TITLE: Sample Collection:  Primate Hair for RNA
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">The pygmy slow loris (Nycticebus pygmaeus) captive population is declining at a rate that jeopardizes their future in the Species Survival Plan (SSP).  Experts agree that diet is somehow compromising reproduction, and this study will investigate dietary estrogenicity. 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 slow lorises. We will adapt existing biomedical methods to investigate dietary estrogens and reproductive outcomes in captive pygmy slow lorises. 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]
?. [Animal Handling]
During routine handling procedures, fold one piece of masking tape around a tuft of ~20 hairs on the dorsal neck area of the subject. As swiftly and firmly as possible, pluck the tuft of hairs in the masking tape. The most important part of the sample is the hair root at the end of the shaft, which is why it is important to pull rather than cut or shave the hair. Safely release the animal as soon as you complete this step while taking care not to disturb the sample still attached to the tape.
[should take less than 5 minutes]
?. [Sample Storage]
Place this tube inside of a clearly labeled sample bag (also provided), and put that into the envelope with postage, already addressed to Alicia Rich at Otterbein University. Please drop this envelope in the mail as soon as possible (within 24-48 hours of sample collection).
Wash hands thoroughly after releasing the animal and monitor the subject for 20 minutes to ensure there are no negative welfare impacts from the procedure. If you observe any signs of distress then notify the PI immediately.
?. [Sample Storage]
Using the sterile tweezers, gently remove each hair from the tape and put it into the tube. Be very careful not to disrupt the root portion of the hairs, so it is best to grip the tweezers as close to the tape as possible.
?. [Sample Storage]
We have provided you with sterile, RNase-free tubes containing  and sterile tweezers. Label the outside of the tube with today's date (YYYYMMDD), your initials, the time (HH:MM:SS), the sample type (hair or feces), the animal name or ID, and the species code (PSL for pygmy slow loris). While still wearing sterile gloves (or after putting on a new pair), carefully open the tube, taking care not to touch the inside of the cap or the tube.