Progress 08/15/17 to 09/30/19
Outputs
Target Audience:Farm animal producers and reproductive physiologists in industry and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Results from these studies were included in two PhD dissertations. These projects provided opportunities for professional development and specific research training. How have the results been disseminated to communities of interest?Presentation at international scientific meetings and publication in books and journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Significant research effort concerning improved reproduction of animals is occurring at public institutions and in private companies, and it is imperative that research of this type be publicly funded to ensure availability of results to animal agriculture and to private industry. This is particularly important for the Louisiana animal agriculture industry since it is centered around the breeding and marketing of genetically elite animals. Combining technologies such as in vitro fertilization, nuclear transfer, and cryopreservation provides phenomenal opportunities for increased efficiency of livestock production. In vitro embryo production could be timed to increase flexibility in animal production. Oocytes from genetically elite females could be better utilized, leading to a faster rate of genetic improvement due to increased selection intensity. Specific accomplishments under major goal 1, understanding the biology and underlying mechanisms of gamete development, fertilization and embryogenesis include: 1. confirmation that stimulation with FSH prior to OPU collection results in greater developmental competence compared to non-stimulated OPU or slaughterhouse-derived oocytes. RNA seq data with oocytes from these methods of collection identified differentially expressed genes in both the germinal vesicle (GV) and metaphase II (MII) stages which can be investigated as molecular markers of developmental competence; 2. The effect of heat stress on the developmental competence and global gene expression profile of oocytes through the transition from spring to summer under Louisiana conditions were assessed. RNA seq analysis identified the genes that were differentially expressed as a result of heat stress at the GV stage and MII stage. This information provided new insight into the molecular mechanisms of detrimental conditions (heat stress) on bovine oocytes that may help to reveal master regulators controlling oocyte competence; 3. Cattle under the effect of heat stress present reduced fertility. Negative effects of heat stress on the oocyte have been observed at morphological, biochemical, transcriptional, and developmental levels. This study evaluated the effect of in vivo heat-stress during the spring to summer transition on the DNA methylation and DNA hydroxymethylation of bovine oocytes at the GV and MII stage. Total oocytes obtained per cow were higher in cold compared to hot months. Percent of Grade 1 oocytes was higher in cold compared to hot months. There was no difference in percent of Grade 2 oocytes between cold and hot months. There was a lower percent of Grade 3 oocytes in cold compared to hot months. Maturation rate (IVM) was not different between cold and hot months. There was no difference between cold and hot months in DNA methylation and DNA hydroxymethylation of GV stage and MII stage oocytes; 4. DNA methylation is crucial in the epigenetic regulation of mammalian embryogenesis. Using both single cell/embryo reduced representation bisulfite sequencing (RRBS) and single-cell/embryo whole genome bisulfite sequencing (WGBS), stage-specific genome-wide dynamics of DNA methylation was characterized in bovine sperm and immature, in vivo, and in vitro matured oocytes as well as in vivo developed early embryo stages.This study provides the first comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos. These data will serve as an important reference base for embryos produced by assisted reproduction such as in vitro fertilization and cloning as well as models for human early embryo development. Specific accomplishments under major goal 2, refining methods for production of genetically enhanced animals to improve livestock production efficiency include: demonstration that oocytes collected by OPU had decreased mitochondrial density and increased incidence of irregularly-shaped mitochondria as well as decreased vesicle density. FSH oocytes had a decreased density of cortical granules, especially noticeable following maturation. Oocytes recovered by conventional OPU had the poorest developmental rates, beginning with poor cleavage rates. Follicles collected from OPU are a heterogeneous population of follicular sizes, potentially resulting in a high variability in oocyte competence.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Diaz, F. A., E. J. Gutierrez, B. A. Foster, P. T. Hardin, and K. R. Bondioli. 2019. Effect Of In Vivo Heat Stress On DNA Methylation And DNA Hydroxymethylation Of Bovine Oocytes. Reproduction, Fertility and Development 31:183
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Foster BA, Gutierrez EJ, Jiang Z, Bondioli KR. The effect of bovine oocyte retrieval methods on oocyte gene expression and competence. 51th Annual conference of SSR, New Orleans, LA, 2018
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gatenby, L. and K. R. Bondioli. 2019. Decondensation Of Bovine Spermatozoa After Dithiothreitol Treatment. Reproduction, Fertility and Development 31:214.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B. A. Foster, B. A., E. J. Gutierrez, and K. R. Bondioli. 2019. The Effect Of Different Bovine Oocyte Recovery Methods On Oocyte Ultrastructure Pre- And Post-In Vitro Maturation. Reproduction, Fertility and Development 31:133.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Diaz, F. A., E. J. Gutierrez, B. A. Foster, P. T. Hardin, and K. R. Bondioli. 2019. Effect Of In Vivo Heat Stress On DNA Methylation And DNA Hydroxymethylation Of Bovine Oocytes. Reproduction, Fertility and Development 31:183.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Jiang, Z., F. A. Diaz, E. J. Gutierrez, B. A. Foster, P. T. Hardin, and K. R. Bondioli. 2019. Effect Of Heat Stress On Oocyte Developmental Competence And Global Gene Expression Dynamics In Bos Taurus Crossbred Beef Cows. Reproduction, Fertility and Development 31:187.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Effect of heat stress on the epigenetic profile of bovine oocytes and embryos. Louisiana State University Ph D dissertation. Fabian Diaz. 2018.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Characterizing cytoplasmic maturation and improving competence of bovine in vitro matured ooctyes. Louisiana State University Ph D dissertation. Brittany Foster. 2018.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Farm animal producers and reproductive physiologists in industry and acedemia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Results from these studies were included in two MS thesis, one Ph D dissertation, and a honors thesis for an undergraduate student. These projects provided opportunities for professional development and specific research training. How have the results been disseminated to communities of interest?Presentation at international scientific meetings and publication in books and journals. What do you plan to do during the next reporting period to accomplish the goals?For major goal 1, we will continue to analyze the RNA seq data from oocytes and compare these results with RNA seq data being generated from oocytes and embryos in which specific genes have been down regulated and/or knocked out by gene editing techniques. These studies have an overall objective of identifying major genetic drivers of embryo development. For major goal 2, we will continue studying the effect of vitrification of oocytes turning our attention to mitochondria number, distribution, and function post vitrification. Eventually we would like to continue these studies to determine if effects on mitochondria function in the oocyte are reflected later in embryo development. We are also studying Intra Cytoplasmic Sperm Injection with bovine oocytes with the objective of using this as a method of delivering gene editing tools to very early stage embryos.
Impacts
What was accomplished under these goals?
For major goal 1, it was confirmed that stimulation with FSH prior to OPU collection results in greater developmental competence compared to non-stimulatedOPU or slaughterhouse derived oocytes. RNA seq data with oocytes from these methods of collection identified differentially expressed genes in both the germinal vesicle and metaphase II stages which can be investigated as molecular markers of developmental competence. For major goal 2, we demonstrated that exposure to DMSO and Glycerol based vitrification solutions as well as vitrification with these solutions results in minor or no change in epigenetic marks such as DNA methylation and histone acetylation. The effects of cryoprotectants and vitrification are more extensive for cytoplasmic structures such as the meiotic spindle. It was further shown that exposure to cryoprotectant solutions (without vitrification) resulted in zona hardening in mature oocytes with glycerol having a greater effect than DMSO. When oocytes were vitrified with these cryoprotectant solutions, there was no significant effect on zona hardening due primarily to a large increase in variability between oocytes. For gene editing of porcine fibroblast cells, it was shown that CRISPER/Cas9 genomic editing with homology directed repair could be used to target specific changes in the porcine Amyloid Precursor Protein gene in porcine fibroblasts. Several factors including electroporation method and type of donor DNA affected the efficiency of editing.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Bondioli, K.R. Cloning of livestock species. In; Neiman, H and Wrenzycki, C. (eds). Animal Biotechnology 2: Emerging Breeding Technologies. Springer. 2018.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Diaz, F. A., E. J. Gutierrez, E. Cramer, D. Paccamonti, G. Gentry, and K. Bondioli. 2018. Pregnancy rates following low-temperature storage of large equine embryos prior to vitrification. Journal of Equine Veterinary Science. (2018): 12-16.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Xu, W., B. A. Foster, M. Richards, K. R. Bondioli, G. Shah, and C. C. Green. 2018. Characterization of prostate cancer cell progression in zebrafish xenograft model. International journal of oncology 52: 252-260.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Rogers, K. D., B. A. Foster, E. J. Guiterrez, F. A. Diaz, and K. R. Bondioli. 2018. Effects of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Zona Pellucida Hardening in Mature Bovine Oocytes. Reproduction, Fertility and Development 30: 160-160.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Diaz, F. A., E. J. Gutierrez, B. A. Foster, P. T. Hardin, and K. R. Bondioli. 2018. Effect of Heat-Stress on Antral Follicle Count and Serum Concentration of Anti-Mullerian Hormone in Bos taurus Crossbred Beef Cows. Reproduction, Fertility and Development 30: 192-193.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Gutierrez, E. J., F. A. Diaz, B. A. Foster, and K. R. Bondioli. 2018. Effect of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Meiotic Spindle of In Vitro Matured Bovine Oocytes. Reproduction, Fertility and Development 30: 163-163.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Foster,BA, FA Diaz, EJ Gutierrez, KR Bondioli. 2018. The effect of follicular wave phase at time of OPU on bovine oocyte cytoplasmic maturation and developmental competence. Reproduction Fertility and Development 30:218
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Lanjewar, S. N. and K. R. Bondioli. 2018. Optimization of transfection efficiency for CRISPR/Cas9 induced genomic editing in porcine fibroblast cells. Reproduction Fertility and Development. 30:243
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Effects of dimethly sulfoxide and glycerol based vitrification protocols on zona pellucida hardening in mature bovine oocytes. Louisiana State University Masters Thesis. Kaci Rogers. 2018.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
The effect of cryoprotectant exposure and vitrification on epigenetic marks in immature (Germinal Vesicle stage) and mature (Metaphase II stage) oocytes. Louisiana State University Masters Thesis. Emilio Gutierrez. 2018.
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Progress 08/15/17 to 09/30/17
Outputs
Target Audience:Farm animal producers and reproductive physiologists in industry and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Studies conducted as part of this project will become part of a graduate student MS thesis and provide opportunities for professional development and specific research training. How have the results been disseminated to communities of interest?Presentation at international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue to study the effect of vitrification on epigenetic marks and gene expression in oocytes and embryos. Study specific molecular markers determining oocyte developmental competency. Investigate factors effecting CRISPER/Cas 9 genome editing methods in porcine somatic cells.
Impacts
What was accomplished under these goals?
This project isinvestigatingmethods of cryopreservation of oocytes and embryos from domestic species. Successful cryopreservation of gametes is a major component of any advanced breeding program for domestic animals. Studies conducted as part of this project defined some of the parameters affecting the survival of oocytes following cryopreservation. Specifically, bovine oocytes were exposed to different cryoprotectant solutions used in vitrification and the effect of that exposure to epigenetic marks determined. It was found that glycerol based vitrification solutions significantly increased DNA methylation in mature oocytes. Pre-implantation genetic diagnosis is a potential tool for enhancement of genetic improvement in domestic animals. This technology involves taking a small biopsy from an early stage embryo and conductinggenetic analysis for different alleles of key genes. In order to obtain enough material for this analysis, whole genome amplification is conducted which assumes that all parts of the animal genome areequally amplified. To test this assumption we conducted whole genome amplification on embryo biopsies and conducted PCR amplification of several genes. We found that all of the genes we selected for amplification were successfully detected in approximately 70% of the embryo biopsies. These results indicate the refinement of the whole genome amplification method is in order to ensure reliable genome analysis for embryo biopsies.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gutierrez, EJ, FA Diaz, BA Foster, PT Hardin, KR Bondioli. 2017. Effect of dimethyl sulfoxide or glycerol based vitrification protocols on the DNA methylation of bovine cumulus oocyte complexes. Reprod. Fertil. Dev. 29:131
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lanjewar, S. and K. Bondioli. 2017. Feasibility of whole genome amplification of lysed cells from embryo biopsies for pre-implantation genetic diagnosis. Reprod. Fertil. Dev. 29:154.
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