Abstract

Zoo-based cetacean populations can serve as test cases to validate research methodologies and provide proofs of concept for extrapolation of these efforts in situ.¹ Ex situ breeding programs are also opportunities for in-depth studies of a species’ reproductive biology. The beluga (Delphinapterus leucas) has a unique reproductive biology characterized by a strong breeding season, induced ovulation, and long gestation.^2-5 The ability to distinguish between different reproductive states and identify normal/abnormal pregnancies would have tremendous application to both in situ and zoo-based populations, especially if done with a single sample. The objectives of this study were to: 1) describe profiles of steroid hormones, in particular progestagens, androgens, estrogens, and glucocorticoids, during different reproductive outcomes (normal pregnancy [NORM], failure to thrive [FTT], peri-natal loss [PNL], and twin [TW] gestations) in the beluga; 2) investigate if any of these hormones alone or in combination could be a biomarker of pregnancy failure, poor reproductive outcome, and maternal and fetal health for this species.

We analyzed longitudinal serum samples (n=618) from 17 belugas located across the SeaWorld parks (California, Florida, Texas) which represented 42 pregnancies (23 normal, 19 abnormal). Serum was analyzed either raw or ether-extracted using an immunofluorescence assay or enzyme immunoassays.^5-7 Progesterone (P4), progestagens (Pg), testosterone (T), androstenedione (A4), estradiol (E2), and cortisol (F) were quantified across pregnancies with the data grouped as luteal or estrous phase and either stage of pregnancy (EARLY [conception to day 156], MID [day 157 to day 312], and LATE [> day 312 of pregnancy]). Hormone concentrations were compared within and between phases, trimesters, and pregnancy outcomes using linear-mixed models with animal ID set as the random variable.

For P4, concentrations were elevated across pregnancy compared to the luteal phase with no distinction amongst gestational stages (p>0.05) except for PNL where LATE P4 was higher than EARLY and MID (p<0.05). Progesterone during LATE PNL was also higher compared to NORM and TW outcomes (p<0.05). Progestagens and E2 were highest during MID and LATE, and no differences amongst reproductive outcomes existed for either hormone (p<0.05). Testosterone was also increased during MID and LATE compared to EARLY (p<0.05), and concentrations during NORM and FTT outcomes were higher than PNL during EARLY stage (p<0.05). For A4, MID and LATE were also elevated compared to EARLY (p<0.05) for all outcomes except for TW where only MID was elevated (p<0.05). Cortisol concentrations during LATE were increased compared to EARLY (p<0.05). No differences amongst outcomes were observed for A4 or F (p<0.05).

No individual hormone or group of hormones emerged as a predictor of poor reproductive outcome in the beluga. Nonetheless, reference ranges for these hormones during different stages were established. Other pregnancy-related hormones, such as relaxin and prostaglandin F2α, could help distinguish between normal and abnormal pregnancy and need further investigation.

Acknowledgements

The authors would like to thank the veterinary, clinical lab, and animal care staff at all the SeaWorld parks for assistance with sample collection and banking. Species preservation lab technicians Amanda McDonnell and Jacqueline Posy are thanked for assistance with sample processing and analysis.

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