A Message From The SSP Coordinator, Don Lindburg2004 Summary of Reproductive Activities for Giant Pandas
at the Smithsonian’s National Zoological Park (NZP)
During the 2004 breeding season, NZP scientists and managers used endocrinology, vaginal cytology and behavior to assess reproductive status during the peri-ovulatory interval in the female giant panda, Mei Xiang (SB 473). Reproduction also was evaluated in the male, Tian Tian (SB 458).
Reproductive Hormones: Noninvasive urinary hormone monitoring permits hormonal information to be obtained within hours after urine specimens are collected. Over the past 3 years (2002 – 2004), ovarian hormones (estrogen and progesterone byproducts) have been assessed year-round in daily urine specimens collected from Mei Xiang. Additionally, testosterone has been assessed in urine samples obtained from the male Tian Tian to assess reproductive status. Hormonal measurements in Mei Xiang are particularly important for assessing ovarian status during the peri- and post-ovulatory intervals (March – April) and for assisting animal managers in preparing for an impending birth or, in the case of a false pregnancy, signalling that a pregnancy watch is no longer necessary (see below).
In 2004, urinary hormone analysis was conducted to determine optimal time for pairing the male and female for mating. Urinary estrogen increased steadily beginning in mid-April coincident with a subtle, yet distinctive decline in urinary progesterone. Urinary estrogen peaked on May 1st at 1000 h (139.4 ng/mg Cr) and declined that same day by 1600 h (80.2 ng/mg Cr); a concomitant increase in urinary progesterone confirmed that ovulation had occurred. These data confirmed that Mei Xiang experienced a normal ovulatory ovarian cycle, and that ovulation occurred on May 1, 2004.
Vaginal Cytology: Analysis of exfoliated vaginal epithelial cells provides critical information for determining estrus and the optimal time for mating or artificial insemination. A vaginal cytology profile (beginning months before peak estrogen) allows the prediction of estrus and ovulation in a seasonal, monoestrus species like the giant panda. Prior to ovulation, progressive changes in vaginal cell type (from basal to intermediate to superficial) correlate with increasing concentrations of estrogen. Using the trichrome Papanicolaou (PAP) stain, changes in vaginal cell color from basophilic (blue) to acidophilic (pink) to keratizined (orange) also correlate with rising estrogen. At the time of peak estrogen, females are expected to have >70% superficial cells (some females have >90% at peak estrus) and >70% orange cells. Most importantly, changes in cell color (from blue to pink to orange) provide critical information for predicting time of estrus. As the female approaches estrus, there are two obvious chromic shifts. First, from blue to pink, and finally from pink to orange. The first chromic shift (from blue to pink) occurs ~8-9 days before peak estrogen (Durrant et al., 2002, Theriogenology 57). The second chromic shift (from pink to orange) occurs ~2 days before peak estrus. Following ovulation, the cell type and cell color begin to change rapidly within days.
In 2004, the NZP animal care staff used the panda training station to collect vaginal swab samples. The training station permits routine medical procedures (e.g., ultrasound, x-rays and blood collection) to be conducted on both Mei Xiang and Tian Tian without animal stress. Both animals appeared eager to enter the training cage, which remains open providing the animal with the option of exiting the cage at any time during a procedure. Beginning in February, the training station was used to facilitate the collection of vaginal swabs from Mei Xiang. A small sterile cotton swab moistened with sterile saline was used to collect vaginal epithelial cells while Mei Xiang was seated, grasping the bars at top of the cage with her forepaws. The vaginal swab then was rolled on at least two glass slides and sprayed with ‘SprayCite’ fixative. Slides were stained using the PAP stain protocol, and a total of 100 vaginal cells were assessed and categorized for cell type (basal, intermediate, superficial) and color (blue, pink, orange).
The increased duration and frequency of vaginal swabs collected in 2004 (as compared to 2003) resulted in excellent longitudinal vaginal cytology profiles (Fig. 2, 3). The PAP stain was effective for assessing reproductive status and predicting estrus in Mei Xiang. During February, intermediate cells increased from 30% to 90% and remained elevated (>90%) until April 26, during which time superficial cells remained low. Beginning April 27, superficial cells increased markedly from 12% to a peak of 79% 4 days later (May 1st). Interestingly, May 1st also was the day of peak estrogen concentrations in Mei Xiang, demonstrating the high correlation between elevated estrogen and superficial vaginal epithelial cells. Although fluctuations in cell colors were observed from February to April, basophilic cells increased to 91% on April 5th, then declined thereafter as acidophilic and keratinized cell types markedly increased. The keratinized (orange) cells reached 71% on May 1st (the day of peak estrogen and superficial cells) and remained elevated until May 6th. The first (from blue to pink, April 21st) and second chromic shifts (from pink to orange, April 27th) occurred 10 and 4 days before peak estrogen, respectively.
These results confirm how vaginal cytology, used in combination with endocrine and behavioral monitoring, provides a useful tool for giant panda reproductive management.
Behavioral Measures: On April 26th, Mei Xiang began scent marking at twice the rate she displayed in 2003. Scent marking frequency increased more rapidly this year compared to 2003. Over the next several days, she continued to scent mark at a high rate, pace and bath in her pool. When not engaged in these behaviors, Mei Xiang rested in her favorite willow tree. Reproductive activity began on April 30th and peak activity continued on May 1st and May 2nd. Initially, Tian Tian attempted to mount Mei Xiang on numerous occasions, but she responded by collapsing and/or remaining positioned on her abdomen or side. During mounting attempts, Mei Xiang exhibited tail-up behavior, but not lordosis. Mei Xiang continued to exhibit solicitous behavior, whereby she repeatedly backed into Tian Tian with her tail raised, but by late afternoon (1600 h) on May 1st his libido ceased. Mei Xiang continued to exhibit estrus behaviors through May 8th.
Mei Xiang’s estrus was characterized by highly solicitous behaviors (e.g., chirping, tail up, backwards walking and, to a lesser extent, bleating) that peaked as urinary estrogens were declining. Lower intensity behaviors, like scent marking, characterized the pre-ovulatory interval, which is similar to what has been reported in other giant pandas. However, scent marking and bleating behavior frequencies exceeded rates observed in previous years, whereas the rate of tail up behavior was 50% lower than we observed in 2003. Post-ovulatory receptive behaviors lasted for approximately 6 days, which was longer than observed in 2003.
Mei Xiang failed to exhibit appropriate lordosis posture during Tian Tian’s mounting attempts. We are unsure whether this is related to her relative immaturity, lack of appropriate sexual stimulation or some other factor. Our overall assessment, however, is that Mei Xiang appears to be behaviorally normal for a developing female giant panda. Whereas Tian Tian is highly spermic and exhibits strong libido, his abrupt cessation of sexual interest during the peri-ovulatory interval is a concern. Modifications in the timing of when animals are paired for breeding may help to overcome this problem in the future.
Intravaginal Artificial Insemination: After unsuccessful mating attempts, Tian Tian’s libido ceased, but Mei Xiang continued to exhibit solicitous behaviors. Therefore, Mei Xiang was vaginally inseminated late afternoon (1630 h) on May 2nd using Tian Tian’s cryopreserved semen (collected May, 2003). The vaginal insemination technique was chosen because, unlike intrauterine artificial insemination which requires that the female be anesthetized, Mei Xiang could be inseminated while she was fully conscious and well positioned within her training cage. To achieve high numbers of sperm needed to mimic a natural breeding, the majority of Tian Tian’s cryopreserved sperm was thawed (24 straws, ~1 billion motile sperm) for vaginal insemination. Post-thaw evaluations revealed excellent sperm motility (70%), forward progression (3.5; on a 0-5 scale with a 5 rating being best) and intact acrosomes (77%). Sperm was thawed, washed, processed and concentrated to 2 ml for vaginal deposition. Mei Xiang was positioned within the training cage in a sitting position, the insemination catheter (provided by the Chengdu Research Base) was placed into Mei Xiang’s vagina, and thawed sperm was transferred from a sterile syringe through the catheter into the vagina. Hormone data, vaginal cytology and reproductive behaviors suggest that artificial insemination was achieved near the time of ovulation (~19 h post-ovulation). Therefore, the outcome of this procedure should provide important information about the efficacy of vaginal insemination for achieving pregnancy in a non-anesthetized female giant panda.
Male Reproductive Examination and Semen Assessment: Following the 2004 breeding season, a reproductive examination was conducted on Tian Tian (now at 6.5 years of age) to assess sperm status and to collect semen for cryopreservation. During the examination (May 13, 2004), anesthesia was induced with ketamine hydrochloride (1,280 mg; 10 mg/kg) and maintained with intravenous ketamine (100 mg) and isoflurane administered by facemask. Testes measurements were similar (length, 9.2 cm; width, 5.6 cm; testes volume, 302.2 cm3) to measurements obtained in 2003. Semen collected by electroejaculation (2.6 or 3.2 cm diameter rectal probe) was of excellent quality and comparable to the ejaculate collected in 2003 (Table 1). Ejaculate traits in 2004 were: semen volume, 1.43 ml; sperm concentration, 1,742 x 106/ml; total sperm/ejaculate, 2,491 x 106; sperm motility, 80%; and sperm forward progression, 3.5 (out of possible 5.0).
Sperm Cryopreservation for the Genome Resource Bank (GRB): We have developed reliable methods for cryopreserving giant panda sperm for storage in our GRB (frozen repository of biological materials). Semen was diluted in TEST egg yolk cryodiluent containing 5% glycerol (cryoprotectant) at 37C. Diluted semen sample was placed in a container filled with 300 ml water, and then cooled slowly in a refrigerator to 5C over a 3-hour interval. Samples were loaded in 0.25 ml straws and frozen over liquid nitrogen vapor using the 2-step cryomethod. Thirty-four straws were cryopreserved and stored in the GRB.
Plans for the remainder of 2004: We will continue to assess urinary progesterone excretion in daily urine samples from Mei Xiang because we expect to observe a secondary rise in urinary progesterone excretion (approximately 75 - 125 days post-ovulation). Urinary progesterone serves as a valuable sentinel measure because when levels decline to baseline (40-50 days after the secondary rise) this either signals an impending birth or, in the case of a false pregnancy, indicates that the pregnancy watch is no longer necessary.
