Ultrasound Description of Follicular Development in the Louisiana Pinesnake (Pituophis ruthveni, Stull 1929)

SIMPLE SUMMARY: A detailed knowledge of reproductive biology is of paramount importance for establishing ex situ conservation programs. This paper describes in detail the follicular morphology, ovarian cycle and reproductive anatomy of the female Louisiana pinesnake (Pituophis ruthveni), which is a threatened North American colubrid. Using ultrasound, we described 7 different stages of the follicles and developed markers of progressive follicular maturation for this species, moreover, we dissected two specimens to describe the macroscopic anatomy of the female reproductive system. Furthermore, we identified the correspondence of the sexual activity peak with a particular follicular stage. This data provides parameters for evaluating growth patterns of ovarian follicles in relation to maturation, ovulation, fertility, and reabsorption that can be useful for conservation of this species as well future conservation programs, commercial breeders, and clinicians monitoring reproductive health and breeding of snakes. ABSTRACT: Accurate monitoring of reproductive activity is necessary for success of captive breeding and recovery of endangered species. Using ultrasonography, we aimed to describe the stages of follicle development of the endangered Louisiana pinesnake (Pituophis ruthveni). Ultrasound procedures were performed weekly for 11 females during the 2020 reproductive season by submerging the last half of an unanesthetized female in water and using a 3.0–10.0 MHz linear array transducer placed and moved along the gastrosteges to explore the whole reproductive tract. The presence of follicles, their size, echogenicity, and stage of development was assessed. We observed small, round, anechoic, linearly aligned previtellogenic follicles in the coelom at the beginning of the reproductive season and found that structures dramatically increased in size and shifted in echogenicity as follicles matured and developed before and after ovulation. We classified follicles according to ultrasonographic appearance into 7 different follicle categories: previtellogenic, early vitellogenic, vitellogenic, preovulatory, peri-ovulatory, post ovulatory, and shelled. Using ultrasound, we developed markers of progressive follicular maturation for the Louisiana pinesnake and identified signs of abnormal development and post ovulatory follicle reabsorption. Detailed description of follicular maturation will be useful to improve captive breeding successes, identify mechanisms of reproductive failure, and develop artificial insemination.