A Message From The SSP Coordinator, Don Lindburg
Dr. Barbara Durrant, C.R.E.S.
SEMEN COLLECTION, EVALUATION AND PRESERVATION
Semen collected by electroejaculation is evaluated for motility , swimming speed, concentration, volume, and is stained for viability .Both short-term and long-term storage methods are in development. Short-term storage is useful for sperm shipment and for insemination of a female over several days. Semen is extended in a nutrient buffer and stored at 4 oC for up to one week without significant loss of viability. Long-term storage, or cryopreservation, involves careful cooling, freezing and thawing to maintain acceptable viability. Storage in liquid nitrogen (-196°C) suspends metabolic activity in sperm cells indefinitely and is used to preserve genetic material of valuable animals for infusion into the population in the future.
The Reproductive Physiology Laboratory staff has conducted semen preservation studies in San Diego, China and Mexico City, comparing multiple cool rates, freeze rates, and post-thaw treatments. Through these studies we have improved cryopreservation protocols useful for all panda researchers. Sperm stored at 4 degrees C for several days has been used for artificial insemination. Researchers from Wolong Giant Panda Breeding Center in China have visited the Laboratory for instruction in semen preservation techniques.
VAGINAL CYTOLOGY
The vagina is a target organ for the hormone estrogen. Therefore, the cells lining the vagina change in response to fluctuating estrogen levels in the bloodstream. San Diego's female panda, Bai Yun, has been trained by her keepers to allow us to collect vaginal cells on a small cotton swab. .Bai Yun is rewarded for her cooperation with apple, carrot, and yam treats. Cells are fixed to a microscope slide, stained and analyzed for size, color, abundance, and nucleus integrity .Estrus can be monitored through this non- invasive procedure, and changes in vaginal cells alert researchers about 10 days prior to impending ovulation.
Researchers in China and Mexico City have initiated vaginal cytology studies using the techniques developed in San Diego. Data from three females in Mexico City and several females in Wolong, China have been combined with data from Bai Yun to create a profile of vaginal cytology that can be used to monitor estrus in any giant panda.
This graph depicts the change in color phase of vaginal cells over time during estrus. The cells stain predominately blue prior to the first chromic shift, pink thereafter, and yellow once the second chromic shift occurs. Shortly after the second chromic shift, the female ovulates and is soon ready for natural mating or artificial insemination.
This image shows blue phase cells found in the vaginal canal. These cells are present prior to the first chromic shift that signals the onset of estrus.
This image shows pink phase cells found in the vaginal canal after the first chromic shift.
This image shows yellow phase cells found in the vaginal canal after the second chromic shift. Yellow cells are present within a few days of ovulation.
ARTIFICIAL INSEMINATION
In 1998 it was necessary to artificially inseminate Bai Yun due to lack of interest from the male, Shi Shi. Semen was collected from the male, and half of it was used immediately for artificial insemination. The remainder was stored at 4°C for 24 hours before it was used for a second insemination of Bai Yun. Unfortunately, Bai Yun did not give birth to a cub last year, but there are convincing data that she did become pregnant. The procedure was repeated in 1999 when Bai Yun was inseminated on three consecutive days with fresh and refrigerated semen.
Mexico City's male panda, Liang Liang, is related to all three of their zoo's females, therefore, he is not allowed to breed them. In an attempt to produce cubs from unrelated males, CRES reproductive physiologists imported frozen semen from males in London and Berlin. The semen was taken to Mexico City where CRES researchers performed artificial insemination of all three female pandas. Semen thawing and artificial insemination techniques were shared with Mexico City zoo staff.
