Progress 07/01/24 to 02/28/25

Outputs
Target Audience:The target audience for our bovine Intra Vaginal Embryo Culture (IVC) device includes producers currently using conventional bovine Assisted Reproductive Technology (ART). These technologies include Artificial Insemination (AI), Embryo Transfer and Flushing (ET), and In Vitro Production (IVP). The most recent IETS report showedthat only 21% of the eggs collected produce a viable embryo. On average, only fourembryos are produced per procedure. Based on this data, many producers will have no viable embryos. Our project is geared towards improving this reproductive technology inefficiency. There is much interest from the interactions we have had as described below. This project is bringing a new form of cattle ART to the market. While IVC has been used successfully in humans, it was only attempted about 25 years ago in cows, and the embryos were arrested at the early cleavage stage. We are the 1st to have made a blastocyst-stage embryo in one of our early prototype trials. Given this backdrop, we are creating a technology that can be easily used by the producers and/or veterinarians who work with them. Target audience: Large animal reproductive veterinarians Beef producers Dairy producers Show cow participants Each audience has used IVP to some degree and is interested in trying new technology to improve the rapid enhancement of their herd through better breeding technology. Since July 2024 we have been in front of several diverse Ag audiences. In August we were one of the five finalists in the Elevate Ventures Rally-IN 2024 Ag and food innovations competition. This conference had over 3000 attendees in Indianapolis with a significant number related to Ag innovation. We participated in the quarterly meetings of Agrinovus Indiana, an Ag not-for-profit in our state. Here we meet face to face with various stakeholders. We presented a talk about the intersection of human and animal medicine at one of the recent meetings. Our company was featured in Hoosier Ag Today, a statewide publication this Fall. Earlier this year we were one of the Farm Bureau 2024 Ag Innovation Challenge Companies (we made the final four!) and we keep nurturing our relationship, as the Farm Bureau touches so many people in Agriculture. We have been working with LARTA on customer discovery, for the eventual Phase II SBIR grant that we will submit in early 2025. We were invited to apply to the 2025 SXSW Ag innovation conference in Austin, TX and we are waiting to hear back from them as I write this update. Lastly, we just found out that our company is one of the finalists for the Indiana Mira Awards for Ag Innovation. This is quite an honor for us. We continue to be a portfolio company of AgLaunch365. Through the Ag Ventures Alliance and the Farmers Fund, we have received investments from farmers, who will be our eventual users. They will be doing paid trials of our technology. It is hoped that this will be in Spring, 2025 in OK and TN. Changes/Problems:The only change that we have is the trial of the semen separation device to compare it to density gradient centrifugation. This is a minor protocol change and because the device has two culture chambers we can make a direct comparison with the same lot of eggs and semen. This was approved by Section Director Bob Smith, DVM. We will still use the density gradient separation per the initial protocol. What opportunities for training and professional development has the project provided?We have been working with several biomedical engineering students at CalPoly on early iterations of the newly designed semen separation device. This gives them some real-world experience with device design and development. How have the results been disseminated to communities of interest?At several of the events listed previously, we have presented some preliminary data. We are working with AgLaunch 365 and they are aware of our progress. What do you plan to do during the next reporting period to accomplish the goals? Technical Objective 1. We plan to use the device in cattle. The device has 2 embryo culture chambers. This will allow us to test the semen separation device and compare it to the density gradient centrifugation. During December 2024 we will polish the aluminum molds and use sonic cleaning to reduce the debris that we noted in our quality control testing of the 1st set of devices. This should improve the embryo growth. We plan to compare the initial polystyrene devices with polypropylene devices. The gad permeability is different for both, and we will begin the production of these polypropylene devices in early 2025. Technical Objective 2. We will confirm that the balling gun applicator works well to deliver the CIDR attached IVC device to the cow vagina. It is more streamlined than other potential options. Technical Objective 3. This will be the most important step, to confirm a pregnancy. We will synch 2-3 recipients so that we may do fresh transfers of embryos per routine. We will be able to cryopreserve embryos as well as document the quality of the IVC derived embryos. We will also be working with LARTA to submit the Phase II SBIR grant. We will hopefully be presenting at SXSW and we will be at the Mira Awards Ceremony in February 2025 as one of the Ag Innovation Finalists.

Impacts
What was accomplished under these goals? Technical Objective 1. ReproHealth is creating a bovine intravaginal embryo culture device. This device has several requirements. The device must be made of a plastic similarto conventional IVF polystyrene culture dishes. It must be non-toxic and allow CO2 and O2 to pass through the walls to modulate and support the bicarbonate-buffered embryo culture media pH and cellular respiration. It must be small enough to fit inside the cow vagina without causing vaginal discharge or irritation. Finally, it must be able to be attached to the CIDR, which is commonly used in bovine reproduction and creates a high progesterone environment, which is typically found in early gestation. In July 2025 we began a redesign of the device to include a small hole at the ends to be used to connect the device to the CIDR and we added small wings to the outer chamber to make it easier to seal the device prior to inserting it into the cow vagina. Preliminary manufacturability testing was done in September to begin the production of the Injection Molding molds that would eventually make the devices that we would use in the animal studies and sell to customers. The mold makes all six components of the device: 2 embryo culture wells, 2 lids for the wells, and 2 parts for the outer chamber that seals the device to keep vaginal flora from getting in and contaminating the culture media. There are additional silicone seals on each culture chamber to keep contamination risk low. The aluminum mold that was created produces all six parts in about 45 seconds. We received the 1st set of parts at the end of November and began Quality Control (QC) testing using the 2-cell mouse embryo assay. The human version of the IVC device uses similar QC testing. This was in vitro testing to confirm that the device would grow embryos in a CO2 incubator. Briefly, the assay involves taking 1.8 ml of pre-equilibrated culture media and placing it in each culture chamber. 2-cell mouse embryos were then thawed, 8 embryos were placed in each chamber and 4 embryos were placed in a Petri dish to be used as a control. The device was then closed and placed in the outer chamber. The seal felt very secure. The device was placed on its side in a CO2 incubator. In 5 days, we examined the contents. 70% of the 2-cell embryos became blastocysts in the device. This was slightly less than the control. We did note that there was some plastic debris and 'shards' inside the culture media. It is possible that the debris from manufacturing had a negative effect, but still, we had blastocysts development meaning the concept and plastic are working as we hoped. We immediately met with the team at Thunderbird Molding of Elkhart, IN, the manufacturer, regarding this issue. They are working to polish the molds and to make them sterile in clean rooms. This will happen during the month of December 2025. This should be in time for animal trials during January through February 2025. While were waiting for the device molds to be created, a 12-week process, we drew our attention to other factors that may affect embryo production in IVP, specifically semen preparation. I sent a letter to the Section Director, Bob Smith, DVM, to see if we could make this change to the protocol, and he agreed that we could. It will not change the actual grant needs, as the design and prototypes will be minimal in cost and can be shifted from other grant funding sections. Most IVP semen prep is done with density gradient centrifugation (DGC). DGC involves a silica nano-particle colloid suspension with an upper layer of 45% and a lower layer of 90% silica particles. Silica particles are charged and have been shown to bind the charged surface of the spermatozoa. The semen specimen is placed on the top of the colloid gradient (i.e. Percoll, Pure-Sperm, etc.) in a 15 ml conical centrifuge test tube and then centrifuged for 15-20 minutes at 300 x the force of gravity. The g-forces will pull the 'purified' spermatozoa down to the bottom of the tube. It is well known that DGC can damage spermatozoa and lead to poor embryo growth in human IVF. We wanted to develop a semen filter device that would not use silica nano-particles or centrifugation. There are human fertility corollary devices. We designed a simple filter device with a structure that fits into the 15 ml conical test tube. The best semen 'swim up,' and separates themselves from abnormal spermatozoa that have lower potential for fertilization, all without centrifugation. Initial trials are positive. When bovine semen is placed in the 15 ml conical test tube and we use either an etched polycarbonate membrane or a PET (Polyethylene terephthalate) 15 um membrane, we see improved motility of the specimen. This would presumably lead to better fertilization and embryos. About 2 weeks ago we tried this on freshly retrieved bovine oocytes after maturation. Unfortunately, the fertilization was quite poor. We believe this is due to the high concentration of glycerol used as a cryoprotectant of semen. This may be an issue going forward. We will try again and try to remove the glycerol prior to processing. This is a start, however. If we are able to process better specimens, we may be able to improve AI, as well. While not part of this project, we will make a larger device to fit into 50 ml conical tubes for boar semen purification. We tried one sample and found improved motility with extended semen, but this was a single trial. Technical Objective 2. We have been working on how to attach the device to the CIDR. We have used a zip tie and other options to attach the device to the backbone of the CIDR. Using a conventional CIDR applicator it goes into the cow vaginal pretty well with little discomfort. With the newly designed device, we have recently come up with the idea to use a modified balling gun. We added a 1.25 " silicone tube to the end of the gun, and the device fits inside and it attached to the 2 mm hole at the top of the CIDR in the middle of the wings. When we push the plunger of the balling gun, the CIDR is released and the device is placed inside the vagina. This is a more streamlined applicator setup compared to attaching the IVC device to the CIDR backbone. We are currently seeing how well this works. Technical Objective 3. At this time, we have not placed embryos in synched recipients. This is planned for the 1st of the year.

Publications