In Mexico City, Chapultepec Zoo is working together with the Zoological Society of San Diego and Ueno Zoo in Japan on the improvement of assisted techniques for the reproduction of the Giant Panda (Ailuropoda melanoleuca) a highly endangered species native to the People's Republic of China.
The Giant Panda is a seasonally monoestrus species in which the estrus lasts from 9 to 11 days, spontaneously ovulating and being sexually receptive for only 2 or 3 days. In Mexico the Giant Panda females have presented an estrus peak within mid February until mid April. Therefore, accurate monitoring of the estrus cycle to pinpoint the time of ovulation is critical for the timed mating or artificial insemination. Monitoring estrogen (E1G) and progesterone (PdG) in a non-invasive way indicates hormonal changes that are continuously evaluated and corroborated with modifications on the vaginal cytology and behavior, as well as to confirm or discard pregnancy and to predict birth time.
Hormone metabolites levels are analyzed through urine samples. These are collected daily from the enclosures or corridors; when females are reaching the peak it is necessary to collect and process 2 or 3 samples per day. Samples contaminated with feces or diluted with water are discarded. Through the Jaffe method creatinine concentration is obtained in order to discard or analyze samples. The hormone profile technique is based on an ELISA test, adapted for Giant Pandas. Results are analyzed indexed on creatinine concentration of each sample and hormones levels (E1G and PdG) are expressed in ng/mg of creatinine. Hormonal and cytology monitoring techniques have been used since 1999 in Chapultepec Zoo's Giant Panda females. Results have shown that once the estrogen basal levels start to increase (>10 ng/mg Cr), it takes the female around 10 days (+/-2 days) to reach the peak, considering day 0 (probably the ovulating day) when the estrogen peak takes place (except in 2003, when a female reached the peak after 16 days). Once the maximum estrogen level is reached, a sudden decrease can be observed, reaching <20ng/mg Cr in 24 hr and basal levels from day +2.
Just like in the domestic dog, vaginal smears represent a quick, simple method to monitor estrus in females, because changes in the vaginal epithelium are related to estrogen levels. In the Giant Pandas, samples are taken using operant conditioning through positive reinforcement, since the beginning of February if each female's temperament allows it. As they approach their estrus peak, they are more willing to cooperate, therefore collecting samples becomes easier. Swabs are stained using the Papanicolaou technique (PAP, modified by Durrant et al 2000), because with this trichromic stain cellular morphology can be observed and evaluated, as well as cell chromatic shifts that can be appreciated regularly in this species. First, a chromatic shift from basophilic (blue) to acidophilic (pink) can be observed 8 days (+/-2) before ovulation (day -8), which is usually related to a gradual increase of intermediate cells and a second chromatic shift takes place 2 days before ovulation, with an increase of orange superficial cells (keratinized cells) that replace acidophilic cells. Considering maximum values can vary from one individual to another, chromatic variations have been considered as important as morphology in Giant Pandas; these are used as another parameter to determine the time of ovulation.
In Giant Pandas, like in other mammal species, external changes in the genital region can be observed during the reproductive season. As females approaches the peak, there are vulvar changes, mainly in its size, color and urgency. A 0-3 scale is used, using 0 in anestrus. A gradual increase of vulvar tumescence can be observed between days-11 and -9 (before ovulation). These figures will reach their peak on the ovulating day, returning to basal figures on the next days. In males, there is an increase in testicular volume that can be observed only under chemical restraint, when testicles can be measured.
Considering behavior related to reproductive activity, both males and females are monitored, when they are together or on their own. The presence and frequency of typical behaviors of the species are recorded: "anus-genital marking" (rubbing the genital region against any surface), increased activities (locomotion, playing, etc.), more frequent and intense vocalizations (bleats, barks, shrieks, moans, etc.), increased response to other individuals and/or increased receptivity, decrease of appetite, masturbation, etc. During the estrus peak, the female calls using the "sexual chirp", showing lordosis and presenting its tail up. If the couple is compatible, the female presents her vulva and might even move backwards towards the male. These behaviors show an increase in frequency directly related to the estrogen levels.
Monitoring will be performed during all the reproductive season. The first stage of mating will be an indirect one, placing a physical barrier between the animals. However, the animals are close enough to see, smell and listen to each other. This procedure will take place every day, exchanging the female and the male enclosures and therefore stimulating them as much as possible. Individual responses will be evaluated. If there is a mutual interest, that is, a favorable response in both, the next step will be introducing both in the same enclosure. If the female is not ready to accept the male, she will reject him by barking and growling. If the couple is compatible, when the female is ready she will gradually accept the male. If aggressive interactions are observed, the animals will be separated, though some interaction is acceptable as it normally occurs in the wild.
If introduction or natural breeding were not possible, electroejaculation and artificial insemination are performed. Fresh semen will always be the first choice, as viability percentages are much better (live-death percentages, quality and duration of sperm motility) than in frozen semen. Thus it is very important to accurately determine the moment of A.I. and the relation between the evaluated data, taking the hormone profile as the main criterion. Usually two inseminations will take place during days +2 and +3, with 24 hours in between procedures, with a minimum dose of 50 x106 motile sperms. Based on preliminary studies of the LH serum levels of the female Giant Pandas housed at Chapultepec Zoo, it was decided to perform the first insemination on day +2, considering that day +1 is too soon considering sperm viability when frozen semen is used.
Therefore, to achieve reproduction in captivity, it is important to consider the correspondence of hormonal profiles, estrus signs and vaginal cytology and variations of these elements between females. In the male, it is necessary to evaluate semen quality and viability, as well as its preservation and individual differences. Also, the individual reproductive ability has to be taken into consideration, as well as dominance and aggressiveness, reproductive experience and compatibility between pairs.
Once all the reproductive management and procedures have been performed, pregnancy monitoring starts, based mainly on hormonal profiles (PdG metabolites in urine), as well as behavior and psychological changes. Hormonal monitoring will be performed two or three times a week, collecting daily samples to determine variations. Based on hormonal profiles it is not possible to distinguish accurately between pregnancy and pseudo pregnancy, as they can be very similar. However, it can be a very useful tool to establish a possible birth date, in order to be prepared for it. Behavioral monitoring begins 30 days before the first probable day of birth. Changes can be detected as soon as they begin. Behaviors that are modified during this period are: increased or decreased appetite, nest building, vulvar and nipple licking, voluntary isolation. Amongst the physical changes that can be observed are increased mammary gland volume and swelling (70 to 30 days before birth), vulvar swelling (30 to 14 days prior to birth), vulvar dilation (3 to 1 days before birth), vulvar licking (14 to 0 days prior birth), uterine contractions (noticed through the abdominal wall 10 hours before birth). In this species, pregnancy length ranges from 84 to 184 days due to delayed implantation.
The experience and knowledge of different reproductive aspects of the Giant Panda obtained through collaborative research between zoological institutions could play an important role in its conservation, contributing to the maintenance of a greater number of animals in captivity, that could eventually be released to the wild and/or be a valuable pool of genetic material of this endangered species.
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