The Giant Panda has inherent breeding problems in its male population and to overcome these problems technology and assisted reproductive techniques (ART) have been required (2, 8, 9, 10, 18). The use of artificial insemination (AI) has been used for many decades and has been quite successful (8). There has been recent research on the cryopreservation of semen for later use in artificial inseminations (9, 18). Research has also been done on postmortem semen collection from the epididymis of the male (10). There are also several other biotechnologies that are being refined for use in the preservation of endangered species including the Giant Panda (2).
Artificial insemination is the process of delivering motile sperm into a receptive female prior to ovulation. It has had a long and successful use in the Giant Panda in captivity (8). The process of insemination is very similar to that used in bovine. In fact the catheters used in insemination are the commercially produced bovine catheters (8).
The semen is collected from the male by the use of electroejaculation. An electroejactulator has a cylindrical probe that has longitudinal probes sticking off of it. The probe is inserted per rectum and placed over the accessory glands of the male’s reproductive tract. A brief electrical stimulus is applied several times to induced ejaculation. In the Giant Panda a rectal probe with a diameter of 2.6-3.8 cm has been used in successful semen collections (9). The time from stimulation to collection has varied from around nine minutes to over forty minutes. The average collection time is around nine minutes and it is believed the higher length of times were due to faulty probe placement (8). Research has shown that the time interval between semen collections has no affect on the quality of the sperm. Semen collected from a male with a one year interval between collections had the same quality as a male that had an interval of only three months. This means that more collections can be done on a male without jeopardizing his sperm quality (9). To reduce stress on the animal during the procedure and also to increase the safety of the procedure the male is always anesthetized during the procedure. Various anesthetics have been used including ketamine, xylazine, isoflurane, and propofol (9).
As soon as the semen is collected via electroejaculation the female panda is sedated with similar drug cocktails and the insemination procedure is done. The semen is released in the cervical canal. The female panda to be inseminated is monitored for total urinary estrogen and pregnanediol levels throughout the year to accurately determine when AI should be performed. In addition, laparoscopic examination of the female’s ovaries is done to further pinpoint the window for insemination (8). [top of page]
The ability for Giant Panda semen to be frozen and thawed and still viable for fertilization is a key for any hopes of reviving this endangered species. If semen can be preserved for long-term storage it will enable genetic diversity to be maintained and inbreeding depression to be avoided (2, 9). The ability for semen to be appropriately extended would allow for nonsynchronous estrous females to be inseminated and fewer collections done on the male. Even though anesthetics have become quite safe over the years, there is always a risk when sedating an animal. In addition, it would allow for semen to be exchanged between breeding facilities, which would aid in the maintenance of genetic diversity (9).
Research has shown that the sperm of the Giant Panda is quite resilient and resistant to cold shock. This has allowed several protocols to be developed for the freezing of semen (see below).
It has been found that when frozen semen is thawed that motility and viability have not been significantly reduced (there is some reduction) (9). The morphology and acrosomal integrity has also been shown to not have been compromised during the freezing and thawing procedures (10).
Even though the sperm appear to be unaffected from cryopreservation it was unknown until recently whether the sperm maintained their ability to fertilize an oocyte. A recent study found that Giant Panda sperm can undergo capacitation in vitro with or without the use of capacitation accelerators. It was also found that zonae emulsions from bears and cats elicited an acrosomal response from the sperm. The results of this study show that frozen and thawed sperm retain their capacitation ability and also maintain the ability to undergo the acrosome reaction (acrosomal integ).
Even though fresh semen or liquid stored semen is the preferred choice for artificial insemination when it’s available, cryopreserved semen is still a viable option for insemination. It allows for breeding facilities around the world to use the semen to maintain genetic diversity. It will also allow wild pandas to be collected in the field and then brought back to breeding facilities (2, 9). [top of page]
When an animal dies the cells of the animal do not instantly become necrotic. Until cells exhaust their metabolic provisions they are viable. This is true for sperm found in the postmortem animal. Mature sperm collect in the cauda epididymis of an animal and if this sperm could be extracted it could be used for artificial insemination. Such a procedure was done on Chu-Lin four hours postmortem. The male died of natural causes and his testicles were removed postmortem. The epididymis of both testicles was dissected to collect any sperm inside. One testicle had moderate hypoplasia and there were only a few non-motile sperm found inside its epididymis. The other testicle was healthy and normal and motile sperm was collected from its cauda epididymis (10).
The sperm was extended, cooled and some was frozen. A comparison between the fresh, cooled and frozen-thawed was done and it was found that the fresh sperm samples were very similar in characteristics to those found in electroejaculated samples. The frozen-thawed sperm had lower motility than the electroejaculated sample. In addition to motility, several other in vitro assays were performed on the different sperm samples. It was found that the morphology, acrosomal integrity, and sperm viability (using a live-dead stain) of the sperm collected from the cauda epididymis were all normal indicating that postmortem extraction can be done and the sperm collected can be cryopreserved for future AI (10). [top of page]
In addition to artificial insemination there has been some development of other technologies to assist in reproduction. There has been interest in using a process of transferring somatic nuclei into enucleated recipient oocytes. This would allow development of endangered embryos in more common and accessible animals. Giant Panda nuclei have been transferred into enucleated rabbit oocytes and blastocysts have been recovered after this nuclear transfer showing promise that this technology might give another way to restore the panda population. There are several other technologies being considered for use in endangered animals other than just in the Giant Panda. Technologies such as cloning, germline preservation/mouse transplantation, and gonad rescuing techniques are all in infancy stages and may be used in the future to restore populations of endangered species (2). [top of page]