Factors affecting date of implantation, parturition, and den entry estimated from activity and body temperature in free-ranging brown bears.

Knowledge of factors influencing the timing of reproduction is important for animal conservation and management. Brown bears (Ursus arctos) are able to vary the birth date of their cubs in response to their fat stores, but little information is available about the timing of implantation and parturition in free-ranging brown bears. Body temperature and activity of pregnant brown bears is higher during the gestation period than during the rest of hibernation and drops at parturition. We compared mean daily body temperature and activity levels of pregnant and nonpregnant females during preimplantation, gestation, and lactation. Additionally we tested whether age, litter size, primiparity, environmental conditions, and the start of hibernation influence the timing of parturition. The mean date of implantation was 1 December (SD = 12), the mean date of parturition was 26 January (SD = 12), and the mean duration of the gestation period was 56 days (SD = 2). The body temperature of pregnant females was higher during the gestation and lactation periods than that of nonpregnant bears. The body temperature of pregnant females decreased during the gestation period. Activity recordings were also used to determine the date of parturition. The parturition dates calculated with activity and body temperature data did not differ significantly and were the same in 50% of the females. Older females started hibernation earlier. The start of hibernation was earlier during years with favorable environmental conditions. Dates of parturition were later during years with good environmental conditions which was unexpected. We suggest that free-ranging pregnant brown bears in areas with high levels of human activities at the beginning of the denning period, as in our study area, might prioritize investing energy in early denning than in early parturition during years with favorable environmental conditions, as a strategy to prevent disturbances caused by human.

Delayed implantation in giant pandas: the first comprehensive empirical evidence.

Successful conservation of an endangered species relies on a good understanding of its reproductive biology, but there are large knowledge gaps. For example, many questions remain unanswered with regard to gestation and fetal development in the giant panda. We take advantage of a sample size that is unprecedented for this species (n=13) to explore patterns in reproductive development across individuals at the China Conservation and Research Center for the Giant Panda. We use ultrasound techniques on multiple giant pandas for the first time to empirically confirm what has long been suspected that pandas exhibit delayed implantation of the embryo. We also show that the duration of postfetal detection period is remarkably similar across individuals (16.85+/-1.34 days). Detection of fetus by ultrasound was strongly correlated to the peak in urinary progesterone (r=0.96, t=8.48, d.f.=8, P=0.0001) and swelling in the mammary glands (r=0.79, t=3.61, d.f.=8, P=0.007) and vulva (r=0.91, t=6.40, d.f.=8, P=0.0002) of adult females. When controlling for both the duration of the total gestation period and the postfetal detection period, infant birth weight was only significantly predicted by the latter (beta=11.25, s.e.m.=4.98, t=2.26, P=0.05), suggesting that delayed implantation increases flexibility in the timing of birth but is not important in dictating infant growth. This study informs reproductive biology by exploring the little-studied phenomenon of delayed implantation in relationship to physiological changes in pregnant giant panda females.

In vitro maturation of oocytes derived from the brown bear (Ursus arctos).

This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer.

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.

In vitro maturation of follicular oocytes of the Giant Panda (Ailuropoda melanoleuca): a case report.

The Giant Panda is an endangered species that would benefit from biotechnological assistance in reproduction. However, because there are only a few of these animals left in the world, scientists hesitate to use them for research procedures. We were fortunate to obtain ovaries from a Giant Panda that died of hepatic cirrhosis during the nonbreeding season. Oocytes were harvested within 4 h of death by dissecting the ovarian cortex in physiological saline and collecting the cumulus-oocyte complexes from the fluid, and then were classified into large (> 125 microns) and small (100 to 124 microns) follicular oocytes and placed in TCM199 supplemented with FSH (10 micrograms/mL) and LH (20 micrograms/mL). After culture for 22 h at 37 degrees C in air with 5% CO2, response was evaluated by growth of oocytes and presence of the first polar body. Of the 26 large follicular oocytes that were harvested, 12 were considered suitable for IVM, and 14 were degenerated, had a broken zona pellucida or had lost some cytoplasm. Of the 12 cultured oocytes, all grew to a mean diameter of 141.1(SD = +/- 6.7, n = 12), and 4 released the first polar body. None of the small follicular oocytes showed growth or other signs of maturation. We conclude from our preliminary results that it is possible to obtain functional Giant Panda oocytes from ovaries obtained post mortem during the nonbreeding season.