100 Reproductive cycle and pregnancy monitoring in the common hippopotamus (Hippopotamus amphibius) through salivary steroid analyses and transabdominal ultrasonography

The common hippopotamus (Hippopotamus amphibius) is listed as vulnerable to extinction by the IUCN due to a significant decrease in population size, caused by habitat loss and poaching. Ex situ populations can help ensure against species loss, but careful reproductive management is essential to maintain sustainable populations. Hormone monitoring allows for characterisation of the reproductive cycle and gestation, offering insight into timing of receptivity and conception and facilitating pregnancy diagnosis and estimation of parturition date. Fecal steroid analysis has been validated for measuring progestogens in hippos. However, hippos are often housed in groups and frequently defecate in the water, making sample collection and source identification difficult. Salivary steroid analysis has been employed for monitoring reproductive activity in several species, but has not been tested in hippos. Additionally, transabdominal ultrasonography has proven valuable in diagnosing and monitoring pregnancy in many large mammals, but efficacy in the common hippo is unknown. The goals of this project were to (1) validate the use of an enzyme immunoassay to monitor progestogens in hippo saliva, (2) confirm that salivary progestogen profiles accurately reflect reproductive activity, (3) determine if transabdominal ultrasonography can be used to diagnose pregnancy, and, if so, (4) monitor and characterise fetal development via weekly examinations. Saliva (4-7 per week) and fecal (2-7 per week) samples were collected from 7 adult female hippos housed at 3 USA facilities over 3-7 months. Saliva and fecal samples were extracted in ethanol and extracts diluted (1:2 to 1:10 and 1:25 to 1:500, respectively) before evaluation by enzyme immunoassay (Progesterone mini-kit; Arbor Assays). Parallelism was confirmed between serially diluted fecal (r2 = 0.993) and saliva (r2 = 0.990) samples and the standard curve. Inter- and intra-assay coefficients of variation were maintained at <10%. Comparison of fecal and saliva progestogen concentrations revealed a strong correlation between the 2 sample types (r2 = 0.848) and suggested that saliva offers a comparable alternative. Both fecal and saliva extracts exhibited elevated progestogens during luteal phases and gestation. One nulliparous female housed at the Cincinnati Zoo & Botanical Garden (Cincinnati, OH, USA) was trained for voluntary transabdominal ultrasound exams. An Ibex Pro portable ultrasound machine (E.I. Medical Imaging, Loveland, CO, USA) with curvilinear probe (5-2.5 MHz) was used at a scanning depth of 17.8 and 23.4 cm. Intrauterine fluid and possible fetal tissue were observed 79 days following the last confirmed mating. Spine, rib cage, and beating heart were clearly visible at ~156 days of gestation. Ultrasound procedures were continued until the premature birth of a calf at ~181 days (normal hippo gestation ~231 days). Salivary progestogen monitoring and transabdominal ultrasonography appear suitable for tracking reproductive activity and diagnosing and monitoring pregnancy in the common hippo.