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Influences of Delayed Meiotic Resumption on Porcine Oocyte Competency

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Influences of Delayed Meiotic Resumption on Porcine Oocyte Competency Influences of Delayed Meiotic Resumption on Porcine Oocyte Competency A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Veterinary Biomedical Sciences University of Saskatchewan Saskatoon By HAYDER AL-SHANOON ©Copyrights Hayder Al-Shanoon, April 2016. All rights reserved PERMISSION TO USE POSTGRADUATE THESIS In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may take it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Request for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of Department of Veterinary Biomedical Sciences Western College of Veterinary Medicine 52 Campus Drive University of Saskatchewan Saskatoon, Saskatchewan S7N 5B4 Canada i ABSTRACT In pigs, successful in vitro production of embryos (IVP) faces significant challenges due to many defects in various steps of this technique, including: in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos. Poor cytoplasmic maturation of oocytes is considered the main factor for low rate of IVP of pig embryos. One of the main causes of this problem is the insufficient time for cytoplasm of oocytes to be matured due to the loss of communications between cumulus cells and oocytes. Recently, a new strategy emerged to inhibit meiotic resumption and provide more time to accumulate important factors for oocytes competence. The overall hypothesis of this thesis was that delaying meiotic resumption during the in vitro maturation will have a positive effect on oocytes competence for fertilization and embryo development in an in vitro environment. In our first study (Chapter 3), the first experiment was designed to evaluate the effect of natriuretic peptide precursor type C (NPPC, 100-1000 nM), cyclic guanosine monophosphate (cGMP, 2-10 µM), cyclic adenosine 3, 5monophosphate (cAMP, 0.5-2 mM), nitric oxide (NO, 0.5-2 mM), and steroid hormones (testosterone, 500-1000 ng/ml ; progesterone, and estradiol, 100 ng/ml for each) to delay resumption of nuclear maturation during in vitro maturation of porcine cumulus oocyte complexes (COCs). Meiotic resumption was inhibited at an optimum concentration of 1000 nM for NPPC, 1mM for dbcAMP and NO, and 100 ng/ml for estradiol. The other substances (cGMP, testosterone, and progesterone) could not inhibit the meiotic resumption. Using the inhibitory substances, the second experiment evaluated the effect of delaying nuclear maturation for different times during IVM process (2, 6, 18, 22 hrs). Estradiol, NPPC, cAMP, or a combination of these substances were able to inhibit the meiotic resumption for 22 hrs. Three treatments (NPPC + estradiol (NE); cAMP (CA); NPPC + estradiol + cAMP (CANE)) were chosen to delay nuclear maturation (n= 450-500 COCs /group) and evaluate their effects on embryo development. Interestingly, blastocyst rate ranged from 6.0-8.7% and did not differ between the treatment groups or treatments to the control (9.4%, porcine follicular fluid in maturation medium, Pff). The selected treatment could be considered a base to develop a medium without animal source products like Pff. ii As a part of the second experiment (Chapter 4), the effects of inhibitory substances (NE, CA, CANE) on molecular pathway and gene expression profile of in vitro matured pig oocytes was evaluated. Microarray technology was used to assess the genes expression profile associated with different treatments compared to Pff group. The results revealed that 120, 55, and 167 genes were differentially expressed in matured oocytes for NE, CA, or CANE, respectively. The data were analyzed using EASE program to classify genes into different ontology categories. While DAVID and PANTHER programs were used to identify the most significant molecular pathways induced by each treatment. The results showed CA treatments significantly up-regulated mitochondrial ribosomal protein L3 ( MRPL3) and heat shock protein 90kDa alpha (HSP90AA) genes. Also, Janus kinase / signal transducer and activator of transcription (JAK/STAT) and Platelet-derived growth factor (PDGF) pathways were up-regulated. The NE treatments up- regulated several genes: MYC associated factor X (MAX), mitogen-activated protein kinase 9 (MAPK9), and v-rel reticuloendotheliosis viral oncogene homolog (REL) involved in cellular proliferation and the development of oocytes. While CANE was found to up-regulate IGF1 gene. The results of this study showed that using different substances to delay meiotic resumption would change the response of molecular pathways and genes expression of matured oocytes. However, the effects of these treatments groups on embryo development were equivalent to Pff group. In our final study (Chapter 5), in vitro produced pig embryos were vitrified using two different devices: a closed device (French Straws (FS), n=127) or an open device (Open Pull Straw (OPS), n=115). After vitrification and warming, the embryos were graded based on their morphological quality and cultured for 48 hrs. The quality of embryos was also evaluated at 48 hrs of embryo culture. The results showed that almost 85% of the vitrified embryo were quality 1 after the warming procedure for both devices. However, the quality of embryos (Grade 1) slightly decreased in OPS compared to FS after 48 hrs of culture (FS=88, OPS=72). Vitrified-warmed embryos did not grow from morula to blastocyst, suggesting sub-optimal culture conditions or iii cryo-damage affecting the development of embryos. These results indicated that using FS in a vitrification protocol can be used to preserve in vitro produced pig embryos on long-term basis. This thesis was demonstrated that delaying meiotic resumption of pig oocytes using NPPC+estradiol, cAMP or NPPC+estradiol+cAMP treatments can support embryo development by improving oocyte competence through different molecular effects. Also, these in vitro produced embryos could be stored in liquid nitrogen in a closed devices (French Straw). iv ACKNOWLEDGEMENTS First and foremost, I’d like to extend my deepest gratitude to my advisor, Carl Lessard, without whom this dissertation would not have been possible. I cannot thank him enough for all his supporting and encouragement during my study. He allows me to learn and practice research and to make the mistakes that helped me become a better researcher. There are just not enough words to express how much his guidance has meant to me. • I also sincerely thank my committee members Jaswant Singh, John Harding, Michael Dyck and Karen Machin and I would like also to thank my external examiner, Francois Richard from Laval University. • Several people have helped me both personally and technically throughout my graduate career. A special thank you goes to Philippe Godin (summer student), Stephen Tsoi (University of Alberta), Kosala Rajapasksha, Pamela Hind and Crissandra Auckland (Agriculture and Agri-foods Canada). • Also, I would like to acknowledge my funding sources: Iraqi Ministry of Higher Education and Scientific Research (MOHERS), Agriculture and Agri-foods Canada - The Canadian Animal Genetic Resources Program, EmbryoGENE program, Department of Veterinary Biomedical Sciences and Associate Dean, Research • I would extend my thankful to Department of Scholarships and Cultural Relations in MOHERS and Iraqi cultural office – Ottawa especially Dr. Asaad Toma Alomran, the Iraqi cultural attache in Ottawa for all his help and support. Without his involving, I would not be able to continue my study smoothly until finishing my defence. So, I want to appreciate his help. • I am indebted to my parents and for all of their encouragement, love and support over years of my study in Canada while I struggled through the challenges of completing my degree. Finally, I am eternally grateful to my wife not only for the much needed laughs but also for being there through each and every day of this work and never allowing me to give up. Without you, I would never have believed I could do this. v DEDICATION I would like to dedicate this thesis to my wife, for her unyielding love…. without her support, I would not go progress. Without you, I never would have tried vi TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................ i ABSTRACT ................................................................................................................................. ii ACKNOWLEDGMENT............................................................................................................... v DEDICATION ............................................................................................................................. vi TABLE
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