11-and 3-hydroxyprogesterone as a pregnancy tracking hormone-metabolite in 13-lined ground squirrels (ictimodys tridecemlineatus)

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science of BiologyThirteen-lined ground squirrels (13LGS; Ictidomys tridecemlineatus) are small, omnivorous, fossorial, hibernating sciurid rodents. As favored models for studies of hibernation and conebased visual function, they have only recently been bred in captivity on any sort of scale, and only here at UW Oshkosh. 13LGS are reflex ovulators, mating only in spring with a ~28 day gestation period. Increasingly, the UW Oshkosh 13LGS Colony fields client requests for pregnant females, in order to facilitate the first steps of transgenic modification and for embryological studies. These aims require a far better understanding of the captive 13LGS’s reproductive endocrinology than we currently have. This project was to ascertain whether enzyme-linked immunosorbent assay (ELISA) of squirrel fecal samples can be used to noninvasively detect pregnancy in a low-stress manner. To this end, competitive ELISAs for 11- and 3-hydroxyprogesterone were conducted on a group of thirteen females, comprising proven dams bred in the Colony or captured from the wild. Feces were collected thrice weekly during the spring of 2016 and frozen for subsequent steroid hormone extraction. Feces collections ceased as soon as a litter was noted. Competitive ELISA tests against the metabolite 11- and 3- hydroxyprogesterone were run using kits (Arbor Assays, Ann Arbor, MI), setting data against seven different time points between hibernation exit and birth of a litter. An ANOVA with repeated measures was run on the ELISA data to identify any significant differences in fecal hormone-metabolite between these time points. Then a pairwise t-test with a Bonferroni correction was run to determine where the differences lay. A Wilcoxon rank sum test was run on the captive-bred vs. wild-caught animals to determine significant differences; a Spearman’s rank correlation was performed to determine a possible correlation between litter size and progesterone concentration; and a Student’s t-test was run on data from pregnant and nonpregnant animals to determine significant differences. Eleven of 13 dams produced litters in the spring of 2016. Fecal ELISA data from the 2 non-pregnant females demonstrated no rise in fecal hormone-metabolite at any time point over four weeks. In contrast, data from the 11 parous females all demonstrated a pronounced rise in fecal hormone-metabolite, with most animals displaying progesterone withdrawal in the final week of gestation. Compared to baseline fecal hormone-metabolite levels on the day of pairing with a male, this rise was statistically significant halfway through gestation, with a >20-fold increase just a week into gestation. No significant differences were noted based on whether a pregnant female was wild-caught or captive-bred, nor with size of the litter. A technical replicate revealed the apparent decomposition of fecal 11- and 3-hydroxyprogesterone over 14 weeks, despite being stored frozen. This study has yielded a step-by-step protocol by which 11- and 3-hydroxyprogesterone may be monitored non-invasively through competitive ELISA of fecal pellets, permitting the detection of pregnancy at least 2 weeks prior to the birth of a litter. It further confirms the 13LGS as consistent with other sciurids in its status as a placental mammal that undergoes progesterone withdrawal late in gestation