Progress 06/01/14 to 07/31/15
Outputs
Target Audience:Target Audience The primary target audience for this technology is dairy farms that use artificial insemination and timed artificial insemination protocols. This includes farmers, inseminators, and veterinarians. We want to learn from them how to modify our products and understand how they use the technology. We limited the number of end-users for this phase because we wanted to make necessary improvements before scaling up to a larger target audience. Phase I of this project,focused onworking withinsemination technicians, veterinarians,and farmers who have dairy herds of over 1000 cows. Feed back information from the end-users provided guidelines for future product modification in terms of product design and presentation. Evaluation of the data from Phase I of this projectgave feedback on product improvements we need to make going forward. The end-users gave us feedback on the quality of the assay and its practical use. As a result, we made modifications in the portability of the assay,by creating a Styrofoam carrier that has holes to hold the test straws and can be fitted into a carpenter's apron worn around the waist. This way the technician has free hands and can work with the cows, lift the tails, and test easily. The Styrofoam carrier has masking tape next to holes that hold the straw so that the cow id can be written at the time that the assay is done. This allows for easy photography at time of testing which records the color test with the cow id written next to the photo. We also learned that there were difficulties in interpreting color values in the different light conditions and time of day. This data and feedback led us to re-evaluate the filter surface and make changes in the color reading protocol. There are two primary reasons why end-users would have interest to use the assay. The assay has to give quick accurate information as to whether or not a cow is a good risk for insemination. The assay has to fit into the management program of the farm. Farmers want a product that clearly and accurately indicates whether or not a cow is ready for insemination. This product has to be easy to use, giveresults in less than 20 seconds, and be cost effective. Farmers will buy this product if it can identify the group of cows that have greater than 50% chance conception/ insemination, because this is an improvement over the current pregnancy rate which is about 34% for herds of over 1000 that use a timed artificial breeding program. Although dairy farmersare the primary target audience for this technology; as a company interested in commercializing this technology, our target audience also included the distributors who can sell the technology to the end-users. We also engaged the interest of companies who market products for improving conception rates in dairy cows. We seecommercialization as a partnership proposition and getting our technology to the end-user requires engagement of the industry that serves the farmer. Hence, the dairy industry isalso our target audience. We engaged support for research by collaborating with the Animal Science Department at Cornell University who provided technical expertise in evaluating hormone levels in the plasma samples and also knowledge in data analysis. The analysis of the data provides feedback as to the science and statistical value of the data. Additionally we have engaged the interest of particular companies that currently market point-of-care assays for testing fertility in dairy cows. We have signed confidentiality agreements with three companies and are in conversationregarding potential partnership to combine our technology with existing products. We reached out to one company and discovered that our technology can benefit their current market, whichis focused on dairy farms in emerging economies. Farmers in these countries are forming co-operatives to sell products in bulk. They have training programs to educate themselves on new methods to improve production costs. This is a new potential market for bulk distribution The simplicity and low cost of production for our product can provide a missing link, because this product brings a cost effective method to narrow the four day window for fertility to the appropriate 12 hour window when insemination is most effective. By partnered with a distributor who already markets to this group of farmers, we can have entry into a market that we had not considered before. We have also engaged the interest of two potential manufacturing companies,that specialize in pharmaceuticals and diagnostics devices. Both of thesecompanies can provide expertise in product design and engineering specific to the components of our technology. By engaging their interest at this early stage, we hope to develop business relationships that will allow for possible future partnerships in commercial production and product development. Hence our target audience includes end-users who will use the product we develop, production companies who manufacture the products,and distributors who market the products to the end-users. Changes/Problems:1: Change in hypothesis as to which color response would give lower pregnancy rate/ artificial insemination. The project proposal stated that the insemination of cows that give a green color response to the vaginal mucosal assay will have a low pregnancy outcome. We found that the color response that gives a low pregnancy outcome is pale purple; not green. The frequency of the green color response/ total color responses is low ( < 10% of the visual responses were green in a study of 585 cows; yet, if a cow gives a green color response at the time of insemination, there is over a 50% chance she will become pregnant. In contrast, about 20% of the color responses at the time of insemination give pale purple color response and of these, less than 25% will get pregnant. Blue is the dominant color observed when testing the cows and a blue color response appears to correlate with about 40% attaining pregnancy when cow vaginal mucosa is tested at the time of insemination. 2. Problem in protocol for data color evaluation. The color reading system is an important part of this project which is to determine whether or not color interpretation is a viable system for an inseminator to identify cows with fertility problems at the time of artificial insemination. We wanted to know whether or not the color reading system is objective and reliable. Comparing visual interpretations by the end-user to the spectral L a b system developed by Filmetrics offered important feedback as to the reliability of the VACA color reading system. Our protocol chose to compare the data on color interpretation in three different ways: A. Written records were kept of direct visual interpretation by the end-user. A scale has been developed with1=purple, 2=blue, 3-green, 4 = colored ring with clear center, 5 =clear. B. A photograph was taken at the time of testing for each sample and records are kept as to date, time, and cow id. C. Spectral data was measured on a Filmetrics F10-AR spectral reading machine that uses light to differenetiate between green and red absorbency. We measured these samples using an L a b scale that is described in detail below. 3. We changed one procedure in the spectral reading. i. Rather than read the filter immediately after testing, we did the spectral readings on the photograph. This is because the filter surface was much too rough for the sensitivity of the aperture in the Filmetrics F10-AR reader. Reading the color on a smoother surface such as paper allowed for improved resolution. The same type of paper was used each time. All photos were printed on the same printer. ii. Modificationfor interpreting spectral data. Filmetrics F10-AR reader that measures color according to the L a b scale. L refers to luminosity a refers to red ( +) or green ( -) b refers to blue ( + ) or yellow ( -). We used the a spectra values for data evaluation in this study. Selection of the red/green scale for color evaluation is based on total differences in absorbance values for each color category as measured on the L a b color scale in the Filmetrics F10-AR machine in our total data base. Only the red/green color spectra showed significant differences between samples tested. The average value of for the a scale at PGF 1/3 the "a' value measured at either GnRH or TAI. The L value showed no statistical differences and the " b" did not have as much difference between readings for PGF and GnRH or TAI as observed with "a" values. There are problems in color interpretation with the spectral L a b system as applied to the VACA assay. The ROC curve for individual "a" values in the L a b scale showed the area under the ROC curve as 53%, with a sensitivity of 68% and, specificity of 38%. This reflects high variance in the filter surface. The Filmetrics F10-AR READER had a very small aperture when compared with the total surface of the assay filter. which is very rough and absorbed pigment at different intensities. The ROC analysis evaluated just 1 out of 3 measurements made for each value "a" in the L a b scale. Results demonstrated the need to engineer improvements in filter surface and possibly change the pigment. The new swab was identified and tested on 200 cows. The new swab either releases or does not releasie pigment into water. If the pigment is released, the cow is ready for insemination. Modified digital system developed.The great variation in individual digital readings that ranged from -6.000 to +6.000 led to a modified L a b scale to separate visual responses into three different categories: (--) , (++) and (+ -): whereby: (- -) refers to two samples from same cow having absorbance values in the red/green color spectra ( value a) at <-0.4000; (++) refers to two samples from the same cow having absorbance values in the red/green color spectra ( value a) at >-0.4000 and (+ -) refers to two samples from the same cow having one sample ( +) >-0.4000 and the other ( -). <-0.4000. 4. Extra time was required in this study because of delay in providing the plasma estradiol assay. We did not receive the estradiol test kits until April, 2015. The final results were not available until May 2015 and collation of the data could not be completed until this information was available. Hence our data analysis could be be not be completed until June 2015. Data analysis was not completed until July 2015. As a result e this report was not be completed until October, 2015. 5. Changes in Analysis of the Data: Initial data for color interpretations was evaluated against concurrent hormone evaluations measured in plasma samples on the same day that the color evaluation was done on the vaginal mucosa. The hypothesis was that a very distinct difference in color response would be observed between the time of the PGF injection when the majority of cows should have a CL present and high P4 concentration versus the time of the GnRH injection and TAI when cows should have very low P4 concentrations (≤ 1 ng/mL) due to luteal regression and high E2 concentrations due to the presence of a pre-ovulatory follicle. Evaluation done on samples taken concurrently showed no significant differences in frequency of color responses versus concurrent plasma hormonal levels for either progesterone or estradiol or for ultra sound data relating to follicle size. Further analysis revealed an indirect relationship between hormone concentration at the time of PGF ( time of prostaglandin injection) and color response measured at GnRH (time for gonotrophin injection). Color responses in the Oratel Vaginal Mucosal assay showed a correlation between frequency of color response measured at GnRH ( 18 hours before insemination) that was proportional to progesterone levels measured at PGF (72 hours prior insemination). Cows with plasma progesterone levels < 1 ng/mL measured at PGF correlated with increased frequency of (++ ) spectral measurements for value "a" and frequency of pale purple color responses at GnRH. The same group of cows had an average plasma estradiol concentration at GnRH of 1.64 pg/mL compared with 2.6pg/mL estradiol , observed for the other color groups. The lower estradiol values at GnRH also showed less follicle growth between GnRH and TAI. The model is that progesterone defines a pathway for estrogen stimulation of follicle growth which in turn affects P/AI ( or conception rates). The VACA assay appears to pick up color differences measured at GnRH that correspond to differences in plasma progesterone measured at PGF. This interpretation of how hormone levels related to the Vaginal Mucosa Color Assay is a shift from the orginal methodology which only compared color responses to concurrent plasma samples . What opportunities for training and professional development has the project provided? Three interns have been involved in this project so far. Two have learned how to collect blood samples and analyze data statistically. Another intern had the opportunity to improve ultra sound evaluation of the cow and to record data into a spread sheet and conduct statistical evaluation of data. How have the results been disseminated to communities of interest?Results from Phase I of this project have been disseminated to three different groups: a. Summary of results specific to each participating farm have been shared with the farmers. b. Results of this study have been shared under agreements of confidentiality with potential commercial partners who are helping us develop the technology for commercial use. c. A report will be written for publication by the Dept. of Animal Sciences at Cornell University. Date for submission has not yet been determined. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
BACKGROUND Timed insemination programs have been used on dairy farms since the 1990's to synchronize ovulation, so that breeding can all be done at one time. The timed insemination program (TAI) involves administering a sequence of hormone injections to the cow. One of these injections is a prostaglandin injection (PGF), administered 72 hours prior to insemination, which will reduce progesterone levels so that when, followed by an injection of GnRH, about 57 hours later, allows a follicle to grow to a size that can result in ovulation. Synchronizing this process in several cows simultaneously can reduce breeding costs and increase milk production. Current success rate for pregnancy/ insemination (P/AI) using the timed artificial insemination (TAI) protocol has been about 34%. This is because about 20% of the cows in a timed insemination program do not respond to the PGF injection, due to spontaneous low progesterone levels at the time of PGF which means that these cows are out of phase in responding to the hormone injection protocol. Eliminating these cows from the timed insemination protocol 72 hours prior to insemination can save $40/ cow in breeding costs. This selection process can also allow for earlier re-entry of these same cows into a new timed insemination program, thus reducing the time ( 39 days) for a veterinarian to check whether or not the cow is pregnant. As a consequence, knowling which cows to remove from a timed insemination program can improve the rate of conception in the herd by 20%, which in turn leads to better efficiency in milk production. This allows farmers to make management choices in herd size, genetic improvements, which are vital to maintaining profitability in the current low profit margins experienced by the dairy industry. MAJOR IMPACTS ACCOMPLISHED IN PHASE I This project identified a subgroup of cows that responded to the Oratel Diagnostics VACA assaywith a pale purple color which correlated with reduced fertility. When not taking into consideration the color responses for the VACA assay, the overall, pregnancy outcome/ artificial insemination, P/AI during the study was 34.8% (227/652) and was similar for first 2+ AI services (P= 0.71) and across all dairies (P=0.72). When cows were grouped based on digital images of theOratel Diagnostics Vaginal System (VACA) assay, we observed that cows in the light purple (+ +) group at TAI (time of insemination), had reduced fertility (P=0.02) P/AI 22.1% n=131) when compared to cows in blue (- - group; 35%; n=231)and other color ( + -; 38.3%; n=191). When only cows that had a functional CL based on progesterone concentrations at the time of PGF injections were evaluated, differences in P/AI between the digital evaluations were even more dramatic. For 20% of the cows in the (++; n=40) group, P/AI was only 17.5%, which is in sharp contrast to 52.0% and 39.0% P/AI (P <0.01) for the 40.0 and 38.7% of the cows in groups (--) and (+-), respectively. This data was reconfirmed in a subsequent study using an improved visual color scale to evaluate 111 cows at the time of insemination. P/AI (conception risk) measured at time of insemination was 19% for pale purple (n=21, 20% of herd) versus P/AI = 65% for blue color responses (n=66, 60% of the herd) and 38% for aqua color responses (n=24, 22% of the herd). Current data suggests an indirect hormone link between plasma progesterone levels, measured at PGF (72 hours prior to insemination), and color response to the Oratel Diagnostics Vaginal Assay-VACA test measured at GnRH ( 18 hours prior to insemination). Cows having plasma estradiol levels > 2.4pg/mL (measured at GnRH), also had plasma progesterone levels >1ng/mL (measured at PGF). Both spectrophotometric and visual color data revealed that cows having l< 1ng/mL of plasma progesterone at the time of PGF showed a higher frequency of pale purple (++) color responses to the mucosal assay at GnRH. This same group of cows also had less estradiol, (mean 1.64 pg/mL compared to mean 2.6 pg/mL estradiol for cows >1ng/mL progesterone at PGF ( P<0.001). The data suggests that progesterone levels at PGF, affect the rate of follicle growth and production of estradiol at GnRH, which coincides with the observed differences in color frequency between blue and pale purple color responses observed in the Oratel VACA assay as tested at time of insemination. The VACA assay can be a tool to select which cows should and should not be inseminated. Field trials using the Oratel Diagnostics Vaginal System (VACA) demonstrated that it is adaptable to current management systems in existing farms. The time needed to do each test was less than 20 seconds, making this system highly useful as a point of care test. There was never any difficulty in testing a cow. There was a problem involving considerable extra time to locate those cows that needed to be tested when they were not in stalls. We tested cows at three different times in the TAI protocol: at PGF (72 hours prior to insemination), GnRH, (18 hours prior to insemination), and TAI ( at the time of insemination). It would be preferable to only test at TAI because, at this time all cows are already in stalls waiting for insemination. In our final product we propose using a progesterone test on the day before the PGF injection in order to identify cows with low progesterone levels, who would not benefit from the timed insemination protocol. The VACA assay would test all the cows scheduled for insemination at TAI, so those cows with (++) or pale purple color responses, thatalso correlate with a low P/AI ( < 20%), can be identified and rescheduled for a later breeding program. This system can save $17/cow net of costs for assays and TAI treatment programs. Additionally the system can reduce average calving intervals by 20% because any cow not ready for insemination can be re-introduced into the breeding program within a week, thus allowing for reduction in the average wait time between inseminations. Such a protocol brings significant savings in time and cost to the dairy farm. Data for the visual assay which read colors according to a color scale defined as 1,2,3,4,5 in Phase I of this project showed a lack of specificity for identifying estrus phase: (P=0.41) in P/AI for the 5 visual color categories as defined in the protocol and tested on 580 cows. We learned that part of the reason for the confusion in color scale was that the filter surface did not allow for separation of hydrophobic and hydrophilic components of the assay. We now have a new swab that releases the hydrophilic component into water and this allows for a binary system for color evaluation that is either blue or clear. We have tested this new system on 200 cows and learned that it can easily be read either as a blue or not blue test or quantitatively at one wavelength in a spectrophotometer. For our company, results from this Phase I SBIR study generated a whole new business strategy. We made contact with a company that markets milk progesterone assays and soon learned that combining our technologies into one kit can enhance marketing opportunities for both companies. The company marketing milk progesterone assays has existing markets in emerging economies, which have some of the fastest growing dairy industries in the world such as in Africa, and in India and China. The Oratel Diagnostics technology provides a simple -easy-to-use color test which, when combined with a milk progesterone assay, can improve P/AI ( conception risk) by identifying those cows not ready for insemination. By combining the two technologies together, both companies gain advantage in marketing opportunities and in improvements to their respective technologies.
Publications
- Type:
Other
Status:
Other
Year Published:
2015
Citation:
none
|
Progress 06/01/15 to 07/31/15
Outputs
Target Audience:The target audience for this technology during this reporting period has been researchers and experts who can evaluate this technology and give us feedback on the science and potential economic value of this assay. The data was shared with one researcher who is an expert in the economics of cow reproductive physiology, who independently evaluated the data in phase I of this SBIR project. This feedback will be important to our business strategy and help us decide the next steps. We also shared a pamphlet that gives an overview of the technology with a workshop held at Cornell Center for Material Research at Cornell University. We feel that academic exposure and support will help to promote this new technology. Our project was also published on the Hubfuz website in the Spring of 2015 so that readers interested in Oratel Diagnostics can learn about this new technology. Hubfuz is an organization that promotes connections between New York State technology companies and manufacturers and product development companies. Additionally we have reached out to potential partners who can help us commercialize this technology. Under confidentiality agreements, we have shared some of the data and discussed how our technology might fit into their markets and enhance existing products they sell. We are exploring possible opportunities to make partnership relationships in order to engineer improvements in our technology and combine our technology with products that already exist in the market. Changes/Problems:The project experienced one major delay which was due to limited availability of RIA estradiol assays. The company that produces these assays could not send sufficient quantity until May 2015. As a consequence we could not complete the master sheet needed for data analysis until June 2015. Unexpected outcomes:The original proposal stated that a sub group of cows would show a "green" color response that was sub-optimal for acheiving pregnancy in a given insemination. Our data showed the color response for a sub group with sub-optimal fertility status to have red defined in the digital system as (+ +) and not green defined in the digital system as (- -). The reason for this different outcome reflects a change in the assay preparation. Earlier tests on cows had a 2-step process, whereby both pigments were added to the filter surface after exposure to the mucosal tissue. This process added time for reading the color. In this study, we added the pigments to the filter surface before exposure to the cow mucosal tissue in order to reduce the wait time for color evaluation. By developing a 1-step assay that has both pigments appied to the filter surface before exposuure to the mucosa, the color response became instant and could be read within 10 seconds. This sequence change resulted in a change as to how the color looks on the filter surface, which could be due to a change in the "charge" of the filter surface when both pigments are added prior to mucosal exposure. Color Evaluation issues The visual system, that has a scale of 1,2,3,4,5 , did not yield signigicant differences in P/AI ratios for the herd of 714 cows evaluated in our study. The visual color response reflects two different chemical reactions: acid-base equilibirum reaction for the anthocyanin, which relates to the blue- red colors and the " charge" changes in quercetin when it is attracted to the deglyocsylated mucin which relates to the green-clear response. These two chemical systems are difficult to anaylze visually. A digital system that separates color anaysis onto reading only one group of colors on a negative/positive axis appears to offer better differentiation. Our data showed that "a" values with positve readings > -0.399 correlated with a sub group of cows ( 23.6% of the herd) that had 22.1% conception rates compared with the group average of 34.8% for 714 cows evaluated. As a result of comparing the visual system to the digital system, we were able to gain more insight into the chemistry of the VACA system and thereby gain further insights as to what improvements still need to be engineered to make this a simple -easy- to use system. It appears that a digital reader is necessary... not only to bring clarity to the specific colors sensitive to the assay, but also to bring objectivity to the reader. A machine that reads in constant light provides more reliability when compared with human eyes reading in different times of day and locations in a barn. For future work, we would like to use a different reader that can be used directly on the sample and has a larger aperature. We have located such machines and priced them for possible testing in future studies. 6. Additional modifications: Anaylsis of the progesterone data in our study gave a new insight into modifications of the VACA assay and how it can be applied in farms that use the Ov-Synch program. Our evaluation of plasma progesterone hormone profiles for the herd idenitified two groups: <1ng/mL in plasma samples at time of PGF injection and the other group as >1ng/ml at the time of the PGF injection that respond with different P/AI ratios for TAI treatment. When comparing color responses to the VACA assay in each group we found that the group that has <-0.399 for "a" value in the Filmetrics scale on the day of TAI and has > 1ng/mL of plasma progesterone at the time PGF showed a 52.0% pregnancy rate for the the cows that showed a (- -) response to the filmetrics assay. Furthermore, the group of cows <1ng/ml progesterone at PGF (20% of the herd), showed "a" responses > -0.399 had than 17.5% conception rate. For cows going through an Ov-Synch program, it might be useful to apply this knowledge and consider the possibility of a rapid milk progesterone test in combination with the VACA assay to more accurately define which cows will not benefit from a TAI protocol. What opportunities for training and professional development has the project provided? The primary investigator gained new experience in analyzing data and generating new insights to help improve the engineering of the product. Analysis of the data led to further insights into the chemistry of the color changes and this knowledge will be useful in making improvements in stablizing the color response and improving color resolution. How have the results been disseminated to communities of interest?A pamphlet was prepared and prototype of assay was shared with a Workshop at Cornell in the Center for Material Sciences to demonstrate how the filter surface is useful for testing mucosal tissue. The preliminary results of the project were shared with participant farmers on May12,2015. Two presentations made under confidential agreements were made to potential partnerships to discuss possible opportunities for collaboration for commercializing this technology. What do you plan to do during the next reporting period to accomplish the goals?1. Prepare a final report to share with the farmers on the results and discuss next steps. 2. Prepare a final report that explains how the study provides information for making product improvements and testing these improvements in field trials. 3. Conduct further analysis of the data to determine whether or not the data can decode any physiological relationships between color response and reproduction physiology not evident in the first round of data analysis. 3. Prepare a business proposal for developing a partnership to commercialize this technology.
Impacts
What was accomplished under these goals?
Out of 829 cows enrolled, 741 cows were inseminated; 680 cows has visual color evaluation for the VACA assay done at the time of the last prostaglandin F2a (PGF) and at the time of the Gonadotrophin Releasing Hormone (GnRH) injection according to the protocol; at the time of insemnation (AI). Spectral evaluation of the VACA assay using a Filmetrics F10-AR color reader was available for 575 cows. From the subgroup of cows (314), blood samples and ovarian information were collected coincident with the VACA assay. Ovaries were examined by transrectal ultra sonography (TUS) to record the size and position of all follicules > 4mm and all corpora lutea ( CL). Blood samples were used to estimate circulating concentrations of progesterone (P4: ng/mL) and estradiol (E2: pg/mL) at Cornell University. Individual cow records were maintained and retrieved from Dairy Comp 305. For this study, cows were grouped based on objective measurements from digital images of the VACA assay or on-site visual evaluation of the VACA assay by a trained technician. The digital system: (Filmetrics F10-AR) uses a modified L a b reading system that measures "a" values for spectral evaluation of red, (defined as +) and green, (defined as -), read on a scale ranging between -6.000 to +6.000. In this study (++) refers to "a" as red color and (- -) refers to "a" as green color and (+ -) refers to "a" as a mixture of red/green. Two readings per VACA assay were available. When the "a" for both readings was > -0.399 the outcome was positive (++); when "a" for both readings was <-0.399, the outcome was negative (- -), whereas cows with one reading < -0.399 > were "intermediate" (+ -). The range for the reading scale in this study was between +6.000 and -6.000. Selection of the cutoff "a" value in the L a b color reading system was based on data from the current study. The visual color system was based on a 5 color scale: 1-pink/purple 2-blue, 3-green 4-bluish/green 5-clear. Overall, P/AI (or conception risk) during the study was 34.8% (227/652) and was similar for first vs.2+ AI services (P= 0.71) and across all dairies (P=0.72). When cows were grouped based on digital images of the VACA assay, we observed that cows in the (+ +) group at TAI, had reduced (P=0.02) P/AI 22.1% n=131) when compared to cows in (- - group; 35%; n=231)and ( + -; 38.3%; n=191). Interestingly, we could not explain these differences in P/AI based on group physiological data collected because there are no differences in groups in the proportions of cows with non-regressed CL at TAI ( P=0.47),concentrations of P4 at TAI (P=0.92), follicle size at TAI (P=0.95), and concentrations of E2 evaluated at the time of GnRH (P=0.44) or at TAI(P=0.95). When only cows that had a functional CL based on P4 concentrations at the time of PGF injections were evaluated, differences in P/AI between the "a" groups were even more dramatic. For the cows in the (++) group; n=40, which was 20% of the group with a functional CL, P/AI was only 17.5%, which is in sharp contrast to 52.0% and 39.0% P/AI (P <0.01) for the 40.0 and 38.7% of the cows in groups (--) and (+-), respectively. As for the whole group of cows (with or without a CL), we could not explain the differences in P/AI between groups with the ovarian and hormone data as we analyzed them. The observed differences in P/AI without a clear link to physiological parameters at the time of AI or GnRH is intriguing and suggest that differences in fertility among "a" value groups are due to biological and physiological differences between cows unrelated to the steroid hormone environment and follicular dynamics or else that they are either too subtle or complex to be detected with the methodologies we used in the current study. When fertility was evaluated for subgroups of cows created based on the visual color response there were no differences (P=0.41) in P/AI between the 5 color groups. In spite of some obvious numerical differences for some groups, there was not a group with a major reduction in P/AI as was shown for the "a" value system. Likely, the current study is underpowered to detect statistical differences because some of the groups in the different visual categories has a low proportion of cows that could not be evaluated satistically. It is important to note that because we used the current data set to define the criteria to create groups based on "a" values our, results should be validated with another study in which cows are prospectively classified in groups based on the digital images before evaluating their fertility after insemination. This study generated data that identified a subgroup of about 24% of the herd with P/AI values < 22% with using two different testing protocols: the Digital VACA system that uses "a" values in a L a b color reader at the time of insemination, and the combination of a plasma progesterone test at PGF followed by the digital "a" value reading prior to insemination. Our goal in developing this technology is to provide a decision making tool to help farmers improve pregnancy risk in dairy cows. Data in this phase of our project suggests that our methodology can identify a subgroup of cows that will yield lower P/AI ratios. Data also revealed that a digital system for evaluating colors gives better differentiation in the sub groups than a visual system. Our next step is to apply the knowledge we have gained from this study to refine the chemistry of our testing surface to develop a better resolution in visual color differentiation and use a portable color reader that can read spectrally color values at point of testing. We also plan to do further analysis of the data from this phase of the study to gain insight into possible physiological factors affecting the color response observed in the assay.
Publications
- Type:
Websites
Status:
Submitted
Year Published:
2015
Citation:
http://fuzehub.com/wp-content/uploads/2015/03/Oratel-Diagnostics-Case-Study.pdf
|
Progress 06/01/14 to 05/31/15
Outputs
Target Audience: The target audience for our technology are dairy farms that use artificial insemination and timed AI programs. We have been working with both insemination technicians, veterinarians, and farmers who have dairy herds of over 1000 cows. One goal of this project is to evaluate how end-users respond to the product and what modifications we need to make to make the assay practical and commercially viable. All the testing of the assay has been done by end-users who have given us feedback on the quality of the assay and its practical use. As a result, we have made modifications in the portability of the assay by creating a Styrofoam carrier that has holes to hold the test straws and can be fitted into a carpenters apron worn around the waist. This way the technician has free hands and can work with the cows, lift the tails, and test easily. The Styrofoam carrier has masking tape next to holes that hold the straws.This allows for easy photography and direct recording of the color test using a felt pen to write cow ids on the masking tape. Our current efforts in reaching out to our target audience has been to learn from them how to modify our products and understand how they use the technology. We have limited the number of end-users for this phase because we want to make necessary improvements before we scale up to a larger target audience. We have learned how end users view the product. There are two primary reasons why end-users would be interested in the assay. The assay appears to have value in identifying cows that are chronic repeaters. An early identification of these cows on the day of insemination saves on semen costs and on the wait period before the next vet check. This allows for early intervention in treating these cows so that they can get back into a functional reproductive cycle earlier. This assay also give assurance to the inseminator as to whether or not the timing for insemination is appropriate. These appear to be two primary reasons why end- users would ant to use this product. Now that we have completed our field work for this study, we note that the end-users continue to want to use our product. We are providing them with a limited supply so that we can get continued feedback on how they use this product. Changes/Problems: Changes and problems The Study has been following the protocol in principle. There is one change in our hypothesis. We proposed that the insemination of cows that give a green color response to the vaginal mucosal assay will have a low pregnancy outcome. We found that the color response that gives a low pregnancy outcome is purple; not green. The frequency of the green color response is low ( < 10% of the responses are green in a study of 701 cows; yet, if a cow gives a green color response at the time of insemination there is over a 40% chance she will become pregnant in our study. In contrast, about 20% of the color responses at the time of insemination give purple color response and of these less than 25% will get pregnant. Blue is the dominant color observed when testing the cows and it appears to correlate with a greater than 40% of attaining pregnancy when present at the time of insemination. in order to evaluate the significance of these data we need to understand how hormones change in the individual cows. Our study includes plasma evaluation of both progesterone and estradiol. The progesterone values are currently being conducted by the Animal Science Department at Cornell University. We have a delay in the estradiol evaluation because the kits to test our samples will not be available until late January 2015. We are asking for a six month extension so that all the testing can be completed and there is sifficient time to evaluate the results statistically. it is possible that we can complete these test earlier, but inorder to guarantee results in a timely fashion, we are requesting a 6 month extension. As a result, we have modified our hypothesis to identifying the sub group of cows that give a purple color response at the time of insemination as the subgroup that has sub-fertile conditions. We will compare ultra sound data and hormone data in these cows and evaluate how fertility in these cows compares to cows with blue color response to the VACA assay. Our study faces many challenges in measuring color. One issue in this study is how to evaluate color consistency and color interpretations. There are different light conditions in the barn depending on the time of day. Different people interpret colors differently. What system can we develop that accounts for these variables? We have chosen to compare the data on color interpretation in three different ways: 1. Records are kept of direct visual interpretation by the end-user. A scale has been developed wit h1=purple, 2=blue, 3-green, 4 = colored ring with clear center, 5 =clear. 2. A photograph is taken at the time of testing for each sample and records are kept as to date, time, and cow id. 3. Each photo is evaluated by a Filmetrics machine that measures color according to a L a b scale. L relates to luminosity a relates to red ( +) or green ( -) b relates to blue ( + ) or yellow ( -). Our data will compare different color readings by end-users on the same sample, on different samples from the same cow , and samples as measured over time. We want to determine how consistent and reliable the color reading system is. The color reading system is an important part of this project which is to determine whether or not color interpretations is a viable system to identify cows with fertility problems at the time of artificial insemination. We want to know whether or not the color reading system is objective and reliable. A comparison of the visual interpretations by the end user to the L a b system developed by Filmetrics will offer important feedback on the reliability of the VACA color reading system. What opportunities for training and professional development has the project provided? Three interns have been involved in this project so far. Two have learned how to collect blood samples and record data systematically. Another intern had the opportunity to learn how improve ultra sound evaluation of the cow and to record data onto a spread sheet. How have the results been disseminated to communities of interest? We are still collecting results and will have a report at the end of the study. The farmers have expressed interest in the results. One farm has viewed some of the results in a particular cow pen that has a high percentage of cows with fertility problems. It was noted that a higher percentage of these cows are repeats in the breeding program and that they also have a higher frequency of purple color results compared to blue color responses. Preliminary data suggests that purple color responses is associated with less than 20% pregnancy outcome and blue color response is associated with between 36% and 40% pregnancy outcome, depending of the farm. Differences in the values between cow pens and between farms led one farmer to invite a nutritional consultant to the farm to evaluate the feeding program for the cow pen that has the lowed conception rate. What do you plan to do during the next reporting period to accomplish the goals? 1. collate all data onto spread sheet. 2. Evaluate consistency in color recording data 3. record color responses as follows: visual interpretation according to a scale of 1 = purple ; 2 = blue; 3=green 4= colored ring with clear center ;5 = clear photo record of color response Filmetrics spectral analysis of photo color for L a b where L stands for luminosity, a is for blue ( +) , yellow (-) and b is for Red (+) and green (-). 4. evaluate consistency of color responses for visual interpretation , photo record, and spectral data 5. Complete plasma progesterone evaluation and Estradiol evaluation. These steps will be done in contract with Cornell Animal Science Department. It will take an additional four months to complete this phase, because the kits for evaluating estradiol are delayed. the progesterone will be completed by Dec. 30, 2014. 6.Statistical evaluation of color responses versus ultra sound data, plasma hormones ( progesterone, estradiol) for data collected on day of PGF shot, GnRH shot, and TAI and compare this data to pregnancy outcome in 700 cows.
Impacts
What was accomplished under these goals?
The technical objectives are: a. Conduct feasibility testing of VACA on dairy cows in commercial operations. b. Determine if there is a statistically valid relationship between VACA color response and cycle phases as measured by hormone levels and follicle growth as measured by ultra sound (TUS) What has been accomplished under these goals? What has been accomplished under these goals? a. As of Nov. 24, 2014, the study has collected 3 samples / cow for 585 cows for each of the following : sample name time at PGF shot time at GnRH shot time at TAI (insemination) VACA color tests 701 cows 701cows 701 cows ultra sound evaluation of left and righ ovary 297 cows 297 cows 297 cows plasma blood 265 cows 265 cows 265 cows b. Data has been collected by 2 different end-users who are the inseminators and herdsmen on the farms and by 2 certified veterinarians who routinely check the cows with ultra sound machines. Tests were conducted on 4 commercial dairy farms. The study adapted to the farms program and milking schedule. No one had any trouble conducting the assay or reading the colors. The use of the test, time taken to conduct the test, and methods for administering the assay fit into the farm routine and management system. There were no adjustments made on the farm for our assay. Every operator was able to conduct the test, read the color response and record the results. c. The data has been collated and stored in a master sheet that includes additional information from Dairy Comp. file for each cow which includes breeding date, days in milk, previous breeding dates, age, lactation cycle, body score, etc. This will be evaluated against color response data, ultra sound data for evaluation of left and right ovary, and plasma levels of progesterone and estradiol . Four different commercial dairy farms participated in this study. Each farm has over 1500 herd size. a. As of Nov. 1, 2014, the study has collected 3 samples / cow for 585 cows for each of the following : sample name time at PGF shot time at GnRH shot time at TAI (insemination) VACA color tests 585 cows 585cows 585 cows ultra sound evaluation of left and righ ovary 297 cows 297 cows 297 cows plasma blood 265 cows 265 cows 265 cows b. Data has been collected by 2 different end-users who are the inseminators and herdsmen on the farms and by 2 certified veterinarians who routinely check the cows with ultra sound machines. Tests were conducted on 4 commercial dairy farms. The study adapted to the farms program and milking schedule. No one had any trouble conducting the assay or reading the colors. The use of the test, time taken to conduct the test, and methods for administering the assay fit into the farm routine and management system. There were no adjustments made on the farm for our assay. Every operator was able to conduct the test, read the color response and record the results. c. The data has been collated and stored in a master sheet that includes additional information from Dairy Comp. file for each cow which includes breeding date, days in milk, previous breeding dates, age, lactation cycle, body score, etc. This will be evaluated against color response data, ultra sound data for evaluation of left and right ovary, and plasma levels of progesterone and estradiol . Four different commercial dairy farms participated in this study. Each farm has over 1500 herd size. 2. original records are kept in a separate file. 3. Data is recorded in the master spread sheet. II. Each sample is photographed and kept in files according to cow id, date, and time III The photographed samples will be read by a Filmetrica Reflectometer according to the L a b color reading system Data from color system will be evaluated for consistency . a. color readings made by multiple operators on the same sample. b. Multiple samples taken from the same cow at the same time. c. Samples tested over time. d. Multiple photos of the same sample e. Consistency between color readings made by the reflectometer and the visual readings. Preliminary Results Results for pregnancy outcome versus color response at TAI ( timed artificial insemination) show the following patterns for the color response for 359 cows tested for pregnancy in this study. In this population of cows the overall conceptionrate as evaluated at 39 days after insemination was 37% for 359 cows. Using the VACA color assay a subpopulation of cows that gave a purple color response at TAI showed 24% conception as evaluated at 39 days after TAi. There were 67 cows that gave this color response out of the 359 herd. This is about 19% of the herd with a 24% pregnancy rate. table { }td { padding: 0px; color: black; font-size: 12pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri,sans-serif; vertical-align: bottom; border: medium none; white-space: nowrap; }.xl64 { background: none repeat scroll 0% 0% yellow; }.xl65 { }.xl66 { background: none repeat scroll 0% 0% rgb(141, 180, 226); }.xl67 { background: none repeat scroll 0% 0% rgb(230, 184, 183); }.xl68 { color: white; background: none repeat scroll 0% 0% rgb(83, 141, 213); }.xl69 { color: white; background: none repeat scroll 0% 0% rgb(226, 107, 10); }.xl70 { color: white; background: none repeat scroll 0% 0% rgb(118, 147, 60); } color value pregnant not pregnant total number of cows % cows pregnant pregnant 1 16 51 67 0.24 24% purple color 2 85 130 215 0.4 40% blue color 3 13 18 31 0.42 42% aqua or green 4 2 11 13 number too small 5 18 15 33 0.55 total 134 225 359 a. As of Nov 1, 2014:Collected 3 samples/ cow for each of the following :ultra sound data on both ovaries for Cl and Follicle size, blood samples to be processed for progesterone and estradiol, and color responses to vaginal mucosal swab assay as recorded visually and by photograph on 265 cows at time of PGF (prostaglandin shot) at time of GnRH injection and at time to TAI ( timed artifical insemination. Data has been collated and stored in spread sheet and linked to information on each cow in Dairy comp. Tests were conducted in four different commercial dairy farms with over 1500 size herd. Blood samples have been collected, centrifuged and stored in freezer. processing for progesterone and estradiol have yet to be done. b. The data that will be evaluated has been collated onto a spread sheet. We have also collected data on the VACA assay ( vaginal anthocyanin color assay) for 701 cows with photographs and recorded color interpretations at day of PGF shot; day of GnRH shot; and day at TAI. The color analysis using Filmetrics technology and statistical evaluation of the data will be done in December 2014.
Publications
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Progress 06/01/14 to 01/31/15
Outputs
Target Audience: The target audience for our technology are dairy farms that use artificial insemination and timed AI programs. We have been working with both insemination technicians, veterinarians, and farmers who have dairy herds of over 1000 cows. One goal of this project is to evaluate how end-users respond to the product and what modifications we need to make to make the assay practical and commercially viable. All the testing of the assay has been done by end-users who have given us feedback on the quality of the assay and its practical use. As a result, we have made modifications in the portability of the assay by creating a Styrofoam carrier that has holes to hold the test straws and can be fitted into a carpenters apron worn around the waist. This way the technician has free hands and can work with the cows, lift the tails, and test easily. The Styrofoam carrier has masking tape next to holes that hold the straws.This allows for easy photography and direct recording of the color test using a felt pen to write cow ids on the masking tape. Our current efforts in reaching out to our target audience has been to learn from them how to modify our products and understand how they use the technology. We have limited the number of end-users for this phase because we want to make necessary improvements before we scale up to a larger target audience. We have learned how end users view the product. There are two primary reasons why end-users would be interested in the assay. The assay appears to have value in identifying cows that are chronic repeaters. An early identification of these cows on the day of insemination saves on semen costs and on the wait period before the next vet check. This allows for early intervention in treating these cows so that they can get back into a functional reproductive cycle earlier. This assay also give assurance to the inseminator as to whether or not the timing for insemination is appropriate. These appear to be two primary reasons why end- users would ant to use this product. Now that we have completed our field work for this study, we note that the end-users continue to want to use our product. We are providing them with a limited supply so that we can get continued feedback on how they use this product. Changes/Problems: Changes and problems The Study has been following the protocol in principle. There is one change in our hypothesis. We proposed that the insemination of cows that give a green color response to the vaginal mucosal assay will have a low pregnancy outcome. We found that the color response that gives a low pregnancy outcome is purple; not green. The frequency of the green color response is low ( < 10% of the responses are green in a study of 701 cows; yet, if a cow gives a green color response at the time of insemination there is over a 40% chance she will become pregnant in our study. In contrast, about 20% of the color responses at the time of insemination give purple color response and of these less than 25% will get pregnant. Blue is the dominant color observed when testing the cows and it appears to correlate with a greater than 40% of attaining pregnancy when present at the time of insemination. in order to evaluate the significance of these data we need to understand how hormones change in the individual cows. Our study includes plasma evaluation of both progesterone and estradiol. The progesterone values are currently being conducted by the Animal Science Department at Cornell University. We have a delay in the estradiol evaluation because the kits to test our samples will not be available until late January 2015. We are asking for a six month extension so that all the testing can be completed and there is sifficient time to evaluate the results statistically. it is possible that we can complete these test earlier, but inorder to guarantee results in a timely fashion, we are requesting a 6 month extension. As a result, we have modified our hypothesis to identifying the sub group of cows that give a purple color response at the time of insemination as the subgroup that has sub-fertile conditions. We will compare ultra sound data and hormone data in these cows and evaluate how fertility in these cows compares to cows with blue color response to the VACA assay. Our study faces many challenges in measuring color. One issue in this study is how to evaluate color consistency and color interpretations. There are different light conditions in the barn depending on the time of day. Different people interpret colors differently. What system can we develop that accounts for these variables? We have chosen to compare the data on color interpretation in three different ways: 1. Records are kept of direct visual interpretation by the end-user. A scale has been developed wit h1=purple, 2=blue, 3-green, 4 = colored ring with clear center, 5 =clear. 2. A photograph is taken at the time of testing for each sample and records are kept as to date, time, and cow id. 3. Each photo is evaluated by a Filmetrics machine that measures color according to a L a b scale. L relates to luminosity a relates to red ( +) or green ( -) b relates to blue ( + ) or yellow ( -). Our data will compare different color readings by end-users on the same sample, on different samples from the same cow , and samples as measured over time. We want to determine how consistent and reliable the color reading system is. The color reading system is an important part of this project which is to determine whether or not color interpretations is a viable system to identify cows with fertility problems at the time of artificial insemination. We want to know whether or not the color reading system is objective and reliable. A comparison of the visual interpretations by the end user to the L a b system developed by Filmetrics will offer important feedback on the reliability of the VACA color reading system. What opportunities for training and professional development has the project provided? Three interns have been involved in this project so far. Two have learned how to collect blood samples and record data systematically. Another intern had the opportunity to learn how improve ultra sound evaluation of the cow and to record data onto a spread sheet. How have the results been disseminated to communities of interest? We are still collecting results and will have a report at the end of the study. The farmers have expressed interest in the results. One farm has viewed some of the results in a particular cow pen that has a high percentage of cows with fertility problems. It was noted that a higher percentage of these cows are repeats in the breeding program and that they also have a higher frequency of purple color results compared to blue color responses. Preliminary data suggests that purple color responses is associated with less than 20% pregnancy outcome and blue color response is associated with between 36% and 40% pregnancy outcome, depending of the farm. Differences in the values between cow pens and between farms led one farmer to invite a nutritional consultant to the farm to evaluate the feeding program for the cow pen that has the lowed conception rate. What do you plan to do during the next reporting period to accomplish the goals? 1. collate all data onto spread sheet. 2. Evaluate consistency in color recording data 3. record color responses as follows: visual interpretation according to a scale of 1 = purple ; 2 = blue; 3=green 4= colored ring with clear center ;5 = clear photo record of color response Filmetrics spectral analysis of photo color for L a b where L stands for luminosity, a is for blue ( +) , yellow (-) and b is for Red (+) and green (-). 4. evaluate consistency of color responses for visual interpretation , photo record, and spectral data 5. Complete plasma progesterone evaluation and Estradiol evaluation. These steps will be done in contract with Cornell Animal Science Department. It will take an additional four months to complete this phase, because the kits for evaluating estradiol are delayed. the progesterone will be completed by Dec. 30, 2014. 6.Statistical evaluation of color responses versus ultra sound data, plasma hormones ( progesterone, estradiol) for data collected on day of PGF shot, GnRH shot, and TAI and compare this data to pregnancy outcome in 700 cows.
Impacts
What was accomplished under these goals?
The technical objectives are: a. Conduct feasibility testing of VACA on dairy cows in commercial operations. b. Determine if there is a statistically valid relationship between VACA color response and cycle phases as measured by hormone levels and follicle growth as measured by ultra sound (TUS) What has been accomplished under these goals? What has been accomplished under these goals? a. As of Nov. 24, 2014, the study has collected 3 samples / cow for 585 cows for each of the following : sample name time at PGF shot time at GnRH shot time at TAI (insemination) VACA color tests 701 cows 701cows 701 cows ultra sound evaluation of left and righ ovary 297 cows 297 cows 297 cows plasma blood 265 cows 265 cows 265 cows b. Data has been collected by 2 different end-users who are the inseminators and herdsmen on the farms and by 2 certified veterinarians who routinely check the cows with ultra sound machines. Tests were conducted on 4 commercial dairy farms. The study adapted to the farms program and milking schedule. No one had any trouble conducting the assay or reading the colors. The use of the test, time taken to conduct the test, and methods for administering the assay fit into the farm routine and management system. There were no adjustments made on the farm for our assay. Every operator was able to conduct the test, read the color response and record the results. c. The data has been collated and stored in a master sheet that includes additional information from Dairy Comp. file for each cow which includes breeding date, days in milk, previous breeding dates, age, lactation cycle, body score, etc. This will be evaluated against color response data, ultra sound data for evaluation of left and right ovary, and plasma levels of progesterone and estradiol . Four different commercial dairy farms participated in this study. Each farm has over 1500 herd size. a. As of Nov. 1, 2014, the study has collected 3 samples / cow for 585 cows for each of the following : sample name time at PGF shot time at GnRH shot time at TAI (insemination) VACA color tests 585 cows 585cows 585 cows ultra sound evaluation of left and righ ovary 297 cows 297 cows 297 cows plasma blood 265 cows 265 cows 265 cows b. Data has been collected by 2 different end-users who are the inseminators and herdsmen on the farms and by 2 certified veterinarians who routinely check the cows with ultra sound machines. Tests were conducted on 4 commercial dairy farms. The study adapted to the farms program and milking schedule. No one had any trouble conducting the assay or reading the colors. The use of the test, time taken to conduct the test, and methods for administering the assay fit into the farm routine and management system. There were no adjustments made on the farm for our assay. Every operator was able to conduct the test, read the color response and record the results. c. The data has been collated and stored in a master sheet that includes additional information from Dairy Comp. file for each cow which includes breeding date, days in milk, previous breeding dates, age, lactation cycle, body score, etc. This will be evaluated against color response data, ultra sound data for evaluation of left and right ovary, and plasma levels of progesterone and estradiol . Four different commercial dairy farms participated in this study. Each farm has over 1500 herd size. 2. original records are kept in a separate file. 3. Data is recorded in the master spread sheet. II. Each sample is photographed and kept in files according to cow id, date, and time III The photographed samples will be read by a Filmetrica Reflectometer according to the L a b color reading system Data from color system will be evaluated for consistency . a. color readings made by multiple operators on the same sample. b. Multiple samples taken from the same cow at the same time. c. Samples tested over time. d. Multiple photos of the same sample e. Consistency between color readings made by the reflectometer and the visual readings. Preliminary Results Results for pregnancy outcome versus color response at TAI ( timed artificial insemination) show the following patterns for the color response for 359 cows tested for pregnancy in this study. In this population of cows the overall conceptionrate as evaluated at 39 days after insemination was 37% for 359 cows. Using the VACA color assay a subpopulation of cows that gave a purple color response at TAI showed 24% conception as evaluated at 39 days after TAi. There were 67 cows that gave this color response out of the 359 herd. This is about 19% of the herd with a 24% pregnancy rate. table { }td { padding: 0px; color: black; font-size: 12pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri,sans-serif; vertical-align: bottom; border: medium none; white-space: nowrap; }.xl64 { background: none repeat scroll 0% 0% yellow; }.xl65 { }.xl66 { background: none repeat scroll 0% 0% rgb(141, 180, 226); }.xl67 { background: none repeat scroll 0% 0% rgb(230, 184, 183); }.xl68 { color: white; background: none repeat scroll 0% 0% rgb(83, 141, 213); }.xl69 { color: white; background: none repeat scroll 0% 0% rgb(226, 107, 10); }.xl70 { color: white; background: none repeat scroll 0% 0% rgb(118, 147, 60); } color value pregnant not pregnant total number of cows % cows pregnant pregnant 1 16 51 67 0.24 24% purple color 2 85 130 215 0.4 40% blue color 3 13 18 31 0.42 42% aqua or green 4 2 11 13 number too small 5 18 15 33 0.55 total 134 225 359 a. As of Nov 1, 2014:Collected 3 samples/ cow for each of the following :ultra sound data on both ovaries for Cl and Follicle size, blood samples to be processed for progesterone and estradiol, and color responses to vaginal mucosal swab assay as recorded visually and by photograph on 265 cows at time of PGF (prostaglandin shot) at time of GnRH injection and at time to TAI ( timed artifical insemination. Data has been collated and stored in spread sheet and linked to information on each cow in Dairy comp. Tests were conducted in four different commercial dairy farms with over 1500 size herd. Blood samples have been collected, centrifuged and stored in freezer. processing for progesterone and estradiol have yet to be done. b. The data that will be evaluated has been collated onto a spread sheet. We have also collected data on the VACA assay ( vaginal anthocyanin color assay) for 701 cows with photographs and recorded color interpretations at day of PGF shot; day of GnRH shot; and day at TAI. The color analysis using Filmetrics technology and statistical evaluation of the data will be done in December 2014.
Publications
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