Evidence that bears are induced ovulators

Theriogenology 61 (2004) 1163–1169

Evidence that bears are induced ovulators William R. Boonea,*, Becky B. Keckb,1, Jeffery C. Catlinc, Kevin J. Caseyd, Edna T. Boonee, Penny S. Dyef, Randy J. Schuettg, Toshio Tsubotah,2, Janice C. Bahrh a

Reproductive Endocrinology & Infertility, Greenville Hospital System, 890 W. Faris Road, Suite 470, Greenville, SC 29605, USA b Clemson University, Department of Animal and Veterinary Sciences, Clemson, SC 29634, USA c Cook OB/GYN, Inc., 1100 W. Morgan Street, Spencer, IN 47460, USA d Bear Country USA, HCR 33, Box 1110, Rapid City, SD 57701, USA e 107 Cardinal Court, Simpsonville, SC 29681, USA f Dakota Hills Veterinary Clinic, 1571 E. Highway 44, Rapid City, SD 57701, USA g Pewaukee Veterinary Service, SC, W240 N3425, Pewaukee Road (Highway J), Pewaukee, WI 53072, USA h Department of Animal Science, University of Illinois, Room 328, 1207 W. Gregory Drive, Urbana, IL 61801-3838, USA Received 12 November 2002; accepted 4 July 2003

Abstract The objective of this study was to determine if black bears are induced ovulators. We conducted a single experiment with two replicates; each replicate was divided into two arms: females exposed to male bears and females without male exposure. We used laparoscopy to examine ovaries for corpora lutea and measured serum progesterone concentrations. Six of the seven isolated females failed to ovulate, while seven of the eight females exposed to males produced one to four corpora lutea. Furthermore, isolated females had significantly lower progesterone concentrations than females exposed to males. Thus, our data suggest that the American black bear is an induced ovulator. These results may aid biologists in their efforts to reproduce ursids in controlled environments. # 2003 Elsevier Inc. All rights reserved. Keywords: Induced ovulation; Black bear; Reproduction; Corpora lutea; Progesterone

* Corresponding author. Tel.: þ1-864-455-5979; fax: þ1-864-455-8492. E-mail address: [email protected] (W.R. Boone). 1 Present address: Horry-Georgetown Technical College, P.O. Box 261966, Conway, SC 29528-6066, USA. 2 Present address: Laboratory of Zoo and Wildlife Medicine, Devision of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan.

0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.07.016

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1. Introduction Ovulation, the production of oocytes, can occur spontaneously, or can be induced through physical stimulus. Most mammalian species are spontaneous ovulators. Spontaneous ovulation is a process in which ovulation occurs as a result of a hormonal sequence of events at a specific time in the reproductive cycle and is independent of mating [1]. In induced ovulation, the act of copulation initiates a series of neural events that pass to the brain and lead to the release of one or more oocytes. Several species are known to be induced ovulators, including the short-tailed shrew (Blarina brevicauda), 13-lined ground squirrel (Citellus tridecemlineatus), short-tailed field vole (Microtus agrestis), domestic cat (Felis catus), rabbit (Oryctolagus cuniculus), ferret (Mustela putorius), mink (Mustela vison), raccoon (Procyon lotor), and llama (Lama glama) [2]. Whether animals should be classified as induced versus spontaneous ovulators is not always clear. Although bears are assumed to be induced ovulators, factual evidence has not been established [3]. Wimsatt proposed that this question of induced ovulation ‘‘. . . could easily be resolved in captive bears; one need only to isolate females during the period when ovulation normally occurs and examine the ovaries thereafter for the presence of large follicles or corpora lutea. It would be essential to the experiment not only that sexually mature females be used, but that they had not born cubs the preceding winter or they had been deprived of their cubs in early spring’’ [3]. The objective of this study was to confirm preliminary data concerning induced ovulation among bears [4]. In this report, we related mating behavior to ovulatory events in American black bears (Ursus americanus).

2. Materials and methods Bear Country USA, is a drive-through park in Rapid City, SD, USA. Inside this park are approximately 80 female and 110 male American black bears that are housed in an area of approximately 30 hectares. We performed two replicates in this study. In the first replicate, eight female bears were randomly selected and placed in one of two protocols. Four females were allowed to roam with the 80 female and 110 male bears in the 30-hectare pen, while four females were isolated from male contact in a 220 m2 pen approximately 100 m from the edge of the 30-hectare pen. Between replicates, a 4-year respite gave all bears time to re-equilibrate to the 30-hectare pen. We repeated the replicate with the use of seven, randomly-assigned, female bears; four from the previous replicate and three new bears. The same protocol was used for the second replicate with the exception that three females were isolated and four were allowed to mate. Because of limited resources, we used the females in a nonsurgical embryo transfer study in conjunction with this induced ovulation study. If the females had reproductive tracts that we could not palpate per rectum because of their small size, the females were

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eliminated from the study. Because four of the eight females were replaced and because there were four years in between these replicates, the animals were simply re-randomized into one of the two arms of the study. The females (n ¼ 11) selected for this study were mature (4 year of age) and weighed 116:2  5:6 kg (mean  S:E:M:). (Female bears with compromised body weight and reproductive status fail to complete the reproductive cycle and lose cubs before birth [5,6].) Only bears that previously produced cub(s) were eligible for this study. To insure that the bears had the opportunity to ovulate, caretakers removed all cubs from their mothers in March. Furthermore, to confirm that the bears selected were actively breeding, visual detection of estrus, similar to that reported for cattle, was used [7]. In addition, mounting was a prerequisite for acceptance into the study. Estrus detection started in mid-May with the first mating and ended in late June or early July after approximately 1 week of no observed matings. All bears in the study that were housed with the males in the 30-hectare pen were aroused daily at 10:00 h and observed for signs of estrus (sexual receptivity), which included mating. The arousal was performed by driving a park vehicle near the female bears. We continued to observe the bears until 20:00 h, with intermittent observations, as is customary when observing cattle for detection of estrus. Because the female American black bear is polygamous and because estrus is

Table 1 Body weight and reproductive end points in nonmated and mated American black bears Replicate

Bear identification

Weight (kg)

Number of matings (within 30–60 min)

Estrus duration (days)

Interval from last observed mating to observation of CL (days)

Number of CL

Serum progesterone (ng/ml)a

1 1 1 1 1 1 1 1

A B C D E F G H

129 111 107 132 111 91 102 123

N/Ab N/A N/A N/A 2c 4c 4c 3c

N/R N/R N/R N/R N/R N/R N/R N/R

Not Not Not Not 9 20 30 35

0 0 0 0 1 2 1 4

1.50 0.95 0.75 0.95 0.70 1.75 2.50 2.70

2 2 2 2 2 2 2

B D F C I J K

106 149 115 90 80 148 149

N/Rd N/R N/R N/R N/R N/R N/R

N/A N/A N/A 11e 10e 1e 2e

Not mated Not mated Not mated 27 27 63 37

2 0 0 N/Vf 2 3 2

0.88 0.32 0.37 2.27 1.39 1.90 1.82

a

mated mated mated mated

Mated females had higher progesterone concentration than nonmated females (P ¼ 0:029). Not applicable. c Number of matings within 30–60 min of observations. d Not recorded. e Number of days in which matings occurred, as determined from opportunistic observations. f Ovaries were not visible. b

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several days in duration (Table 1), we were confident that we could detect estrus during the day-light hours and so we did not perform night-time observations. Similar regimens are used for estrus detection in cattle. Females were considered to be in estrus if they remained still when mounted. The females that were isolated from males were not aroused for estrus detection. Breeding behavior in the American black bear has been described in detail elsewhere [8]. In brief, bears undergo a breeding pattern similar to many other mammalian species (e.g. cattle, sheep, cats), which can be divided into three standard segments: courtship, mating, and refractory period. Courtship begins with an obvious movement by the male toward the female and ends when the male mounts the female. Courtship includes posturing by the male, urogenital sniffing of the female, as well as auditory emissions to the female. If the female is responsive, she will undergo lordosis, and is subsequently mounted by the male. Mating includes mounting and pelvic thrusts that are often followed by a flutter. The refractory period begins with a dismount by the male or a ‘‘roll-out’’ by the female to break the union. During the refractory period, the male is no longer interested in the female and the female is less receptive to a male. Estrus in the American black bear has many aspects. Prior to an intensive study designed to study this topic, we attempted to gather preliminary data. The first year, our objective was to determine if the female mated more than once and, if so, was she monogamous or polygamous (Table 1). Having determined that American black bears mate more than once and with more than one male, we did not pursue these data any further during the first year. During the second year of this study, we turned our attention to the length of the estrus cycle for individual females (Table 1). In July, we moved the bears from their respected pens to the laboratory where we performed phlebotomy and laparoscopy. To restrain the bears, we sedated them with either ketamine hydrochloride (4 mg/kg, i.m.; Ketaset1, Fort Dodge Laboratories, Fort Dodge, IA, USA) and xylazine (1 mg/kg, i.m.; Rompun1, Miles, Inc., Shawnee Mission, KS, USA) or tiletamine/zolazepam HCL (8 mg/kg, i.m.; Telazol1, Fort Dodge Laboratories) and xylazine. (We selected the drug regimes based upon cost and availability.) Bears were transported, via stretcher, to the on-site laboratory. In the laboratory, bears were intubated and placed on isoflurane (1–5% Forane1, Ohmeda, Guayama, Puerto Rico). Heart rate and respiration were monitored every 5 min. The laparoscopic procedure in the American black bear has been described in detail elsewhere [9]. Briefly, after surgically preparing the abdomen, we placed a pneumoperitoneum needle through the umbilicus and filled the abdominal cavity with CO2. A trocar and sleeve were placed through the umbilicus. The trocar was removed and a laparoscope was passed through the sleeve. In addition to the laparoscope, a retractor and a grasper were inserted similarly into the lower abdominal quadrants to retract bowel and facilitate a better view of the ovaries. Blood was drawn from an intravenous catheter with serum being separated and frozen at 20 8C and shipped (with dry ice), to the laboratory for radioimmunoassay of progesterone as previously described [10,11]. Intra- and inter-assay coefficients of variation were 4.1 and 10.4%, respectively. The difference in mean progesterone concentrations between mated and nonmated bears was assessed with Analysis of Covariance (covariate ¼ bear).

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Fig. 1. Corpora luteum (CL) on an ovary of an American black bear. The ovary and oviduct (OV) are encased in a bursa (2).

3. Results Laparoscopy revealed six of seven isolated bears failed to ovulate (Table 1). Bear B did not ovulate in the first trial, but, while isolated in the second trial, produced two corpora luteum (CL). In contrast, seven of eight females allowed to mate with males in the open pen had one to four CL per animal (Fig. 1). Progesterone concentrations for isolated bears were 0:82  0:15 ng/ml (mean  S:E:M:) compared to 1:82  0:30 ng/ml for bears allowed to mate (P ¼ 0:029).

4. Discussion Using laparoscopy and radioimmunoassay, we demonstrated that most American black bears are induced ovulators. A few bears appear to release ova without neurological response provided by the mating process, but the proportion of the bears that do so is unknown. Similar results are reported for felids, which are classified as induced ovulators [12–14]. It would appear that most bears, like most cats, require vaginal touch to induce ovulation. While the isolated females did not come in direct contact with males, the short distance (approximately 100 m) between these two groups of bears would allow for olfactory and auditory cognition. Because bears are known to have a keen sense of smell [15], we speculated that the isolated females were aware of males in the area. Progesterone concentration remained near baseline among nonmated females (0.32– 1.50 ng/ml). Others report similar baseline results for serum progesterone concentrations from pre-bred, estrus, and lactating adult black bears (<1.0 ng/ml [6,9]), for brown bears that were 210–300 days pre-parturition (0.4–1.1 ng/ml [16]), and for nonpregnant polar

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bears (<1.0 ng/ml [10]). In our study, as post-mating interval increased, progesterone concentrations increased among mated females (0.70–2.70 ng/ml). Similarly, elevated progesterone concentrations appear among pregnant bears, regardless of the species [5,6,10,16–18]. Furthermore, our findings were confirmed by observing the number of CL. In isolated bears, seven of eight bears had no detectable CL, whereas in mated females, at least one CL was present in each bear when ovaries were observable. By observing reproductive tracts from feral animals killed between late May to midDecember, Wimsatt speculated that bears were induced ovulators. However, Wimsatt’s observations were based upon indirect evidence. Using factors directly associated with ovulation, i.e. progesterone concentrations and number of CL, our report is the first to verify that mating is required to initiate ovulation. While not directly observing CL, Sato et al. have confirmed in Japanese black bears (Ursus thibetanus japonicus) similar differences in progesterone concentrations between isolated and mated bears [18]. On the basis of current results, two future studies need to be performed. One, a study to determine what proportion of the bears are spontaneous ovulators should be conducted. Two, even though we repeated this study to reduce potential year-to-year variation, the number of bears used was small. We would encourage others to repeat our study, possibly with different bear species, to verify or to disprove our results. Our preliminary data indicated that the estrus cycle in the American black bear has many aspects that need to be explored. While it appears that the black bear has a single estrus period that occurs in late spring, this may not be the case. Furthermore, the estrus cycle may last for more than 1 week. During this time, the female is receptive to more than one male and will mate multiple times during a day. What is not known is the number of copulations that are necessary to induce ovulation, if multiple partners will produce offspring from different sires, or if the estrus characteristics that we have observed are altered depending upon the male population present. The factors that determine the length of the estrus, the numbers of matings per day, and the males with which the female is willing to breed are unknown. Further studies are needed to address these questions. In conclusion, we confirmed that the majority of nonmated bears failed to ovulate, whereas most mated bears ovulated and produce corpora lutea. Thus, the American black bear can be considered an induced ovulator.

Acknowledgements Bear Country USA, Rapid City, SD provided animals and facilities for this research. United States Surgical Corporation, Norwalk, CT, provided laparoscopic equipment. Greenville Hospital System, Greenville, SC, provided a supportive environment in which to conduct this research.

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