Literature Review

The development and assessment of wild or captive animal population management strategies are based on reproductively important parameters such as reproductive seasonality, estrous cyclicity and frequency, gestation length, age at reproductive maturity, growth rates, reproductive longevity, and factors affecting the calving interval. Most of our present knowledge concerning many of these reproductive parameters of killer whales, Orcinus orca, and other cetaceans are estimates which have been obtained through post mortem analysis of reproductive tracts and ovaries from stranded, entangled or harvested animals. (Nishiwaki and Handa, 1958; Jonsgard and Lyshoel, 1970; Christensen, 1984) Reproductive information has more recently been obtained through longitudinal observations of select stable wild populations of Orcinus spp. located off the coast of British Columbia and Washington State (Bigg, 2; Balcomb et al., 1982) and of captive populations (Sweeney, 1980; Bigg, 1982).Based on tooth growth layers, Christensen (1984) suggested a length at sexual maturity of 15 ft. and age of 8 years for females and for males between 18 and 20 ft. and 15 years. Based on growth rates of captive animals, Bigg (1982) estimated the age at which sexually mature lengths were reached was at 7 yrs and 10-12 yrs for females and males, respectively. Matkin and Leatherwood (1986) concluded that reproductive activities fluctuated seasonally, but the occurrence of this season varied on a regional level. Mating has been observed to occur throughout the year (Jonsgard and Lyshoel, 1970), from May to July in the North Pacific (Nishiwaki and Handa, 1958) and from September to January for the North Atlantic. Gestation has been estimated at 12 months (Jonsgard and Lyshoel, 1970) and more recently at 15 or 16 months (Perrin, ed., 1982; Matkin and Leatherwood, 1986) Calving intervals were approximated at a minimum of 3 yrs for wild stock (Christensen, 1984; Bigg, 1982; Balcomb, 1980).However, Sweeney (1980) noted that a captive pair of killer whales has conceived within four months of a parturition on three separate occasions, but normal rearing of the calves after each birth was not accomplished.

While longitudinal observational surveys with wild and captive populations and post-mortem analyses have yielded a wealth of information concerning social and behavioral interactions, accurate estimations of many of the above-mentioned reproductive parameters can be determined only by a combination of observational and physiological monitoring. Although the utility of comparing captive animal behavior to wild populations is wisely debatable, providing adequate technology is available, captive populations provide an undeniable opportunity for precise determinations of many reproductive physiologic events. These captive animal investigational determinations can then be carefully applied toward interpretation of wild population observations and data collection. The development of radioimmunoassays (RIA) has provided reproductive physiologists an accurate method for determining circulating levels of reproductive hormones (Lasley, 1980). When used with captive cetaceans, this technique could provide the necessary vehicle for determining reproductive physiologic values which can then be utilized toward a more complete understanding of wild populations and for the development of more efficient and intensive captive breeding programs (Kirkevold B.C., 1986; Schroeder, 1989).

RIA was first utilized in marine mammals by Harrison and Ridgway (1971). They determined that testosterone levels in adult male Tursiops truncatus were elevated during seasons which corresponded to the highest frequency of calving. Richkind and Ridgway (1975) were the first to evaluate reproductive endocrinological events in captive female Tursiops. They determined the effects of FSH administered intramuscularly and intravascularly on estrogen, corticosteroids and progesterone levels in gravid and non-gravid dolphins. Sawyer Steffen et al., (1983) evaluated baseline serum levels of progesterone and total immunoreactive estrogens from captive intact and ovariectomized Tursiops and newly captured wild adult females. They also attempted to induce ovulation using pregnant mares' serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). They concluded that levels of progesterone >3ng/ml for an extended period of time were indicative of pregnancy; Tursiops truncatus is capable of spontaneous ovulation in captivity; and that captive females can be induced to ovulate using PMSG and HCG. Baseline levels of progesterone have been reported to be <lng/ml with the average around 0.3ng/ml for T. truncatus (Sawyer-Steffen and Kirby, 1980; Sawyer-Steffen et al., 1983; Kirby and Ridgway, 1984), and D. delphis (Kirby and Ridgway, 1984). The 25-30 day progesterone episodic fluctuations ranged from baseline to 22ng/ml for T. truncatus and baseline to 15.5ng/ml for D. delphis. Wells (1984) and Kirby et al. (in prep) found similar baseline levels in Stenella longirostris, Orcinus orca, Lagenorhynchus obliquidens and Globicephala macrorhyncha. Wells (1984) and Kirby and Ridgway (1984) also found variable ovulation rates in T. truncatus, D. delphis and S. longirostris including: polyestrous, seasonal polyestrous, and one to two years of anestrus followed by "normal" cyclic patterns. Schneyer et al. (1985) validated commercially available human radioimmunoassay (RIA) kits for follicle stimulating hormone (FSH) and luteinizing hormone (LH) for use with dolphins. By determining serum progesterone levels from weekly, 3 yr longitudinal serum samples, Schroeder (1989) demonstrated variations from seasonal polyestrous to anestrus for 6 female bottlenose dolphins. Andrews et al. (1989) used biweekly to monthly serum progesterone levels from 10 killer whales to determine a mean gestation length of 515 ± 7 days, anestrus intervals and an approximate estrous cycle length of 45 days.

Longitudinal analysis of weekly serum samples has provided information which when appropriately combined with wild animal observations will increase the ability to properly assess and manage wild populations of those species. However, when trying to establish controlled or artificial breeding programs for captive populations, sampling frequencies are needed that will more precisely delineate estrous events in the breeding colony. Since daily sequential blood sampling is generally not considered a viable methodology, the use of serum analyses in order to define estrous events is seriously limited. The ability to measure reproductive hormone metabolites in the urine of many exotic hoofstock and primates provides reproductive managers with a mechanism for characterization of the daily hormonal fluctuations necessary for the development of efficient captive breeding programs (Lasley, 1985; Loskutoff et al., 1982; Loskutoff et al., 1986). Inspired by the desire to understand and improve the reproductive efficiency of captive killer whales, Sea World managers trained their whales to voluntarily provide daily urine samples. The first two years of these samples were used by Walker et al. (1988) to validate the efficacy of monitoring reproductive hormone metabolites in the urine. This research demonstrated that two common progesterone metabolites, pregnanediol-3-glucuronide, 20alpha-hydroxyprogesterone and estrone metabolites, estrone-conjugates, could be used as indicators of reproductive physiologic activity. Once these hormonal assays were validated, Walker et. al. (1988) were then able to determine a follicular cycle length of approximately 7 weeks and a gestation length of approximately 17 months.

Project Overview

The goal of this research is to utilize urinary RIA analysis of reproductive steroid hormone metabolites of progesterone and estrone in order to describe the reproductive activity of 6 captive killer whales. The results of these urinary analyses will provide insight towards defining reproductive seasonality, estrous cycle variability, and postpartum anestrus periods. We will also combine behavioral and hormonal data for determination of conception dates. Daily to weekly urine samples were collected rom 6 captive killer whales located at 4 Sea World locations from fall of 1985 to fall of 1989 for 3 whales. First, onlv weekly urine samples throughout the sampling period from all of the whales were analyzed for total immunoreactive levels of a progesterone metabolite, pregnanediol-3-glucuronide (PdG). The results from these initial analyses were used to determine seasonality and gestation lengths. Next, urine samples were analyzed on a daily basis during periods of estrous cycle activity (as determined by the weekly analysis) for total immunoreactive levels of PdG and an estrone metabolite estrone-sulfate (ES) in order to determine intra- and interindividual estrous cycle variations and conception dates.

The immunoreactive levels of the steroid metabolites were determined by radioimmunoassay (RIA) and adjusted for urinary concentration variances based on urine creatinine (cr) levels. Assays were validated by demonstrating parallelism between standards and a dose response curve. The interand intra-assay coefficient of variation (CV) for the PdG assay ranged from 27% to 29% and 5.8% and 7.8% respectively. The inter- and intra-assay CV for the ES assay was 14.5% and 5.9%, respectively. Finally, the interassy CV for the cr assay was 16.2%.

Selected Results and Discussion

In an effort to demonstrate the utility of the assays and to provide a limited view of the results, a brief discussion about the assay results from postpartum urine sample will be presented. During this study period, collection of urine samples occurred during three different postpartum situations. These situations are: a stillbirth, no suckling, early neonatal death, limited suckling attempts, and 3 successful births and subsequent suckling. The first detectable postpartum luteal phase (as determined by PdG) was considered to indicate the resumption of estrous activity. The cow which had a stillborn calf returned to estrous activity in 122 days. The cow which had a live calf, with non-successful suckling, returned to estrous activity in 53 days. (Figure 1).

Figure 1.

The cows with successful births and subsequent suckling had anestrus periods of 356 and 482 days. A third whale in this category had an anestrus period of at least 288 days before the urine sampling was discontinued. She was anestrous through spring and early summer, a time period when she had cycled before. The difference in mean anestrus periods between cows with suckling calves (375-33 days, ± 98.4) and cows without suckling calves (X=88.5 days, + 33.5) was 286.8 days.

In general, lactational alteration of reproductive function is believed to be caused by suckling stimuli which suppress gonadotropin (particularly luteinizing hormone) secretion, preventing normal follicular maturation and ovulation (McNeilly, 1988). The change of reproductive activity associated with suckling is species dependent, and within species considerable animal variation occurs. Generally, the domestic pig remains anestrus through the first four to eight weeks of lactation. Ewes with suckling lambs as compared to lactating ewes without lambs will have a delay in estrous activity of at least 3 weeks. Cows without calves will have a postpartum interval of anestrus of approximately 20 to 70 days. This postpartum anestrus period may be delayed for up to 150 days postpartum in cows with suckling calves. (McNeilly, 1988) Most horses have a postpartum estrus termed a "foal heat" which occurs between 5 and 12 days postpartum, but lactational anestrus may begin after parturition or subsequent to a nonconceptive foal heat. This anestrus may continue until the foal is weaned at 4 to 6 months (Ginther, 1979). Based on the 286 day anestrus difference observed between whales with suckling calves and whales without, it would appear that suckling (which also helps to maintain lactation) plays an important role in regulating the calving interval of killer whales. In our study, the calving interval between nonsuccessful calf rearing (stillbirth or unsuccessful nursing) was approximately 3 years (1060 days and 997 days). If both of these females had been with males during the beginning of their first postpartum estrous activity (assuming the estrous cycles were fertile and that they conceived on their second estrus), their calving interval would have been approximately 19 and 21 months, respectively. The calving interval observed in one whale with a successful suckling period followed by conception was 3 years 2 months. The minimum calving interval observed for wild killer whales by Balcomb et al. (1980) was three years. However, the average birth rate for wild population was estimated at 8.59 years (Balcomb et al., 1980) and 10.30 years (Biggs et al., 1982). Sweeney (1980) reported observing captive killer whales conceiving within 4 months of having early postpartum or peripartum neonatal death. The reduced calving interval of captive whales as compared to wild whale calving intervals is probably explained to some extent by nutrition and environmental difference, as Matkin and Leatherwood (1986) suggested. A decreased reproductive productivity in response to adverse or seasonal nutritional and environmental conditions is well documented among wild and domestic animals (Bronson, 1988). Another possible explanation for the calving interval variation might be that early postpartum or peripartum neonatal calf mortality might go unnoticed by wild killer whale observers. This would increase the minimum calving interval of 3 years by at least 19 months (based on our results) to almost 5 years and in conjunction with the nutritional/environmental factors might help explain the captive/wild differences.

The evidence presented indicates suckling suppression of estrous activity has important ramifications for optimizing reproduction of captive killer whales and for clarifying interpretations of calving interval obtained from wild populations.

Acknowledgments

We thank the training and animal care laboratory staff at Sea World of California, Texas, Florida and Ohio for making this research possible. We thank Dee Smith for her help with the RIAs and Laura S. Chenault-Robeck for her assistance and for providing helpful criticism of this manuscript. Support was provided by Nixon Griffis Fund for Zoological Research. This is Sea World Technical Contribution Number 9001-C.

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