Source: UNIVERSITY OF MISSOURI submitted to NRP
DEVELOPMENT OF DEFINED CONDITIONS FOR PIG EMBRYO CULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0207652
Grant No.
2006-35203-17282
Cumulative Award Amt.
(N/A)
Proposal No.
2006-00883
Multistate No.
(N/A)
Project Start Date
Aug 1, 2006
Project End Date
Jul 31, 2009
Grant Year
2006
Program Code
[41.0]- Animal Reproduction
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
ANIMAL SCIENCES
Non Technical Summary
Culture of cells or creation of embryos in the laboratory generally requires the addition of fetal calf serum or bovine serum albumin to the culture medium. Unfortunately each lot or batch of serum is different and this results in different degrees of development of the cells or embryos. In addition, culture in serum containing medium has resulted in animals that are abnormal at birth (large offspring syndrome). Studies that have evaluated gene expression in the early pig embryo have identified a number of genes that code for cell surface receptors. Many of these increase in abundance during the first week of embryo development. We hypothesize that addition of the ligands for these receptors will permit us to remove serum from the culture medium and retain or improve the present rate of development and embryo quality. So, the objectives are to add individually or in combination a series of ligands to culture medium which does not contain any added serum. Once an optimum combination of ligands is identified, then we will make a direct comparison to in vivo cultured embryos. Improvements in the culture system will impact production agriculture, as we will have a defined, and hence reproducible, system to create embryos for research and potentially commercial production. Also we will have a system to improve methods to create transgenic and cloned embryos.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30135101050100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
The original objectives were to develop a culture system, capable of promoting high rates of embryo development, that is composed of purified molecules such that the composition of the medium is known it its entirety, i.e. no BSA or FCS. Our revised objectives are basically the same, but we will not be able to do the differential methylation hybridization. Compounds to be tested include: Laminin, Low Density Lipoprotein (LDL), Epidermal Growth Factor (EGF), Glycine, Fibronectin, Platelet Derived Growth Factor (PDGF), Progesterone, and Lysophosphatidic acid (LPA). Subsequent experiments will compare the development in vitro to that obtained in vivo and evaluate embryo quality, as well as produce offspring to check for gross abnormalities.
Project Methods
The mRNA for receptors that increase in abundance during development from the germinal vesicle stage to the blastocyst stage have been identified in previous work. We intend to produce embryos in vitro in conditions where BSA and fetal calf serum have been removed. To this culture system we will add purified ligands for the receptors that have been previously described. The rates of development will be compared. In addition the quality of the blastocysts will be determined by evaluating the number of inner cell mass nuclei and trophectoderm nuclei. The best system will be used to compare directly to in vivo produced and cultured embryos.

Progress 08/01/06 to 07/31/09

Outputs
OUTPUTS: The goal of the project was to identify and characterize molecules that could substitute for BSA in culture media for the development of pig embryos. We identified two molecules, LDL and NMDA, that could partially substitute for BSA. The data generated was published as abstracts and we are in the process of finalizing our first manuscript. PARTICIPANTS: Lee Spate performed most of the experiments. Dr Prather oversaw all data collection and analysis. Clifton Murphy helped with surgeries and oocyte aspiration. TARGET AUDIENCES: Data were presented at scientific meetings and will be submitted to peer reviewed journals for publication. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The true impact of the research finding will only be know as the research community and industry either adopt the new information and use it to further improve the development of pig embryos in vitro or if they ignore it. We anticipate that this is really the next step toward a completely defined culture system for pig embryo production.

Publications

  • Spate, L.D., B.K. Bauer, C.N. Murphy, R.S. Prather. 2009. N-methyl-D-aspartic acid can be used to partially replace bovine serum albumin in culture medium. International Embryo Transfer Society annual meeting, Reprod. Fertil. & Devel. 21:164.
  • Telugu, B.P.V.L., L. Spate, R.S. Prather, J.A. Green. 2009. Acid peptidase activity released from in vitro produced porcine embryos. Mol. Reprod. Dev. 76:417-428. PMID 18937336.


Progress 08/01/07 to 07/31/08

Outputs
OUTPUTS: One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop a chemically defined media based off the embryos biological and physiologic make up. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3 fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mM) would improve development of embryos in an already chemically defined medium. Oocytes were matured in vitro and fertilized in vitro. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: 1) PZM3, 2) PZM4, or 3) PZM4+0.5 mM NMDA. Embryos were cultured until day 7. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure. The percentage developed to blastocyst was 1) 47.5%, 2) 29.6% and 3) 36.1% respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was 1) 25.8, 2) 19.6 and 3) 22.9, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number, however, with the addition of 0.5mM NMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media. PARTICIPANTS: Lee Spate (Research Specialist) performed most of the experiments. He was assisted by K. Walker (DVM student working on a research project), C. McHughes (a MS student), Clifton Murphy (a post-postdoc), and Bethany Bauer (a MS student). Dr. Prather oversaw and directed the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results generated over the past year show that Ionotropic receptors may be present in early embryos and have a major impact on development. These receptors interact with glutamate and glycine, other compounds that are important for embryo culture. These results shed light on a receptor that has otherwise been ignored, and this moves us one step closer to developing defined conditions for embryo development.

Publications

  • Spate, L.D., K. Walker, C. McHughes, R.S. Prather. 2008. Addition of low density lipoprotein to chemically defined protein embryo culture medium can partially replace bovine serum albumin. International Embryo Transfer Society annual meeting. Reproduction, Fertility & Development 20:136-137.


Progress 08/01/06 to 07/31/07

Outputs
Embryo culture media typically contain undefined biological such as bovine serum albumin (BSA). Our goal is to develop chemically defined culture media that is based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos as various stages of development and determined that the message for the Low Density Lipoprotein Receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of Low Density Lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous-oocyte complexes (COC) identified and the COCs were matured for 44 hours in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in Modified Tris buffered Medium and cocultured with 0.25X 106/mL frozen thawed porcine semen for 5 hours. The presumptive zygotes were then transferred to either Porcine Zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 30 hours cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3+20 ug/mL LDL, 3. PZM4, 4. PZM4+10 ug/mL LDL, 5. PZM4+20 ug/mL LDL, 6. PZM4+50 ug/mL LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a,b,c P<0.05). The percentage blastocyst was 51.3+/-0.09a, 51.6+/-0.09 a, 33.1+/-0.99 c, 35.8+/-0.09 c, 36.9+/-0.09 c, and 41.3+/-0.06 b for treatments 1-6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 ug/mL of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts and preliminary results suggest that the ICM to Trophectoderm ratio in the High LDL treatment group is closer to the ratio found in in vivo produced embryos.

Impacts
These results should impact all areas of in vitro production of pig embryos. Thus application of this knowledge should improve embryo transfer, cloning, and transgenic animal production.

Publications

  • No publications reported this period