2007 Pregnancy Watch Ends Without Cub
Over the past week, Mei Xiang’s progestin levels have declined. Generally, when such a decline occurs with pregnant giant pandas, they give birth. As the progestin levels have been at lower levels for nearly a week, a birth is extremely unlikely. This decline, combined with the absence of a fetus on Mei's weekly ultrasounds, has led us to the conclusion that Mei experienced a pseudopregnancy this year.
The hormonal patterns of non-pregnant and pregnant females following estrus is the same, therefore we are unable to diagnose pregnancy in the giant panda. These hormonal patterns occur during a reproductive period known as the luteal phase, and can be broken down into two stages.
The first stage is characterized by progestin levels rising to about two to three times the normal progestin levels and occurs immediately after estrus. The second stage has a duration of 40 to 50 days, with concentrations at least 20 to 30 times greater than normal, and is the same among pregnant and non-pregnant females. The singular difference is that pregnant females give birth at the end of this stage.
Since we are at the end of the second stage and Mei has not given birth, we now know she was not pregnant. Although this is disappointing, each reproductive cycle of a giant panda is a learning opportunity.
Mei Xiang's progestins continue to be elevated. Stay tuned for more hormone analyses.
Mei Xiang's progestins are still elevated during the secondary phase of her post-ovulatory cycle. It looks as though we are still on track to see her progestins drop in early to mid-July. We will continue hormone analyses and let you know what we see.
Mei Xiang's progestin concentrations are definitely higher. Although in many other species high progestin levels are a good indication of pregnancy, this is not the case for most bears, and that includes the giant panda. The giant panda will produce the same high progestin levels regardless of pregnancy. The first increase occurs immediately after ovulation, at which time the levels of those hormones are about two to three times higher than baseline concentrations (see the chart). The first rise will eventually transition into the secondary rise after 50 to 130 days, where progestin levels are about 20 to 30 times that of baseline. The secondary rise, which we first described in the previous update, lasts about 40 to 50 days. Therefore by noting when the secondary rise begins, we project ahead and find a ten-day window of when it will end. For females that are pregnant, the secondary rise will end with the birth of a cub or cubs. In non-pregnant females, the secondary rise ends with no significant event.
For Mei Xiang, we detected that the secondary rise in progestins began in late May, therefore it is likely to end some time around mid-July. Although we will not know for a while whether Mei is pregnant or not, we do have an estimate of when it is all likely to end. We (and many of her admirers) just hope it ends in a birth(s). We will continue to monitor her hormone levels to track the progress of the secondary rise.
Progestin levels have increased substantially over the past week. In the sample we collected on May 29, the progestin was more than 300 ng/mg Cr. While this could be an meaningless spike in progestin excretion (as we’ve seen in the past—see previous years' pregnancy watches), it could signal the beginning of the secondary rise in progestins that marks the onset of fetal development. Progestins during the secondary rise remain elevated for 40 to 50 days, which suggests that either a pregnancy or pseudopregnancy could be concluded as soon as mid-July. We are confident that we’ll be able to fine tune this estimate with more data gathered in the coming days.
It has been about 50 days since Mei Xiang was inseminated. The duration of pregnancy in giant pandas is highly variable. Females have given birth as early as 80 days and as much as ten months after mating. Therefore, it is very difficult to determine when Mei Xiang will give birth if she is pregnant. Part of the variation in the length of pregnancy is attributed to a reproductive phenomenon known as delayed implantation.
Delayed implantation is common among some families in the order Carnivora, including mustelids (such as ermines and sea otters) and ursids (such as polar bears and giant pandas). Following mating, the female’s egg becomes fertilized and undergoes some cell division to become an embryo. However, at a certain stage, the cell division is slowed greatly or stops altogether. Essentially the embryo and reproductive system are paused. At some point down the line the "play" button is pushed, and everything picks up right where it left off.
We do not yet know what the trigger(s) is (are) that push the play button. It is likely something environmental (extrinsic), but could also very well be something that happens in the body (intrinsic). The National Zoo research program is evaluating environmental factors to determine what extrinsic factors may play a role in affecting the length of pregnancy. However, with each new reproductive cycle, we add to our body of knowledge about the giant panda.
There were few changes in estrogen and progestin levels over the past two weeks. The duration of the first phase of progestin activity following estrus, called the primary rise, is 50 to 180 days. Hormone concentrations will bounce around quite a bit during this phase. Coupled with this phase’s variable length, this makes it difficult to identify the onset of the second phase, which ends 40 to 50 days after the end of the primary rise. As a result, prospective analysis of the trend in progestin concentration changes will be touch and go for the next couple months.
Throughout this time, we will compare current profiles with historical profiles from Mei Xiang to provide the best interpretations available. However, combining these data with behavioral observations will provide the best clue to when the luteal cycle is likely to conclude—with a birth, we hope.
The reproductive cycle of almost all mammalian females can be divided into two phases: the follicular phase, during which the ovary produces and ovulates eggs to be fertilized; and the luteal phase, which begins after ovulation and, among other effects, serves as a preparatory phase for pregnancy.
In most mammals, once the reproductive system has recognized that there is not a fertilized egg and that the female is not pregnant, the luteal phase ends. In human females, menstruation signals the end of the luteal phase. On the other hand, if a female is pregnant, then the luteal phase continues until the female gives birth. Thus, the luteal cycle is usually short when a female is not pregnant and long when she is. The luteal phase is named after the corpus luteum, the body on the ovary that forms following ovulation and produces most of the progesterone.
Just as we use changes in estrogen levels to track a female’s follicular phase, we measure progesterone levels to follow the luteal phase in most mammals. However, in bears, the luteal cycle lasts the same amount of time whether the female is pregnant or not. As a result, it is very difficult to use hormone analysis to determine whether a bear is pregnant. The giant panda is a bear and, like other bears, it has a luteal cycle that is the same whether or not she is pregnant. Also, as in other bear species, the progesterone profile of the luteal cycle has two phases: a primary rise phase and a secondary rise phase. Following estrus the female begins the primary rise of her luteal cycle.
The latest hormone data from Mei Xiang indicate that she has entered primary rise phase of the luteal cycle. The length of this phase in the giant panda is highly variable, lasting anywhere between 80 and 160 days. The secondary rise is characterized by very high progesterone concentrations. Although the length of the primary rise is variable, the secondary rise usually lasts only 40 to 50 days. This smaller time frame is helpful to researchers as it provides a window of when the female might give birth.
If a female is pregnant, she will give birth at the end of the secondary rise, however if she is not then nothing will happen. In both cases, progesterone levels fall rapidly at the end of the luteal phase. So although we cannot use hormone analysis to diagnose pregnancy in the giant panda, we can use the progesterone profile to determine when the secondary rise ends and hope that it ends in a birth.
Because we were confident that Mei had ovulated, Zoo scientists and veterinarians have performed two artificial inseminations on Mei Xiang. The first procedure took place yesterday early evening, and the second this morning. We will continue to monitor her hormones over the next several months.
The hormonal change we have been waiting for—indicating that Mei has entered estrus—was measured in two samples, one collected yesterday afternoon and one collected this morning. We measured decreasing concentrations of estrogens and increasing levels of progestins in these consecutive samples.
Although these data are a good indication that ovulation has likely occurred, our conclusionn was bolstered by other measures, including behavioral observations and vaginal cytologies. On their own, these separate measures are useful in timed breeding. When we consider all three together and the data are aligned, we can identify the time of ovulation with greater certainty.
This process is a collaborative effort among the scientists, keepers, and veterinary staff at the Zoo to ensure that we maximize the reproductive potential of Mei Xiang.
We will continue to monitor Mei's reproductive hormones to track changes in her luteal cycle, which is the next reproductive phase. With pandas, this phase is rather complex, but hormonal assessment is still useful. In future updates over the next few months we will explain this phenomenon.
We were able to get a good sample this morning. The estrogen concentration from this morning's sample (about 118 ng/mg Cr.) is up from yeserday afternoon's estrogen concentrations. There was also a slight increase in progestins, but not as much as we would expect to see following ovulation. So we are still in a holding pattern.
This morning we saw some exciting behaviors that indicated Mei Xiang may be in in estrus but analysis of her hormones tells us she has yet to ovulate. An additional sample was collected yesterday afternoon and when averaged with another sample collected the same day, the estrogen concentration for April 1 was about 92 ng/mg Cr. The one sample that has been analyzed so far today had a value of almost 179 ng/mg Cr.
Often, when monitoring giant pandas' estrus, substantial increases in estrogen concentrations provide further evidence that we are nearing ovulation. Although these data indicate that we are near, we still have a ways to go.
We hope you'll stay tuned—hormonal changes can occur very rapidly.
Mei Xiang’s vaginal cytology continues to change. The superficial cells were at their highest point today. The vaginal cell color is changing from predominately orange to pink. As of today, 44 percent of the cells are orange, and 77 percent are superficial cells. As estrogen continues to increase near peak estrus, we expect to see more than 80 percent superficial cells.
Mei Xiang’s reproductive hormone levels didn’t change much over the past few days. The average estrogen value from March 31 was about 74 ng/mg Cr., and the one sample today had a value of about 76 ng/mg Cr. As noted in yesterday's update, it is not uncommon for a sort of hormonal lull to occur during the periestrous period. However, hormone levels could change at anytime and we are ready to monitor that change.
Up to now, all hormone analyses have been performed at the Zoo’s endocrine lab at the Conservation and Research Center. Panda staff delivered samples from the Zoo to CRC each day. Now, to provide faster turnaround, on Monday morning we will set up a temporary endocrine lab at the Zoo.
New technologies enable lab staff to transport and use our endocrine equipment just about anywhere. Staff successfully set up hormone labs at the Chengdu Research Base in China and at the Chiang Mai Zoo in Thailand to help both facilities monitor giant panda hormones closely during the breeding season to optimize the reproductive potential of their females.
When the Zoo started its giant panda hormone project, one of its goals was to enable other facilities to benefit from hormone analysis and improve giant panda breeding. Working with our colleagues in China and Thailand, this goal is being achieved.
We are also looking at Mei Xiang's vaginal cells, using the Papanicolaou (PAP) stain. Her cytology is changing as the urinary estrogen increases. The vaginal cell color is changing from blue to pink to orange, and cell type is changing from intermediate cells to superficial cells. As of April 1, 68 percent of the cells are orange, and 58 percent are superficial cells. As estrogen continues to increase near peak estrus, we expect to see more than 80 percent orange superficial cells.
You can see images of these cells directly below, and changes in the percentage of cell type and color further below. You'll see changes occurred on March 27, when we collected the first sample in which estrogens were above baseline.
|Basal cell||Intermediate cell||Superficial cell|
After hormone analyses were conducted yesterday, we collected additional urine samples. For better analysis, we averaged all the hormone values from these samples into a single point on the graph shown below.
We collected another sample this morning, and the estrogen concentration was only slightly higher than yesterday's average estrogen concentrations. Slight changes in estrogen concentrations during the giant panda's periestrous period are not unusual. Within the first few days of the periestrus, estrogen concentrations increase almost four- to fivefold. Often there are a few days during the periestrous period when estrogen concentrations vary little. On other days, estrogens vary greatly. We will continue to analyze Mei’s urinary hormones to assess her fertility.
After analyzing two samples, one from overnight and one from this morning, we see that the estrogen concentrations from those samples are indeed higher than they were yesterday and seem to be on track with estrogen trends from the previous years.
The timing of the onset of the Mei's periestrous period this year is also comparable to that of previous years. In 2002 her estrogens peaked on April 26, in 2003 they peaked on April 3, in 2004 they peaked on May 10, and in 2005, they peaked on May 9.
Reports from some of China's panda breeding centers indicate that some females there have already ovulated this year. The onset of the periestrous period varies from year to year and from female to female, but it almost always occurs between February and July.
What triggers the onset of the panda's periestrus is not well known. At the large breeding facilities in China that house many females, estrus can occur any time during the breeding season. These females are exposed to the same environmental conditions yet the timing of estrus varies, suggesting that factors other than day length and temperature may play a role. The Zoo’s research in collaboration with our Chinese colleagues may provide insight into what these factors might be.
Mei Xiang has entered the periestrous interval, which is characterized by the growth and maturation of follicles in the ovary. The interval ends with ovulation—the expulsion of an egg or eggs from a follicle or follicles into the female reproductive tract. As soon as a female ovulates, she is fertile and ready to breed. During the periestrous interval, estrogens rise as a result of the growth of the bodies that produce them, which are the follicles.
To determine that follicular growth has begun and a giant panda will soon be fertile, we look for a clues in the pattern of hormonal changes.
The first clue is a rise in estrogens above what is known as baseline levels. While this may seem obvious, estrogens may sometimes rise above baseline levels even if the female has not begun follicular growth. These periods of elevated estrogens are often short-lived, and may only occur in one sample. So, to help tell whether an increase in estrogen in a sample indicates follicular growth, we look at two other factors. First, we look for a drop in progestins. Often, when follicular growth begins in a giant panda, there is a drop in progestin levels in the same sample that shows an increase in estrogens.
Second, we look for a continued trend, waiting to see if the next sample also shows elevated estrogen levels. This year, the first sample in which estrogens were above baseline was collected on March 27. As you can see in the chart below, the next two samples, collected on March 28 and 29, indicated such a trend, as estrogens were higher each day. Based on this, we believe Mei Xiang’s periestrous interval has begun
Mei's periestrous interval in 2002 lasted ten days, in 2003 it lasted eight days, in 2004 it was 11 days long, and in 2005 it took ten days from estrogen baseline to peak concentrations.
Considering the data on Mei's past periestrous intervals and the current hormone data, we expect this periestrous interval to end some time in the middle to end of next week. Hormone levels are a direct measure of ovarian activity, so they are the most accurate and consistent means of pinpointing the time of ovulation. We will be watching for a peak in estrogen levels, followed by a steep fall, to determine that she has ovulated. Knowing this is critical to timing an artificial insemination.
We will be posting more frequent hormone updates over the next week to keep you informed about Mei's reproductive state.
There is still a lack of activity in Mei Xiang's reproductive hormones. However, recent analyses of urine samples collected from Tian Tian for testosterone concentrations indicate that he is reproductively ready. Testosterone is produced by the testes in the male during spermatogenesis (the process of making sperm). Therefore, by measuring testosterone in the urine, we are able to monitor testes function and determine if the male is producing sperm. Testosterone production in the male giant panda follows a seasonal trend—testosterone levels are high for only part of the year, just prior to the breeding season.
Since females have only one estrus during the breeding season and that estrus can occur anytime in a four-month period, males need to be ready to mate even before the breeding season begins just in case a female's estrus occurs early. Additionally, once the testosterone is produced by the testes, it travels through the blood stream and causes behavioral and physiological changes that prepare the male for breeding. One of these changes is an increase in muscle mass, which may be a sign of fitness that females find attractive. So, although we often focus attention on the reproductive function of the female, male reproductive function is just as important.
The estrogen and progestin levels measured from the urine collected from Mei Xiang over the past two weeks indicate that she has yet to begin her follicular phase. The follicular phase is a reproductive term used to characterize the period from the onset of follicular growth to the time of ovulation. During this period, follicles within the ovary grow eggs. Additionally, follicles produce estrogen. As the follicles grow to support the growth of the egg so does their production of estrogen. Therefore, by measuring estrogen we are able to gauge follicular growth and egg development.
When the eggs are mature, the follicle releases the egg in the event known as ovulation, and estrogen production drops off substantially. We can estimate the time of ovulation based on when a sharp drop in estrogen levels is measured. Upon ovulation the female is fertile for as long as the egg is viable. Egg viability is variable among mammalian species, but it is believed to be as brief as 36 to 48 hours for the giant panda.
As the breeding season has begun we should expect hormonal changes to begin fairly soon (within the next two months).
Mei Xiang’s reproductive hormone levels have changed very little over the past couple of weeks. Typically the brief breeding season for giant pandas can occur at anytime between February and May. However, most estrous events occur in March and April.
In the wild, a number of factors, including the amount of time a panda is exposed to daylight and the availability of bamboo, play a role in the onset of estrus during the breeding season. In zoos and breeding centers, we are not sure about what the causal factors are that trigger estrus during the same time period. At any one of the main breeding facilities in China that house a number of females, estrus for those pandas occurs at anytime during that four-month period. Most females will ovulate in March and April, but at each facility there are females that will cycle as early as February and as late as May. Therefore, it is very difficult to say when Mei Xiang will come into estrus—if she does cycle, chances are it will happen in the next two months.
Mei Xiang's hormones remain at baseline levels. People may wonder why we are monitoring Mei's hormones the way we do. We assess Mei's hormones in her urine because the concentrations of estrogen and progestin are the best indications of when her breeding season will begin. The values derived from her urine, which we collect daily, provide a clear index of physiological changes. They also allow us to rapidly evaluate her reproductive status, particularly when used in conjunction with observations of her behavior and study of her vaginal cells during the breeding season.
Giant pandas are a monoestrous species—only one ovulation event occurs during the breeding season. This is preceded by an increase in estrogen activity that can last between ten and 14 days. Once we observe the initial increase in Mei's estrogen activity, we will begin daily analyses to help us identify the time of ovulation. We will continue to monitor Mei’s reproductive hormones and provide updates.
Mei Xiang’s reproductive hormones remain at baseline levels. Although giant pandas can come into estrus as early as January or February, it is rare. The earliest Mei Xiang came into estrus was early March in 2005 and the latest was early May in 2004. We do not expect to see changes in her reproductive hormones for a little while longer. We will continue to measure her hormones regularly so that we can monitor changes when they occur.
Mei Xiang’s reproductive hormones are at baseline levels. Last year, her reproductive hormones remained at baseline and she did not cycle. Many mammals do not come into heat when they are nursing young, a phenomenon called lactational anestrus.
Last year we documented lactational anestrus in the giant panda for the first time by measuring cortisol in the urine collected from Mei Xiang. Cortisol has been shown to be necessary to maintain lactation in other mammals, and elevated levels of cortisol can suppress pituitary gland activity. The pituitary gland is responsible for secreting the hormones that stimulate reproductive hormone production, which then drive behavioral and physiological changes associated with mating, creating a sort of domino effect. Mei Xiang’s elevated cortisol levels last year most likely prevented that first domino from falling and, therefore, she did not come into heat.
Now, we hope that once Tai Shan is weaned and she stops lactating the block on her reproductive system will be lifted. We will continue to monitor her reproductive hormones as well as her cortisol levels closely over the next few months to assess her reproductive status.