Source: MEMBRANE PROTECTIVE TECHNOLOGIES submitted to NRP
IMPROVING BOVINE EMBRYO QUALITY AND YIELD WITH NOVEL MEDIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1019552
Grant No.
2019-33610-29786
Cumulative Award Amt.
$100,000.00
Proposal No.
2019-00909
Multistate No.
(N/A)
Project Start Date
Aug 1, 2019
Project End Date
Mar 27, 2020
Grant Year
2019
Program Code
[8.3]- Animal Production & Protection
Recipient Organization
MEMBRANE PROTECTIVE TECHNOLOGIES
6363 E COUNTY ROAD 56
FORT COLLINS,CO 805249319
Performing Department
(N/A)
Non Technical Summary
Embryo transfer is one of the tools a livestock producer can utilize to rapidly increase the genetic gain within the herd, overcome low pregnancy rates during physiological stress or leverage high quality females by producing multiple offspring per female (rather than 1/year). The ability to transfer fresh or frozen embryos is important in dairy and beef cattle reproduction as well as in sheep, goats, deer and humans. Embryos can be produced in a petri dish in a process called in vitro production. Unfortunately, this is a very expensive tool in part because of the low production (~9% of eggs result in a live calf) in this fixed cost production system. That means that it costs approximately the same to produce 9 embryos as 50 embryos. Membrane Protective Technologies' (MPTI) technology GameteGuard® is poised to improve these economics to the benefit of dairy farmer and beef producers. In this Phase I research proposal, MPTI will use its proprietary technology comprised of organic, plant-derived antioxidants called GameteGuard®, to improve the yield of high quality blastocysts (embryos) in an in vitro production system. Collaboration with Colorado State University allows MPTI to assess the benefits of our technology in a well-defined embryo production system. The production of embryos requires multiple steps, transferring the developing embryo into different media. The various media simulate the transition from the site of fertilization in the female (ovary and fallopian tube) to the site of maturation (uterus). MPTI will examine the benefit of various GameteGuard® formulations in each of the four steps of in vitro embryo production.Preliminary research, wherein GameteGuard® was added ONLY to the first, or first and second medium (<16% of the total process) resulted 30 to 150% improvement in blastocyst yield as compared to the control (depending on treatment). Perhaps more importantly, the quality of blastocysts improved. One treatment resulted in 25% more grade 1 blastocysts (the best quality). This is economically important because only high quality blastocysts can be successfully frozen. These data indicate that GameteGuard® addition will substantially improve the rate of oocyte transition to high quality, freezable embryos. This phase I project will measure the impact of adding GameteGuard® to each of the incubation media in a step followed by quantifying the combined effects in Phase II. The ultimate goal of these research is optimization of a system to produce high quality frozen embryos (the most economically important product) via supplementation with GameteGuard®.The potential economic impact of this low cost technology is eclipsed by the potential gain by improving the embryo yield from 8% to 12% , a potential $14,000 benefit per 100 oocytes in high quality beef cattle. Furthermore, the economic upside of developing a technology that serves as a platform for other species is immense.This project is responsive to USDA strategic goal 2, objective 2.2 by improving the competitive nature of USDA agriculture and FY research priority 1, by improving production efficiency of agriculturally important animals.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30133301030100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3330 - Other beef cattle products;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
Embryo transfer is one of the tools a livestock producer can utilize to rapidly increase the genetic gain within the herd, overcome low pregnancy rates during physiological stress or leverage high quality females by producing multiple offspring per female (rather than 1/year). The ability to transfer fresh or frozen embryos is important in dairy and beef cattle reproduction as well as in sheep, goats, deer and humans. Embryos can be produced in a petri dish in a process called in vitro production. Unfortunately, this is a very expensive tool in part because of the low production (~9% of eggs result in a live calf) in this fixed cost production system. That means that it costs approximately the same to produce 9 embryos as 50 embryos. Membrane Protective Technologies' (MPTI) technology GameteGuard® is poised to improve these economics to the benefit of dairy farmer and beef producers. In this Phase I research proposal, MPTI will use its proprietary technology comprised of organic, plant-derived antioxidants called GameteGuard®, to improve the yield of high quality blastocysts (embryos) in an in vitro production system. Collaboration with Colorado State University allows MPTI to assess the benefits of our technology in a well-defined embryo production system. The production of embryos requires multiple steps, transferring the developing embryo into different media. The various media simulate the transition from the site of fertilization in the female (ovary and fallopian tube) to the site of maturation (uterus). MPTI will examine the benefit of various GameteGuard® formulations in each of the four steps of in vitro embryo production.Preliminary research, wherein GameteGuard® was added ONLY to the first, or first and second medium (<16% of the total process) resulted 30 to 150% improvement in blastocyst yield as compared to the control (depending on treatment). Perhaps more importantly, the quality of blastocysts improved. One treatment resulted in 25% more grade 1 blastocysts (the best quality). This is economically important because only high quality blastocysts can be successfully frozen. These data indicate that GameteGuard® addition will substantially improve the rate of oocyte transition to high quality, freezable embryos. This phase I project will measure the impact of adding GameteGuard® to each of the incubation media in a step followed by quantifying the combined effects in Phase II. The ultimate goal of these research is optimization of a system to produce high quality frozen embryos (the most economically important product) via supplementation with GameteGuard®.The potential economic impact of this low cost technology is eclipsed by the potential gain by improving the embryo yield from 8% to 12% , a potential $14,000 benefit per 100 oocytes in high quality beef cattle. Furthermore, the economic upside of developing a technology that serves as a platform for other species is immense.This project is responsive to USDA strategic goal 2, objective 2.2 by improving the competitive nature of USDA agriculture and FY research priority 1, by improving production efficiency of agriculturally important animals.
Project Methods
The in vitro production process involves 4 distinct stages to transition from oocyte to a blastocyst. Each of these phases is physiologically distinct and will have different appropriate reduction/oxidation balance. This project will identify the optimum antioxidant (GameteGuard®) formulation for each stage. If multiple medium are used within a stage (e.g. maturation), each will be amended with the same GameteGuard® formulation, or will be unamended.The 4 major steps to be tested are:Oocyte Maturation- 23 hrs; Includes HCDMM and IVM media. mature oocytes so they are receptive to fertilizationFertilization- 18 hrs -Includes FCDM medium. Fertilization with spermEmbryo Culture 1- 56 hrs: Includes HCDM-1 and CDM1 media. Progression from early cleavage stages (2-8 cells)Embryo Culture 2- 96 hrs; Includes HCDM2 and CDM2 media. Progression from 8- cell to blastocystPhase I will assess effectiveness of amendment of the media for each step with 1 of 4 different formulations of GameteGuard® (designated GG-1, GG-4, GG-7 and GG-8) with each of the steps. Within a step, the same formulation will be used even if multiple media are utilized. The different GameteGuard® formulations represent different proprietary blends of antioxidants.

Progress 08/01/19 to 03/31/20

Outputs
Target Audience:The target audiences reached during MPTI's Phase I SBIR were companies that either 1)produce media for use in embryo technologies, (2) produce embryos, or (3) professionals and farmers that utilize embryo technologies. The focus was both within the bovine industry as well as emerging porcine industry and the human industry. The efforts included attending a scientific meeting focused on embryos, meeting with companies individually, as well as meeting with individuals (veterinarians, practitioners, scientists) to determine if the technology being developed is useful, commercially relevant, and of interest. Changes/Problems:The only major change within the research was related to an issue with embryocidal plastics. A new lot of plastic eppendorf tubees was purchased. Outside of the grant, plastics were investigated and shown to be embryocidal, up to 100%, during the in vitro production of mouse embryos. The main cause was found to be from plastics shards falling from the plastic and leaching contaminants. The net result of this diversion was a required repetition of 3 rounds of experiment with the supplement that was stored in embryo-safe materials, and a limitation of the final experimental steps (CDM2) due to budget and time constraints. What opportunities for training and professional development has the project provided?This grant served to develop a professional MS. recent graduate, into a fine research technician. In addition, this grant served as an internship for a second recent professional MS student, in fulfilment of one part of her degree. Both women learned new research skills, learned how to execute a robust scientific study, and learned new skills such as fluorescent staining of embryos, and fluorescent microscopy. How have the results been disseminated to communities of interest?Results have been disseminated through verbal communication, webinar-type meetings, and in person data presentation meetings. A poster was developed for sharing at the Rocky Mountain Reproductive Conference but because of the COVID19 outbreak this was not presented. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In vitro embryo production is a rapidly growing industry in the United States and across the world with applications in cattle and other species. Yet, problems with reactive oxygen species (ROS) in the production of embryos still exist. In this study, the improvement in blastocyst quality and quantity, from supplementation of embryo production media with GameteGuard® was quantified. Oocytes were treated with one of a variety of GameteGuard® supplements at each of the four media steps (IVM, FCDM, CDM1, CDM2) of in vitro embryo production. Each medium was amended in isolation to assess the impact and optimum formulation. After blastocysts were produced, they were morphologically evaluated for stage and grade then either fixed for cell count as a measure of quality, or frozen to be evaluated outside of this study. In vitro maturation medium (IVM) amended with GameteGuard-4 resulted in 28% increase (P<0.08) in blastocyst quantity and an average 22% increase (P<0.01) in the highest quality (grade 1) embryos. Chemically defined medium 2 (CDM2) culture medium, like IVM, showed an improvement in embryo quality with GameteGuard® supplementation. The data from this robust study demonstrated (1) statistically significant improvement in economically important parameters (number and quality of embryos) when using GameteGuard®, (2) indicate supplementation requirements differ between each media step, and (3) demonstrated a significant reduction in apoptotic cells/embryo (p<0.05),meeting the points of success outlined in phase I application. In Phase II, media will be amended with the best GameteGuard® formulation for each IVP medium identified in Phase I to create a complete system. It is hypothesized this will result in an increase of >25-35% in quantity and similar increase in quality creating a significant impact in this fixed-cost system.

Publications


    Progress 08/01/19 to 03/27/20

    Outputs
    Target Audience:The target audiences reached during MPTI's Phase I SBIR were companies that either 1)produce media for use in embryo technologies, (2) produce embryos, or (3) professionals and farmers that utilize embryo technologies. The focus was both within the bovine industry as well as emerging porcine industry and the human industry. The efforts included attending a scientific meeting focused on embryos, meeting with companies individually, as well as meeting with individuals (veterinarians, practitioners, scientists) to determine if the technology being developed is useful, commercially relevant, and of interest. Changes/Problems:The only major change within the research was related to an issue with embryocidal plastics. A new lot of plastic eppendorf tubees was purchased. Outside of the grant, plastics were investigated and shown to be embryocidal, up to 100%, during the in vitro production of mouse embryos. The main cause was found to be from plastics shards falling from the plastic and leaching contaminants. The net result of this diversion was a required repetition of 3 rounds of experiment with the supplement that was stored in embryo-safe materials, and a limitation of the final experimental steps (CDM2) due to budget and time constraints. What opportunities for training and professional development has the project provided?This grant served to develop a professional MS. recent graduate, into a fine research technician. In addition, this grant served as an internship for a second recent professional MS student, in fulfilment of one part of her degree. Both women learned new research skills, learned how to execute a robust scientific study, and learned new skills such as fluorescent staining of embryos, and fluorescent microscopy. How have the results been disseminated to communities of interest?Results have been disseminated through verbal communication, webinar-type meetings, and in person data presentation meetings. A poster was developed for sharing at the Rocky Mountain Reproductive Conference but because of the COVID19 outbreak this was not presented. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? In vitro embryo production is a rapidly growing industry in the United States and across the world with applications in cattle and other species. Yet, problems with reactive oxygen species (ROS) in the production of embryos still exist. In this study, the improvement in blastocyst quality and quantity, from supplementation of embryo production media with GameteGuard® was quantified. Oocytes were treated with one of a variety of GameteGuard® supplements at each of the four media steps (IVM, FCDM, CDM1, CDM2) of in vitro embryo production. Each medium was amended in isolation to assess the impact and optimum formulation. After blastocysts were produced, they were morphologically evaluated for stage and grade then either fixed for cell count as a measure of quality, or frozen to be evaluated outside of this study. In vitro maturation medium (IVM) amended with GameteGuard-4 resulted in 28% increase (P<0.08) in blastocyst quantity and an average 22% increase (P<0.01) in the highest quality (grade 1) embryos. Chemically defined medium 2 (CDM2) culture medium, like IVM, showed an improvement in embryo quality with GameteGuard® supplementation. The data from this robust study demonstrated (1) statistically significant improvement in economically important parameters (number and quality of embryos) when using GameteGuard®, (2) indicate supplementation requirements differ between each media step, and (3) demonstrated a significant reduction in apoptotic cells/embryo (p<0.05),meeting the points of success outlined in phase I application. In Phase II, media will be amended with the best GameteGuard® formulation for each IVP medium identified in Phase I to create a complete system. It is hypothesized this will result in an increase of >25-35% in quantity and similar increase in quality creating a significant impact in this fixed-cost system.

    Publications