned to normal within 6 hours after challenge (LPS by hour p < 0.0001). Rectal temperature increased after LPS and returned to normal 6 hours after challenge (LPS by hour p = < 0.0001). Dorset ewes displayed higher mean temperature at each hour (p = 0.0284); however, Suffolk ewes exhibited a greater change in temperature from hour zero in response to LPS (p = 0.0314). LPS Suffolk ewes displayed more behavior changes than LPS Dorset ewes (p = 0.0029). Real-time PCR was performed for expression pro-inflammatory (CXCL8, IL6, IFNG, C3, TLR4, MRC1, PTGS2) or anti-inflammatory (IL10, SOD2, FOXP3, PPARG) factors. LPS caused an increase in neutrophil chemoattractant, CXCL8 (p = 0.0003), LPS receptor, TLR4 (p = 0.0036), antioxidant enzyme, SOD2 (p < 0.0001), and complement component 3 (p = 0.0028), while the anti-inflammatory transcription factor, PPARG (p = 0.0056), and mannose receptor, MRC1 (p = 0.0028) decreased. Dorset ewes had higher expression of TLR4 (p = 0.003), IL10 (p = 0.0448), PPARG (p = 0.0016), FOXP3 (p = 0.0001), and SOD2 (p = 0.0002). Increased TLR4 expression suggests an enhanced ability to respond to LPS, but other genes work to control the intensity of the immune response. Suffolk ewes had higher expression of IL6 (p = 0.0007), IFNG (p = 0.0153), PTGS2 (p = 0.0002), and C3 (higher expression p = 0.007; greater increase in response to LPS p = 0.0004). These genes are pro-inflammatory, which may explain increased sickness behavior and temperature in Suffolk ewes. Pregnancy status was assessed at 25 dpc with transrectal ultrasound with 45.6% of Dorset LPS, 40% Dorset PBS, 87.5% Suffolk LPS, and 50% Suffolk PBS ewes being pregnant. In summary, acute infections may cause embryonic loss by shifting the environment to be pro-inflammatory and breed specific differences are observed in this response.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2016
Citation:
Wise, Melissa Jill. Potential biomarkers to predict fertility in dairy cattle. West Virginia University, ProQuest Dissertations Publishing.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target Audiences:Scientists working in complementary areas, producers, graduate students, technicians, undergraduate students, and veterinary medicine students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two students received MS degrees How have the results been disseminated to communities of interest?Results reported at national and regional meetings What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Restraint involved in blood sampling appeared to affect the immune response in control as well as treated ewes. Hence, a study will determine whether the routine method of taking jugular samples by restraining the head and withdrawing blood from the jugular vein initiates a "flight or fight response," which results in elevated number of lymphocytes. Lymphocyte concentrations in animals that are restrained, as done previously, will be compared to those from animals with catheters in the jugular vein. The use of catheters will eliminate the need to extend and restrain the head in a manner that could initiate a stress and immune response. Forty animals will be randomly assorted into a 2 X 2 X 2 experimental design. The groups will be divided by type of blood withdrawal (restraint versus catheter), sex (male versus female), and treatment (LPS versus PBS). Catheters will be placed into the jugular vein the day prior to the experiment. On the day of the experiment animals will receive via the jugular vein either 2.5 mL of 0.1% BSA/PBS (controls) or 2.5 mL of 2.5 μg/kg of LPS (Sigma Altrich, St. Louis, MO). Jugular samples (6-8 mL) will be collected in EDTA treated tubes every 30 min post challenge for 180 min, and every hour through 12 h and again at 24h. Leukocyte differentials will be measured to determine if sampling method initiates an acute immune response, and, if so, is it related to sex status (castrated male versus intact female)? Objective 2: Feeding diets high in omega-3/omega-6 PUFAs have been shown to increase pregnancy per artificial insemination and to reduce pregnancy loss. In response to an LPS challenge, studies show animals fed PUFAs have reduced levels of pro-inflammatory cytokines. The objective of this project is not only to identify genes associated with the innate immune response that could cause embryonic loss but also to examine if feeding a diet high in omega-3 polyunsaturated fatty acids (PUFAs) could alter the immune response as well as to examine if there are breed differences in the immune response to LPS. Aim 1: To investigate genes associated with immune mechanisms of early embryonic loss via gram-negative bacterial infections, ewes will receive, via the jugular vein, on the 5th d post coitus (0 dpc) either PBS (3ml) or LPS (2.5 µg/kg) to induce an innate immune response. Rectal temperature and any change in behavior/physical appearance will be recorded hourly through 6h and then at 9 and 12h. Blood will be collected on the day of CIDR , which will be used for synchronization,withdrawal, d4, 0dpc (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24h), and 9, 16, and 25dpc. Total white blood cell counts will be determined. Samples will be centrifuged with plasma being stored for ELISAs and the buffy coat being collected for RNA extraction and gene expression analysis. Progesterone will be measured in plasma from samples collected 10 and 25dpc. Detection of pregnancy will be on 25dpc by transrectal ultrasonography and number of lambs born will be recorded at lambing. To assess the stress and acute phase response after LPS exposure, cortisol and two acute phase proteins (serum amyloid A and haptogloblin) will be measured in plasma at various time points on 0dpc. To examine changes in the innate immune system, real-time PCR will be performed for expression analysis of genes that can be categorized as pro-inflammatory (IL-6, TNF-α, IFN-γ, C3, COX2), anti-inflammatory (IL-10, SOD2), chemokine (IL-8), receptor (TLR4, MRC1) or transcription factor (NF-κB, FOXP3, PPARγ). Aim 2: Whole flaxseed, which is high in alpha-linolenic acid and can be converted into the longer chain omega-3 FAs in the body, will be fed to Dorset ewes. Diets will consist of either 250g whole flaxseed (55% whole flaxseed + 45% sheep ration) or a control (20% corn, 14% soybean meal, 21% soybean oil, 45% sheep ration) diet at 2.2kg per head/day. The control diet will be formulated to match the energy, protein, and fat content of the flaxseed. Aim 2: To examine whether different sheep breeds have a distinct immunological response to LPS, Suffolk ewes will be fed the control diet (20% corn, 14% soybean meal, 21% soybean oil, 45% sheep ration) and treated with LPS or PBS and compared to the Dorset ewes on the control diet and treated with LPSor PBS. Ewes will be on pasture and fed their respective diets starting on the day of CIDR withdrawal through 25dpc. Dorset ewes will be split between diets and treatment giving four groups: flaxseed with PBS (n=10), flaxseed with LPS (n=11), control with PBS (n=10) and control with LPS (n=11). Suffolk ewes being fed only the control diet will be dividedinto PBS (n=16) and LPS (n=16) groups. Objective 3: Inhibition of prostaglandin (eicosanoid) synthesis by treating with flunixin meglumine attenuated the increase in body temperature but had no effect on the corresponding changes in either systemic (jugular) or pelvic area (inferior vena cava) concentrations of TNFα, which increased dramatically, but transiently, after challenge with LPS. The objective of this study will be to determine the pathway of the response from binding of the antigen to Toll-like receptor 4. Animals will be checked for estrus and inseminated twice using a different ram for each natural service. Ewes will be assigned at random on d5 after observed first mating to one of six treatment groups with 10 animals in each group: injection of LPS (control), administration of aspirin prior to and during LPS response, glucocorticoid (dexamethasone), omega-3 fatty acids or a combination of the treatments. Expected outcomes are that LPS will result in inflammatory responses, which will be ameliorated by treatment with aspirin, glucocorticoids, omega-3 fatty acids, or some combination of these treatments. During the experiment, frequent blood samples will be taken based on the outcome of the previous experiments, to assess for changes in SAA, Hp, TNFα, PGFM and progesterone, the hormone primarily associated with maintenance of pregnancy.
Impacts
What was accomplished under these goals?
The effects of proinflammatory pathway triggers on pregnancy success was assessed in early pregnant ewes. Effects of endogenous cytokine activation (via LPS), direct exogenous tumor necrosis factor-α (TNF-a) and TNF-a+Dexamethasone (TNF-a+Dex) administration were compared in regard to pregnancy rate. Dorset×Texel ewes (n = 38) weresynchronized for estrus and bred to fertile rams (d 0). On d 6, ewes were assigned to receive via the jugular 2.5 mL containing either PBS (n =9), 2.5 μg/kg LPS (n = 9), 1 μg/kg TNF-a (n = 10) at 0 and 30min, or 2.5μg/kg of LPS after 3.5 mL containing 0.14 mg/kg BW im Dex (n =10) at −12h and 0h. Behavioral changes and rectal temperatures (BT) were recorded before challenge and hourly for 12h. Jugular blood wascollected before challenge, every 30 min for 3 h, hourly until 12 h, at 24, 36, and 48 h, and on d 10 and d 26 for progesterone. At d 26, pregnancy was examined using ultrasonography. The proportion of ewes pregnant was analyzed by Chi-square and Fisher's exact test with preplanned contrasts. Other data were analyzed by repeated measures ANOVA. Treatment with LPS resulted in peak BT at 4 h (40°C; control 38° C; P< 0.05), increased lethargy, mucosal response, white blood cell (WBC) count (9.8 × 106; control 9.1 × 106; P< 0.05), serum TNF-a (0.9 ng/mL; control 0.2 ng/mL; P< 0.05). Treatment with LPS+Dex resulted in peak BT at 4 h (39°C), but was blunted by Dex. Additionally, Dex blocked mucosal and lethargic responses and attenuated LPS-induced increases in TNF-a and did not improve pregnancy rate compared with LPS alone (P> 0.05). TNF-a treatment increased BT at 1 h (39°C), did not induce lethargy; 5/10 ewes showed a mucosal response; and WBC counts did not change but high concentrations of TNF-a were not sustained. More control and TNF-a ewes (15/19) were pregnant than LPS or LPS+Dex ewes (9/19; P= 0.045). In summary, an inflammatory response was elicited by LPS and resulted in reduced proportion of ewes pregnant regardless of Dex administration. Treatment with TNF-a did not affect proportion of ewes pregnant, perhaps due to low serum TNF- a levels or different pathway components stimulated by LPS. These data show that a sustained inflammatory response to LPS decreased pregnancy rate and may involve a complex cascade of inflammatory mediators elicited by LPS.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Akers Keli Ann. 2014. Changes in body condition from prepartum to postpartum on health and reproduction in dairy cattle. M.S. Thesis, West Virginia University
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Graham, Megan Renee. Effects of Lipopolysaccharide Induced Inflammatory Response on Early Embryo Survival in
Ewes. West Virginia University, ProQuest, UMI Dissertations Publishing,
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Effects of lipopolysaccharide (LPS) induced inflammatory response on early embryo survival in ewes. M. R. Graham, E. C. Bowdridge, S. A. Bowdridge, I. Holaskova, T.H. Elsasser, and R. A. Dailey, West Virginia University, Morgantown
J. Anim. Sci. Vol. 93, Suppl. s3/J. Dairy Sci. Vol. 98, Suppl. 2 p 421
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Changes in Body Condition from Prepartum to Postpartum on Reproduction in Dairy Cattle. M. Wise*, K. Akers, E. Bowdridge, I. Holaskova,R. A. Dailey West Virginia University, Morgantown, WV J.Anim.Sci.Vol 94 Suppl 1 p 21
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Progress 03/01/14 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Based on the results, restraint involved in blood draws might have affected the immune response in control as well as treatment ewes as the control groups followed the same pattern of lymphocyte concentrations. Hence, a study will be completed to determine if the routine method of blood collection by restraining the head and drawing blood from the jugular vein, which may initiate a "flight or fight response," was the cause. Lymphocyte concentrations will be compared in animals that are restrained for blood draws as done previously and from animals with catheters in the jugular vein. In addition, the response will be compared between wethers and ewe lambs to have animals at comparable ages and weights to examine for an effect of sex . Forty animals will be assorted randomly into a 2 X 2 X 2 experimental design. The groups will be divided by blood draw type (restraint versus catheter), gender (male versus female), and by treatment (LPS versus PBS solution). Catheters will be placed into the jugular vein the day prior to the experiment. On the day of the experiment, animals will receive via the jugular vein either 2.5 mL of 0.1% BSA/PBS (controls) or 2.5 mL of 2.5 μg/kg of LPS. Jugular samples (6-8 mL) will be collected in EDTA treated tubes every 30 min post challenge for 180 min, and every hour through 12 h and a sample at 24h. Leukocyte differentials will be measured to determine if sampling method initiates an acute immune response.
Impacts
What was accomplished under these goals?
Thirty-eight Dorset x Texel ewes were synchronized for estrus and bred to fertile rams (day 0). On day 5 or 6, ewes were assigned to receive via the jugular vein either 2.5 mL of 0.1% BSA/PBS (controls, n=9), 2.5 mL of 2.5 μg/kg of LPS (n=9), 5 mL of 1 μg/kg of TNF-α (n=10) in two bolus injections given thirty minutes apart, or 2.5 mL of solution containing 2.5μg/kg of LPS after having received 3.5 mL of solution containing 0.14 mg/kg BW im dexamethasone (Dex, n=10) at -12 and 0 hours. Behavioral changes and rectal temperature were recorded before challenge and every hour for 12 hours post challenge. Blood was collected from the jugular vein before challenge, every 30 minutes for 3 hours, every hour until 12 hours, and once at 24, 36, and 48 hours, and processed soon after collection for total white blood cell counts. A white blood cell differentiation was determined by staining and counting one hundred cells classified among monocyte, lymphocyte, eosinophil, neutrophil, or basophil cell types. Plasma was collected after centrifugation and stored at -80°C. Assays were conducted for TNF-α, SAA, and Hp. Jugular samples were collected in EDTA treated tubes on days 9 or 10 and 25 or 26 pc for determination of concentrations of progesterone. At day 25 or 26 pc, detection for pregnancy was examined ultrasonographically. Treatment of ewes with LPS or LPS+Dex on day 5 or 6 after mating initiated an inflammatory reaction resulting in a change in body temperature for six hours in LPS ewes; the respose was blunted by DEX. The TNF-α was not as effective in eliciting fever response with an elevation only at 1 hour and elicited no mucosal responses. Treatment with DEX blocked the mucosal and lethargic responses but not the leucocytosis and increased concentrations of neutrophils induced by LPS. The TNF did not induce lethargy; only half of those ewes showed a mucosal response; and white blood cell and neutrophil counts did not change as dramatically. Similarly, LPS increased plasma TNF and the reponse was attentuated by DEX. All three treatments yielded an increase in SAA, but only LPS majorily increased Hp concentrations. Interestingly, counts of lymphocytes appeared to decrease comparably in all ewes, while counts of monocytes increased in all ewes, suggestive of a common stressor. The number of lymphocytes returned to starting levels by 24 hours. More control ewes and ewes treated with TNF-α remained pregnant than ewes treated with LPS or LPS+Dex (p=0.05). In summary, an inflammatory response and APR was elicited via treatment with LPS and resulted in reduced pregnancy. Dexamethasone attenuated the inflammatory response indicative of blocking Toll-like receptor 4 but did not increase pregnancy outcome. Likewise, treatment with TNF-α did not affect pregnancy outcome because embryonic loss was induced either by an alternative pathway or the exogenous dose was not sufficient; high concentrations were not sustained with two injections of TNF-α..
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Graham, Megan Renee. Effects of Lipopolysaccharide Induced Inflammatory Response on Early Embryo Survival in Ewes. West Virginia University, ProQuest, UMI Dissertations Publishing, 2014. doc 1616758225
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