TITLE: Dietary Sample for Phytochemical Assays
AUTHORS: Alicia Rich

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">All of the methods and protocols for this project have been reviewed and approved by the Otterbein University Institutional Animal Care and Use Committee, Otterbein University's Environmental Health and Safety Officer, the AZA Prosimian Taxonomic Advisory Group, and the individual collaborating AZA Institutions.</div><div class = "text-block">Many plants produce chemicals known as phytoestrogens that activate estrogen receptors. The phytoestrogen-fertility relationship is well-documented in livestock, rhinoceroses, cheetahs, and pandas, and this study will characterize the pattern in primates, especially pygmy slow lorises, gorillas, and pottos. We will adapt existing biomedical methods to investigate dietary estrogens and reproductive outcomes in captive primates. Gene expression profiles will serve as proxies for in vivo estrogen receptor activity, and our heterologous cell-based receptor reporter assay will quantify in vitro estrogen receptor activation. Generalized linear mixed-effects models will integrate these parameters with reproductive outcomes to test the effect of gene expression profiles and diet estrogenicity on fertility. This long-term project will ultimately provide one of the first deep physiological examinations of phytoestrogen’s proximate and ultimate influences on primate reproduction.</div></div>

[STEPS]
?. [Set aside food]
For one full day, each time you prepare the diet for our target animal, set aside one extra portion of food in a plastic bag or styrofoam crate. Keep this food in the same location or under the same conditions as you would just before you feed it to the primate.
Our goal will be to extract chemicals from the food items when they are in the same state they would be right when the animal eats them. So if they are typically frozen and thawed, that is what we want to happen. If they sit at room temperature, then that is what we will replicate.
For some large herbivores (e.g., gorillas) the total volume of food consumed in one day may be unreasonably large for shipment. In those cases, try to take a representative portion of each food item (e.g. 1/2 or 1/4 of the usual portion for every food, making note of this). Our goal is to have all of the food items in representative portions.
If you have detailed diet documentation of some kind that your zoo uses already and are willing to share it, we would find it very helpful. This is not required though.
?. [Package diet sample for transport]
At the end of the day, prepare all of the food for immediate pickup and transport to our lab or for shipment, depending on your institution's location.
?. [Package diet sample for transport]
Combine all items into the plastic bag provided. Close the bag as shown below.
?. [Package diet sample for transport]
Put the plastic bag and the ice packs (provided by us) into the styrofoam cooler that we sent. Try to position it similar to the image below. Make sure the outside of the styrofoam cooler is labeled with your information (we should have sent it to you this way).
?. [Package diet sample for transport]
Place the styrofoam cooler inside the cardboard box we sent. Either prepare this box for shipment or pickup, depending on your location.
?. [Sample Pickup]
Send a text message or email to Dr. Rich or the other contact you were given to let us know the box is ready. We will arrive shortly to receive the box at the most convenient time/location for you.
?. [Shipment Procedures]
Make sure you include the letter and any other documents we included outside the cooler and inside the box.
?. [Shipment Procedures]
Seal the box shut, and put the labels that we gave you on the outside of the box as shown below.
We are not using dry ice or any other hazardous materials for this shipment, so you will not have that special label shown in the image above.
?. [Shipment Procedures]
Email Dr. Rich and let her know that the box is ready for shipment. She may have already arranged a pickup day and time between you and Fedex. Shipment will automatically be charged to her research account.
?. [Sample Pickup]
We will follow up with you as soon as we have results! Thank you!
?. [Shipment Procedures]
We will follow up with you as soon as we have results! Thank you!