Reproductive Physiology and Development of Artificial Insemination Technology in Killer Whales (Orcinus orca)1

Abstract

Research was conducted to define the basic reproductive physiology of killer whales (Orcinus orca) and to use this knowledge to facilitate the development of artificial insemination procedures. The specific objectives were 1) to determine the excretory dynamics of urinary LH and ovarian steroid metabolites during the estrous cycle; 2) to evaluate the effect of an exogenously administered, synthetic progesterone analog on reproductive hormone excretion; 3) to validate the use of transabdominal ultrasound for ovarian evaluation and timing of ovulation; 4) to examine the quality of semen after liquid storage and cryopreservation; and 5) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of 41 follicular phases and 26 complete cycles from five females, estrous cycles were 41 days long and comprised a 17-day follicular phase and a 21-day luteal phase. A consistent temporal relationship was observed between peak estrogen conjugates and the LH surge, the latter of which occurred approximately 0.5 days later. Two animals placed on oral altrenogest (three separate occasions for 30, 17, and 31 days, respectively) excreted peak urinary estrogen concentrations 25 days after withdrawal that were followed by sustained elevations in urinary pregnanediol-3alpha-glucuronide excretion. Mean preovulatory follicle diameter was 3.9 cm (n = 6), and ovulation occurred 38 h (n = 5) after the peak of the LH surge. Based on visual estimates of motility, liquid-stored semen maintained 92% of its raw ejaculate sperm motility index (total progressive motility x kinetic rating [0-5 scale, where 0 = no movement and 5 = rapid progressive movement]) when held at 4 degrees C for 3 days postcollection. Semen cryopreserved using a medium freezing rate demonstrated good postthaw total motility (50%), progressive motility (94%), and kinetic rating (3.5). Insemination during eight estrous cycles resulted in three pregnancies (38%), two from liquid-stored and one from cryopreserved semen. Two calves were delivered after gestation lengths of 552 and 554 days, respectively. These data demonstrate the potential of noninvasive endocrine monitoring combined with serial ultrasonography to improve our understanding of the reproductive biology of cetaceans. This fundamental knowledge was essential for ensuring the first successful conceptions, resulting in live offspring, using artificial insemination in any cetacean species.

Variation of bowhead whale progesterone concentrations across demographic groups and sample matrices

“…There appears to be a strong relationship between urine progesterone and PDG, suggesting a relatively simple, though crude, transformation that links the 2 values (Stanczyk et al 1997, Falk et al 1999. The urine progesterone values were similar to other mammals (Herrick et al 2000, Khan et al 2008) and comparable to the transformed PDG values reported in other cetaceans (Robeck et al 2004(Robeck et al , 2005. Urine data was available for only 2 demographic groups in the present study: immature males and immature females.…”

“…Though urine levels of progesterone have not been reported previously in any cetacean, the progesterone metabolite, pregnanediol-3 alpha-glucuronide (PDG), has been measured in several odontocete species (Walker et al 1988, Kjeld 2001, Robeck et al 2004). There appears to be a strong relationship between urine progesterone and PDG, suggesting a relatively simple, though crude, transformation that links the 2 values (Stanczyk et al 1997, Falk et al 1999.…”

Assessment of luteinizing hormone and prolactin immunoactivity in Asian and African elephant urine using assays validated for serum

“…Serum and urine were collected during spontaneous and GnRH-induced LH surges, which were clearly identified in serum, but not in urine. By contrast, this antibody has proven effective in measuring LH concentrations in urine of other species, including callitrichids (Ziegler et al, 1993(Ziegler et al, , 1996, bottlenose and white-sided dolphins (Robeck et al, 2005), killer whales (Robeck et al, 2004), giant pandas (Kersey, Monfort, Brown, unpubl. ), manatees (Iske, Brown, unpubl.…”

“…Glycerol itself did not interfere with antigen/antibody binding, as determined by similar parallelism and mass recovery analyses of samples spiked with exogenous hormone standards to results from samples without glycerol. Many studies have evaluated urinary protein hormones, like LH and prolactin, often using neat urine (e.g., Czekala et al, 1988;French et al, 1992French et al, , 1999Ziegler et al, 1993;Miller and Soules, 1996;Robeck et al, 2004Robeck et al, , 2005. However, when no immunoactivity was observed in neat samples, either in longitudinal samples or as a result of parallelism tests on urine pools, efforts were made to concentrate samples to ensure the problem was not due to sample dilution.…”

“…Both LH and prolactin are excreted in urine of humans and nonhuman primates and can be quantified using standard immunoassay techniques (LH -Miller and Soules, 1996;French et al, 1999: prolactin -Miller andSoules, 1996;Soltis et al, 2005;Schradin et al, 2003;Ziegler et al, 1996). LH also has been measured in the urine of several marine 0016-6480/$ -see front matter Published by Elsevier Inc. doi:10.1016/j.ygcen.2010.08.007 mammals, including bottlenose dolphins, white-sided dolphins and killer whales and is used to time artificial insemination (Robeck et al, 2004(Robeck et al, , 2005. Thus, the ability to measure protein hormones, like LH and prolactin, in elephant urine would be invaluable for assessing the reproductive status of intractable animals in situ and ex situ.…”