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Diabetic gastroenteropathy is a common complication, which develops in patients with long-term diabetes. The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known for its cytoprotective properties and plays an important role in neuronal development, neuromodulation and neuroprotection. The present study was designed to elucidate, for the first time, the impact of prolonged hyperglycaemia conditions on a population of PACAP-like immunoreactive neurons in selected parts of the porcine gastrointestinal tract. The experiment was conducted on 10 juvenile female pigs assigned to two experimental groups: The DM group (pigs with streptozocin-induced diabetes) and the C group (control pigs). Diabetes conditions were induced by a single intravenous injection of streptozocin. Six weeks after the induction of diabetes, all animals were euthanised and further collected, and fixed fragments of the stomach, duodenum, jejunum, ileum and descending colon were processed using the routine double-labelling immunofluorescence technique. Streptozotocin-induced hyperglycaemia caused a significant increase in the population of PACAP-containing enteric neurons in the porcine stomach, small intestines and descending colon. The recorded changes may result from the direct toxic effect of hyperglycaemia on the ENS neurons, oxidative stress or inflammatory conditions accompanying hyperglycaemia and suggest that PACAP is involved in regulatory processes of the GIT function in the course of diabetes.

<p>Two experimentswere  conducted  using  pigs  at different  life stages to  determine  the  effects  of dietary  tryptophan  and  water  delivered  oregano  essential  oil  on growth performance, rectal temperature, water use,intestinal integrity and gene expression of biomarkers in the face heat or transport stress. In the first experiment, 192 grow-finish pigs were used to investigate the effects of  water  supplementation  of  oregano  essential  oil  (OEO)  on  growth  performance,  water  intake, rectal temperature, intestinal integrity, and expression of genetic biomarkers during an acute heat challenge. Pigs were randomly allotted to 2 X 2 factorial arrangement of treatments with pigs being heat  stressed  or  not  and  being  supplemented  with  OEO  or  not with 8  replicate  pens  of  each treatment  with  6  pigs/pen  (4  barrows,  2  gilts  per  pen).  Water  treatments  were administered immediately, with  dosing at  47 μL/L of OEO. One-half  of  the  pigs  on  each  water  treatment remained under thermoneutral conditions (TN; 21.1C), while the other half was subjected to a 3 d diurnal, acute heat stress (HS) with 12 hours at 33.3 oC (7AM-7PM) and 12 hours at 26.7oC (7PM-7AM). Three days post-HS, temperatures were reduced back to TN for the rest of the study, and pigs remained on their water treatments. Rectal temperatures were collected in the morning and evening  of  the  heat  stress  period  on  one  barrow  and  one  gilt  in  each  pen. Jejunal  tissue was collected for subsequent histological examination and determination of gene expression. All data were  analyzed  using  the  GLM  procedure  of  SAS  (ver.  9.4).  Pigs  subjected  to  heat  stress  had reduced ADG (P < 0.003) and G:F (P < 0.008) during the 3d heat stress compared to pigs reared under thermoneutral conditions. However, post-heatstress, heat stressed pigs had compensatory gain resulting in increased ADG (P < 0.001) and G:F (P < 0.001) compared to thermoneutral reared pigs.   Overall,  there  was an  interaction  (P  <  0.006)  observed  between  water  and  heat  treatment with  OEO  increasing  ADG  in  thermoneutral  pigs  but  not  in  heat  stressed  pigs.  Similarly, interactions  between  water  and  heat  treatment  were  observed  for  ADFI  during  heat  stress  (P  < 0.004),  post  heat  stress  (P  <  0.01),  and  overall  (P  <  0.004)  from  increasing  OEO  intake  in thermoneutral pigs but not in heat stressed pigs. Rectal temperatures were higher (P < 0.001) for heat stressed pigs at the end of d 1 and 2 of the acute heat challenge compared to TN housed pigs. Pigs exposed to HS also used more water than pigs housed in a thermoneutral environment (P < 0.002). There were no differences between villi height, crypt depth or VH:CD between treatment groups  (P  >0.05).  There  was  also  no  difference  in  TP53  and  CDKNA1  gene  expression  among treatments (P > 0.10). In the second experiment, 36 barrows were used in an 18d experiment to investigate the effects of pre-weaning tryptophan supplementation on performance and intestinal integrity following  weaning  with  or  without  transport  stress  at  weaning.  Pigs  were  randomly allotted to 2 X 2 factorial arrangement of treatments of pre-weaning tryptophan supplementation or not and weaning transport or not. Pigs on the tryptophan treatment received 0.35, 0.45, and 0.55 g Trp/d in 5 day intervals, beginning 15 d prior to weaning.Tryptophan was dissolved in chocolate milk and administered by oral gavage with control pigs receiving milk only. At weaning, 4 pigs from each pre-weaning treatmentwere euthanized for collection of jejunal tissue.  Of the remaining pigs, half the pigs oneach treatment were transported for 12 h, and half were moved into individual pens  with  no  transport.  Following  transport,  all  pigs  were  individually  housed  and  provided  ad libitum  access  towater  andfeed  from  a  common  diet.  On  d  3  post-weaning,  all  pigswere euthanized for collection of jejunal tissue. Jejunal tissue was used for histological examination and for determination of gene expression. All data were analyzed using the GLM procedure of SAS (9.4).  No  effects  of  Trp  supplementation  were  observed  on  pre-weaning  (P  >  0.10)  growth. Pig BW and ADFI were unaffected (P > 0.10) by Trp supplementation and transport at weaning. Post-weaning, there was a tendency (P < 0.06) for an effect of transport on ADG as transported pigs lost weight in the 3 d post-weaning period while non-transported pigs gained slightly. Gain:Feed post-weaning  was  lower  (P  <  0.04)  for  transported  pigs  compared  to  non-transported  pigs.  No differences  were  observed  for villus base  and  mid  width,  villus  height,  crypt  depth  or  villus height:crypt   depth.   There   was   a   tendency   for   an   interaction   of   transportation   and   Trp supplementation  (P  <  0.06)  on  villi  base  width  driven  by  an  increased  villus  width  in  non-transported pigs given supplemental Trp but a decrease in villus width in transported pigs given supplemental Trp.These results conclude that these alleviating agents had minimal effects when pigs  were  stressed, however TN grow-finish pigs  benefitted  from  OEO  water  supplementation among growth performance.</p>

<p>Vitamin C inhibits the oxidation of biologically important molecules and may have a potential protective role against cancer, cardiovascular diseases, and aging. Clinically relevant models of vitamin C function are essential for understanding the role of the antioxidant in the pathogenesis of these complex diseases, and its therapeutic potential. In this thesis, we examine ascorbic acid synthesis and deficiency in animal models, and develop these animal models into powerful tools to examine specific questions of vitamin C function. This thesis first presents a review on the existing animal models for antioxidant function in human nutrition, focusing on their clinical relevance in chronic diseases. We concluded that equivocal proof of beneficial effects of high dose antioxidant supplementation has not been established, and further investigations of animal models of antioxidant function are needed to resolve outstanding questions.</p> <p>We then examined the feasibility and efficacy of an alternative vitamin C delivery method using gene therapeutic lentivirus vectors in a guinea pig model of vitamin C function. The guinea pig exhibits an inactivated gulonolactone oxidase gene (<em>Gulo</em>), which is required for endogenous ascorbic acid synthesis, and as such must acquire vitamin C from the diet. Using a lentivirus vector carrying the mouse <em>Gulo</em> under the murine cytomegalovirus (mCMV) promoter, which was previously developed as a part of my undergraduate thesis, we examined the ability of this gene therapeutic vector to mediate the expression of GULO and the production of ascorbic acid in guinea pigs. At a titre of 10¹⁰ viral particles per animal, the life of lentivirus-treated guinea pigs were prolonged by 35 days compared to the scorbutic control, which was given an ascorbic acid-free diet. Ascorbic acid was produced in the liver of the treated guinea pigs, but the amount produced was not sufficient to elevate plasma concentrations or fully correct the metabolic deficiency. We conclude that lenti-mCMV-<em>Gulo</em> is able to mediate the expression of GULO and endogenous production of vitamin C in guinea pigs.</p> <p>To test the role of vitamin C in cancer etiology and outcome, we are currently in the process of introgressing the <em>Gulo</em>^-/- inactivation mutation, developed by Maeda <em>et al.</em> in 2000, from the C57BL/6 strain background into the FVB/N strain background. The FVB/N strain is also the background for several models of <em>erbB2/neu</em> overexpression in human breast cancer, associated with increased metastasis and low patient survival rates. Taken together, this thesis develops two animal models of vitamin C function, which can be employed in future applications.</p> / Master of Science (MSc)

The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low. / Master of Science

With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.