Estrous cycle characterisation and artificial insemination using frozen–thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus)

Abstract

The reproductive endocrinology of the bottlenose dolphin, Tursiops truncatus, was characterized to facilitate the development of artificial insemination using cryopreserved spermatozoa. Specific objectives were: (i) to determine the excretory dynamics of urinary luteinizing hormone (LH) and ovarian steroid metabolites during the estrous cycle; (ii) to evaluate the effect of an exogenously administered synthetic progesterone analog (altrenogest) on reproductive hormone excretion; (iii) to correlate follicular growth and ovulation (as determined by transabdominal ultrasound) to urinary LH and ovarian steroid metabolites; (iv) examine the in vivo fertilisation capacity of cryopreserved semen, and (v) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of natural estrous cycles (2 consecutive cycles) and nine post altrenogest cycles in ten females, estrous cycles were found to be 36 days long and comprised of an 8 day and 19 day follicular and luteal phase, respectively. Peak estrogen conjugates (EC; 5.4 6 3.8 ng/mg creatinine (Cr)) occurred 8 h prior to the LH surge (70.9 6 115.7 ng/mg Cr). The time of ovulation, as determined by ultrasonography, occurred 32.1 6 8.9 h and 24.3 6 7.0 h after the onset of the LH surge and LH peak, respectively. Mean preovulatory follicular diameter and circumference were 2.1 6 0.5 cm and 6.5 6 1.5 cm, respectively. Of the 27 estrous synchronisation attempts, 13 resulted in an ovulatory cycle, with ovulation occurring 21 days post-altrenogest treatment. Intrauterine (4 of 5) and intracornual (1 of 3) inseminations conducted across eight estrous cycles resulted in five pregnancies (63%), one pregnancy resulted from the use of liquid stored semen, whereas four were achieved using cryopreserved semen. These data provide new information on female bottlenose dolphin reproductive physiology, and demonstrate that the combination of endocrine monitoring and serial ultrasonography contributed to successful AI using liquid-stored and cryopreserved semen.

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

“…In the bottlenose dolphin, AI was developed after basic research was conducted to characterize the species' reproductive physiology (Robeck et al 2005). Longitudinal studies enabled the prediction of ovulation and characterization of female reproductive cycles through transabdominal ultrasonic evaluation of the ovary (Brook et al 2000) and urinary hormone measurement (Robeck et al 2005).…”

“…In the bottlenose dolphin, AI was developed after basic research was conducted to characterize the species' reproductive physiology (Robeck et al 2005). Longitudinal studies enabled the prediction of ovulation and characterization of female reproductive cycles through transabdominal ultrasonic evaluation of the ovary (Brook et al 2000) and urinary hormone measurement (Robeck et al 2005). These techniques were combined with sperm cryopreservation (Robeck & O'Brien 2004) and hysteroscopic insemination, which resulted in the first bottlenose dolphin calf being born by way of AI (Robeck et al 2005).…”

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.…”