1	Research Internship Michael D. Kleinhenz Department of Animal Sciences Columbus, OH
2	Department Background Department of Animal Sciences Part of the College of Food, Agriculture and Environmental Sciences Mission Statement The Mission of the Department of Animal Sciences is to discover and communicate knowledge about animals and their products. The delivery of this mission is directed to the students of The Ohio State University, the citizens of Ohio and other parts of the world, the scientific community, stakeholders of the department, and others who are interested in animals used for food and fiber, recreation and companion purposes.
3	Background Information Dr. Ottobre’s research Professor of Animal Sciences Reproductive Physiology Vascular Endothelial Growth Factor (VEGF)
4	Background Information Ann Ottobre Dr. Ottobre’s Lab technician Direct supervisor Conducted in lab training
5	Activities and Responsibilities Bleeding vaccination trial sheep Sectioning ovaries Staining ovary section slides Counting follicles Library research
6	Bleeding Sheep 20 Sheep at the OARDC Sheep Center in Wooster Bleed on Tuesday and Friday Collected ~5cc’s of blood Used blood for Radioimmunoassay and ELSIA
7	Bleeding Sheep Collected blood out of the jugular vein Used a 20 gauge 1 inch needle on a 6cc syringe Placed the blood in test tubes with 2 drops of Heparin Put the tube of blood on ice for transport back to Columbus
8	Processing Blood Spun blood down in centrifuge Drew off the plasma over the red blood cells Placed the plasma in vials for storage
9	Processing Blood
10	Plasma Uses ELISA test to determine VEGF vaccine titers in the vaccine trial sheep Radioimmunoassay Determine progesterone levels of vaccine sheep Progesterone levels determined using competitive binding to progesterone antibodies sites on the walls of test tube.
11	Radioimmunoassay I-125 isotope used Isotope bonded to progesterone Calibrators of known progesterone levels are used to develop a curve on logit-log paper Samples are compared to the calibration curve to determine progesterone levels Concentration of 1ng/mL determined if sheep has reached puberty
12	Radioimmunoassay
13	Radioimmunoassay
14	Radioimmunoassay
15	Preparing Ovaries Harvest ovaries from Intrabursal injection sheep Preservation Kahle’s Solution Alcohols to dehydrate Xylene Paraplast (paraffin)
16	Sectioning Ovaries Section ovary using a Microtome 10 µm thick section Placed section in warm water bath Flattened out section Gelatin in water aided in section adhering to the slide Float section onto slide and dry
17	Sectioning Ovaries
18	Staining Ovary Slides Used so structures on the ovary section can be visualized and follicles counted Steps Xylene used to remove paraffin from section Alcohol steps rehydrated the sections for water soluble stains Schiff’s Reagent, Weigert-Lillie alum hematoxylin and picric acid with methyl blue Redehydrate using alcohol and xylene
19	Staining Ovaries
20	Follicle Counting Types and definitions of follicles Primordial – intact oocyte with visible nucleolus and a flat, single layer of granulosa cells Primary – intact oocyte with a visible nucleolus and a single layer of cuboidal granulosa cells Secondary – intact oocyte with a visible nucleolus and having two or more layers of cuboidal granulosa cells
21	Follicle Counting
22	Follicle Counting VEGF Intrabursal Trial Sheep Primary and secondary follicles were counted Follicle counts were compared to the untreated or control ovary of the sheep An increase in the number of primary and secondary follicles supports the theory that the VEGF does play a role in follicle development
23	Follicle Counting
24	Follicle Counting VEGF-Antibody Intrabursal Trail Sheep Primordial follicles counted Decrease in the number of primordial follicles
25	Library Research Literature search for information Use of OSCAR resources and on-line databases Items researched Procedure for intramuscular fat determination BAX antibody for immunohistochemistry In vitro follicle culture
26	Skill-a-thon 4-H and FFA skill-a-thon Ohio State Fair Dairy Question Facilitator Quality Assurance Dog skill-a-thon facilitator Dog diseases
27	Skills Developed How to handle and bleed sheep Counting follicles and histology Literature research Better communication skills
28	Quality of Experience This internship experience was educational, enjoyable, and interesting