Artificial insemination of bottlenose dolphins (Tursiops truncatus): A trial with simple instruments based on criteria for estrous behaviors linked to changes in estradiol levels and follicle development

Abstract

Herein, we report a case of pregnancy of a female bottlenose dolphin (Tursiops truncatus) that was subjected to artificial insemination (AI) in water based on its estrous behavior using simple instruments. AI was performed on this female dolphin once or twice daily for 4 days at the detection of estrous behavior, such as floating horizontally and showing reduced responsiveness, likely indicating the appropriate timing for AI. The female was placed in supine a position in the water to position the genital slit above the water surface. A Nélaton catheter (Fr. 10, 40 cm length), with its tip modified, was inserted approximately 20 cm into the vagina through the genital slit, and 1–2 ml of fresh semen was injected. The AI procedure was performed within 1 min by two technicians. Thus, this AI method may be a new choice for artificial reproduction, as pregnancy success can be achieved with relatively less cost, less difficulty, and less invasive treatments of cetaceans.

Assisted reproductive techniques have been applied to captive animals [1]. Artificial insemination (AI) is a useful method for achieving systematic breeding management for cetaceans, which are aquatic mammals and, thus, cannot be readily transported between facilities. The application of the conventional AI method to bottlenose dolphins (Tursiops truncatus) generally requires holding female animals, bringing them ashore, and injecting sperm into their uteri with a catheter using an endoscope [2,3,4]. However, this conventional method of moving animals from water to land and holding them can burden both animals and their keepers. It is difficult to use endoscopes, which can improve the success rate of AI, as they are expensive and require operating skills. The optimal timing of insemination is usually estimated by ovulatory detection or by determining peak levels of estrogen and luteinizing hormone [2, 5,6,7]; however, the high-frequency ultrasound examination of aquatic mammals can be a burden to investigators, and hormonal evaluation can take a long time to obtain diagnostic results. In this study, we report a case of pregnancy success in a female bottlenose dolphin in a trial of AI with simple instruments while kept in water, based on the criteria for estrous behavior. In addition, we demonstrate that the observation of estrous behavior is a valid indicator for estimating the optimal timing of AI.

The animals used in this study were treated in accordance with the Code of Ethics for Breeding Animal Experiments prepared by Marine Palace, Inc. and approved by the Oita University Animal Ethics Committee (No. 175101). The test individuals were three mature female bottlenose dolphins (ID-16: 272 cm body length, 10 years estimated age, and nulliparous; ID-10: 274 cm body length, 12 years estimated age, and parous; and ID-17: 287 cm body length, 11 years estimated age, and nulliparous) and one mature male (ID-Athens: 299 cm body length, 13 years estimated age, and proven sire). The dolphins were kept at Tsukumi Irukajima in Oita Prefecture, Japan, and housed separately in fenced open-sea pens (females: 20 m × 15 m × 3.5 m depth; ID-Athens: 10 m × 10 m × 4 m depth). The average ambient temperature in 2017, when the AI was performed, was 16.0°C (a maximum of 27.5°C in July and a minimum of 3.0°C in January), the average seawater temperature was 18.5°C (a maximum of 26.2°C in August and a minimum of 12.1°C in February), and the day length was the same as in the natural environment. From October 2014 to August 2015, estrous behavior was observed in ID-16 during feeding or standard training sessions, such as floating horizontally on the water surface (Fig. 1A), sinking and rearing vertically in the water, keeping eyes closed and mouth slightly open, showing reduced responsiveness, which was similar to that reported in previous studies [2, 8], and mating when cohabiting with a male. In addition to daily behavioral observations by 1–4 investigators, blood samples were collected from ID-16 almost once weekly, and the serum estradiol concentrations (E₂) were measured. E₂ showed periodic peaks that were synchronized with the onset of estrous behavior (mean duration of sexual behaviors of 4.3 days, with a maximum of 6 days and a minimum of 2 days; Fig. 1C). Based on these results, it was possible to determine estrus based on daily behavioral observations. Behavioral expression because of E₂ elevation during follicular development and after spontaneous ovulation can be used as an indicator of the appropriate timing to conduct AI, which, in this study, was performed using a new method. The instruments used were a food storage plastic bag for semen collection and a two-hole Neraton catheter (Fr. 10, 40 cm length) connected to a 20 ml syringe (Fig. 1B) to inject semen into the female genitalia. The AI was conducted in July 2017 when ID-16 was kept separate from male individuals. When estrous behavior was observed in ID-16, semen was collected in a plastic bag from ID-Athens using the hand massage method [9]. Although semen quality parameters, such as sperm concentration, viability, and motility, were not evaluated, the male had a breeding history by mating. Semen collection was performed once or twice daily during the estrous behavior observed in ID-16. The semen volume collected was 1–2 ml, which was sucked up with a catheter into a 20 ml syringe with 10 ml of air. ID-16 was kept in a supine position in the water, its genital slit was raised above the water surface, and seawater was removed from the genital slit trench to avoid contact between semen and seawater, as described previously [10]. A catheter was inserted approximately 20 cm into the vagina through the slit, and semen was injected. AI was conducted once daily from the 1st to the 3rd day of estrous behavior (Fig. 1D). Female ID-16 could be immobilized voluntarily during blood sampling or body temperature measurements through daily husbandry training, and no dedicated training was required to perform this AI method. On the 4th day, AI was conducted twice because the volume of semen ejaculated was small compared with that during the first AI. On the second AI, 4 h after the first AI, almost the same volume of semen was ejaculated as on the days before. Estrous behavior was observed 2 days after the completion of the 4-day AI treatment. In total, five semen injections were administered over 4 days. The semen injection procedure lasted approximately 1 min. The entire process, from semen collection to AI, took approximately 5 min and was performed by two technicians. After the AI trials, the serum progesterone concentrations (P₄) increased from 0.1 ng/ml at 2 days after the first AI to 5.8 and 16.0 ng/ml at 15 and 27 days after the first AI, respectively. Pregnancy was diagnosed using ultrasonographic examination 75 days after the first AI.

Fig. 1.

(A) A female bottlenose dolphin (ID-16) showing estrous behavior by floating horizontally on the water surface. (B) A syringe and a catheter were used for semen injection. (C) Serum estradiol concentrations and estrous behaviors were observed from October 1, 2014, to August 31, 2015, in the same female (ID-16). (D) Serum estradiol and progesterone concentrations after artificial insemination (AI) in the same female (ID-16). Black arrows show the timings of AI treatments, which were conducted during the expression of estrous behavior.

A detailed investigation was performed on the three mature females to evaluate whether estrous behavior could be used as an indicator of optimal timing to conduct AI. In this study, three females (ID-16, ID-10, and ID-17) were used to examine E₂ and follicle dynamics during the expression of estrous behaviors. Blood collection and the ultrasonographic examination of ovaries were performed on ID-16 and ID-17 once daily from the 1st or 2nd day of the onset of estrous behavior until E₂ decreased to the baseline level. During estrous behavior, which lasted 4 days for ID-16 and 3 days for ID-17, increased E₂ (ID-16: maximum of 94.7 pg/ml; ID-17: maximum of 50.0 pg/ml) and follicle growth (ID-16: maximum diameter of 2.53 cm; ID-17: maximum diameter of 2.40 cm) were observed, as reported earlier [11]. On the day after the end of the expression of estrous behavior, E₂ decreased to the baseline level (ID-16: 13.7 pg/ml; ID-17: 9.4 pg/ml), and a loss of follicles was observed (Fig. 2). For ID-10 and ID-17, blood sample collection was performed once daily during the expression of estrous behaviors throughout a year, in addition to periodic health checks and individual treatments. The average duration of the expression of estrous behavior in the two dolphins was 4.9 days (a maximum of 10 days and a minimum of 2 days). In ID-10, estrus was observed 5 times from April to September 2021 (Fig. 3), with an average interval of 27.5 days (a maximum of 31 days and a minimum of 26 days). The average duration of the expression of estrous behavior was 6.2 days (a maximum of 10 days and a minimum of 3 days), with an increase in E₂ (average peak concentration of 46.4 pg/ml, with a maximum peak of 56.2 pg/ml in September and a minimum peak of 32.6 pg/ml in June). After September, there was no expression of estrous behavior until March of the following year, suggesting that the animal was in an anestrous period [12]. In ID-17, estrus was observed 4 times between April and November 2020. The estrous interval in ID-17 was longer than that in ID-10, with an average interval of 55 days (a maximum of 56 days and a minimum of 54 days) from July to November, excluding the period when the cycle was extended from April to July. The average duration of the expression of estrous behavior was 3.3 days (a maximum of 4 days and a minimum of 2 days), with an increase in E₂ (average peak concentration of 48.6 pg/ml, with a maximum peak of 61.3 pg/ml in July and a minimum peak of 33.2 pg/ml in September). Estrous behavior was observed from April 22 to July 12, and the estrous interval extended to 81 days. During this period, P₄ increased to 14.3 ng/ml in June. False pregnancy cases have been reported in female bottlenose dolphins, with the disappearance of cyclic E₂ elevation and a prolonged luteal phase with high P₄, possibly because of stimulation from a cohabiting immature male [12]. Notably, in this study, ID-17 cohabited with a male individual (ID-24) on April 4. Reproductive behaviors, such as chasing and mounting, followed by mating, reported previously as estrous behaviors [8], were observed during this cohabitation period, which may have stimulated ID-17 into false pregnancy. This cohabitation and mating behavior of ID-24 with ID-17 and other females, observed until January 2022, did not result in pregnancy in the females. The average serum testosterone concentrations in ID-24 were 0.24 ng/ml from April to November 2020 (maximum concentration of 1.1 ng/ml in July and below the detection limit in October). Therefore, ID-24 was considered immature based on a previous report that circulating testosterone levels in an immature male are 0.8 ± 0.1 ng/ml [13]. On July 5, P₄ in ID-17 decreased to the baseline level; subsequently, the expression of estrous behavior resumed with an increase in E₂. No expression of estrous behavior was observed from November to April, suggesting that ID-17 was in an anestrous period similar to that observed for ID-10 (Fig. 3).

Fig. 2.

Changes in serum estradiol levels and follicle sizes measured in two female dolphins, (A) ID-16, and (B) ID-17, during their estrous cycles in 2018. Photographs show the images of follicles observed by an ultrasonographic examination at the time indicated by white arrows. Black arrows indicate the time when the follicles disappeared.

Fig. 3.

Serum estradiol and progesterone concentrations and estrous behaviors of two female dolphins, (A) ID-10, and (B) ID-17, as observed throughout 2021 and 2020, respectively.

In this study, pregnancy was achieved by a simple intravaginal injection of fresh semen using a commercial catheter with the female kept in water. However, it remains unclear how far the catheter reached and at which site semen was inseminated into the female genitalia. In a previous study, Orbach et al. concluded that the penis tip did not penetrate the cervix and was blocked by the vaginal folds [14]. The penis length of ID-Athens was 15 cm from the base to the tip, and the cervical length of female bottlenose dolphins was ~24 cm deep [4]. In this study, the injection of the catheter to approximately 20 cm may have resulted in ejaculation in the vagina. However, Yamamoto et al. reported that the conception rate of intrauterine insemination was 60–67%, whereas the conception rate of intravaginal insemination was 0% [5]. Determining the ideal position for ejaculation to obtain stable fertility results using this simple AI method will be a challenge in the future. Estrous behavior appeared along with an elevation in E₂ and follicle growth and ended with a decrease in E₂. Therefore, the disappearance of follicles was considered an indicator of the optimal time to perform AI. The results obtained in this study suggest that pregnancy can be achieved by performing AI on consecutive days after the onset of estrous behavior without the detection of ovulation. However, as there are individual differences in the behavior, intensity, and duration of estrous behavior, individual characteristics must be understood.

In conclusion, the successful AI trial performed in this study used only simple tools and did not require expensive medical equipment or elaborate techniques. The optimal timing of AI was determined immediately based on the evaluation of natural estrus through behavioral changes, which can eliminate the personal burden of detecting ovulation using ultrasonography or the loss of time in obtaining the results of hormonal diagnoses. This AI method is less invasive compared with the conventional endoscopic AI method, as female dolphins can stay in water during AI operations, and no dedicated training is required. The AI procedure takes less than a minute when performed by two people and does not require much manpower; therefore, it can be carried out daily as long as estrous behavior is expressed. Thus, the AI trial performed in this study is cheaper, easier, and less invasive than the conventional endoscopic AI method and, therefore, can be a new choice for artificial reproduction in cetaceans.

Methods

During the study, ambient and seawater temperatures were measured daily at 0800 h. Approximately 3 ml blood samples were collected from the caudal fin vein of animals voluntarily immobilized by husbandry training to investigate peripheral hormone dynamics. The blood samples were transported on ice in a chill box to a commercial laboratory (Rintec Co., Ltd., Fukuoka, Japan), and E₂ and P₄ were measured using a chemiluminescence immunoassay. Follicle size was examined using an M-Turbo ultrasound system (Fujifilm SonoSite, Tokyo, Japan) equipped with a C60/5-2 (2–5 MHz) convex probe attached to the abdominal body surface of the dolphins while they were in a supine position on the sea surface [15].

The behavior of the female bottlenose dolphins was observed daily by 1–4 investigators during feeding and husbandry training between 0800 and 1600 h. The estrous behaviors observed included floating horizontally on the water surface (Fig. 1A), standing on sticks on the water surface or in the water, slowing responses to other stimuli, keeping eyes open and mouth slightly open, and extremely reduced responsiveness to food and holding a fish in their mouth. AI was conducted on ID-16 when the female exhibited estrous behavior. The instrument used was a 20 ml plastic syringe (Terumo, Tokyo, Japan) connected to a two-hole Neraton catheter (Fr. 10, 40 cm length; Terumo) (Fig. 1B). The tip of the catheter was cut and modified into a one-hole opening to avoid air contamination during semen aspiration and was rounded to prevent damage to the vaginal mucosa. Semen was collected in a plastic bag from a male bottlenose dolphin using the hand massage method immediately before AI. Semen was drawn into the catheter using the syringe. Air (~10 ml) was filled in the syringe in advance to inject semen into the female genitalia without wasting. The female was placed in the supine position in the water. The genital slit was raised above the sea surface, and the water present in the genital slit was removed by hand to avoid contact between semen and seawater. A catheter was inserted approximately 20 cm into the vagina through the slit, and semen was injected.

Conflict of interests

The authors declare no conflict of interest.