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Final year project submitted to the University of Nottingham in partial fulfilment of the requirements for BSc Animal Science
The giant panda have been a world renowned endangered species since 1986. It was endangered due to poaching and habitat fragmentation. The isolation of different wild populations in the mountain ranges due to habitat fragmentation is still a major factor that endangers the giant pandas today. The giant panda has a dietary requirement that is completely different to other Ursids even though it has a simple carnivorous/omnivorous digestive system. The giant panda spends most of its day foraging and only digests about 20% of their diet. To aid ingestion, the giant panda has unique elongated radial sesamoid which acts as a false thumb. The low natural birth rates can be highly due to the reproductive system of the giant panda. The female giant panda has a reproductive system that is similar to the pig’s which allows for natural twinning to occur. Like other Ursids, the female giant panda also exhibits delayed implantation. Moreover, the females give birth to underdeveloped young cubs which are only 1/10 of the mother’s body weight. The male giant pandas have extremely small L-shaped (or S-shaped) penises. The giant panda uses vocal, visual and olfactory cues in the wild to recognise conspecifics, especially near breeding seasons. To prevent further habitat fragmentation, the Chinese government have now set up many different nature reserves on the giant panda natural habitats. To further increase the total number of giant pandas, breeding programmes were carried out in various captive environments. Due to the success in the breeding programmes, the ex-situ population can be viewed as a sustainable population and may be reintroduced back to the wild. Taking examples from large species that have been successfully reintroduced, it may take the giant pandas at least 30 years to achieve the goal of reintroduction. However, the successful reintroduction may prove that the giant pandas have a better ecological value and should be saved.
Acknowledgements Supervisor - Dr Martin Luck (University of Nottingham) Xiang Bo and Xiao Qian (Chengdu Giant Panda Research Base) Gratitude is extended towards the above mentioned people for their guidance and extensive support throughout the project.
Contents 1. Introduction............................................................................. 7
1.1. Classification of Giant Pandas................................................... 1.2. IUCN Red List........................................................................ 1.3. Giant Panda Habitats..............................................................
7 7 7
2. Digestive system......................................................................
2.1. Diet...................................................................................... 2.2. Dentition and Jaw Musculature................................................. 2.3. Digestive Tract....................................................................... 2.4. Other Physiological Features....................................................
10 11 12 13
3. Reproductive System................................................................
3.1.1. Ovary......................................................................... 3.1.2. Oviduct....................................................................... 3.1.3. Uterus......................................................................... 3.1.4. Vagina........................................................................ 3.1.5. External Genitalia......................................................... 3.1.6. Oocyte........................................................................ 3.1.7. Oestrus Behaviour........................................................
15 15 15 16 16 16 16
3.2.1. Testis.......................................................................... 3.2.2. Epididymis................................................................... 3.2.3. Vas Deferens............................................................... 3.2.4. Accessory Sexual Gland................................................. 3.2.5. Penis.......................................................................... 3.2.6. Scrotum...................................................................... 3.2.7. Sperm......................................................................... 3.2.8. Sexual Behaviour..........................................................
19 19 20 20 20 20 21 22
3.3. Reproductive Difficulties..........................................................
4. Recognition of Conspecifics......................................................
4.1. Vocal.................................................................................... 4.2. Olfactory............................................................................... 4.3. Visual................................................................................... 4.4. Spatial Memory......................................................................
25 26 27 27
5. Giant Pandas in Captivities/Reserves.......................................
5.1. Nature Reserves.....................................................................
5.2.1. Main Aims for Captive Breeding......................................
5.3. Breeding Strategies................................................................
5.3.1. Natural Mating............................................................. 5.3.2. Assisted Breeding......................................................... 188.8.131.52. 184.108.40.206. Artificial Insemination (AI)................................... Twin Swapping...................................................
31 33 33 34
5.4. Chengdu Research Base of Giant Panda Breeding (CRBGPB).........
5.4.1. Location...................................................................... 5.4.2. Climate.......................................................................
5.5. Behaviour of Giant Pandas ex-situ............................................
5.5.1. Summer Time.............................................................. 5.5.2. Winter Time................................................................. 5.5.3. Stereotypic Behaviour...................................................
36 37 38
5.6. Breeding Outcomes................................................................
5.6.1. Rising Numbers of Captive Giant Pandas..........................
5.6.2. Male to Female Ratio..................................................... 5.6.3. Genetic Variability........................................................
6. Future of Giant Pandas.............................................................
6.1. Releasing into the Wild............................................................
6.1.1. Rules for Successful Reintroduction.................................
6.2. Examples in other species.......................................................
6.2.1. Père David’s Deer......................................................... 6.2.2. Chinese Alligator.......................................................... 6.2.3. Black Rhino..................................................................
45 45 46
Appendix 1 – Cryopreservation and Thawing Methods (Spindler et al., 2006)............................................................................................. 56
This project aims to understand the physiology of giant pandas and to explore the possibility of releasing captive giant pandas into the wild.
Classification of Giant Pandas
The giant panda (Ailuropoda melanoleuca), also called “Pi xiu”, “Mo” or “ironeating mammal” in ancient Chinese literature (Zhang & Wei, 2006), was first discovered in the Western society by a French Priest and naturalist Armand David in 1869 (Nicholls, 2010).
The fossil data suggested that the giant panda may have originated in late Miocene, expanded in middle Pleistocene and contracted in late Pleistocene (Zhang et al., 2007).
There had been historical debates on whether the giant panda is more similar to the Red Panda, or Lesser Panda (Ailurus fulgens), or is more related to normal bears. With the advance in DNA technologies, it was discovered in 1972, the giant pandas are clearly more associated with bears (Nicholls 2010). Chaudhuri et al., 1988 mentioned that the giant panda is phylogenetically closer to bears than in raccoons.
The giant pandas are well known for their body size (from extremely underdeveloped during birth to around 100kg in adulthood), specialised diet (predominantly seven specific bamboo species and fruits), reproductive
constraints and morphological adaptations, which were to lead people to believe that they are at an “evolutionary dead end” (Zhang et al., 2007).
IUCN Red List
The giant pandas have been on the IUCN since 1986 with a “Rare” status, since then, the giant pandas are “Endangered” on the Red List until now
(www.iucnredlist.org). Although the giant pandas are increasing in number, largely due to captive breeding success in the Chengdu Research Base of Giant Panda Breeding, their natural habitats are still shrinking due to human activities.
Giant Panda Habitats
Giant pandas have been extinct in Hunan, Hubei, Guizhou, Chongqing and Yunnan provinces during 18th to 19th century, which historically the giant panda appeared in southern China, northern Myanmar, northern Vietnam, Laos and Thailand (Zhang et al., 2007; Zhu et al., 2010). Now the giant panda habitat have shrunk to only six mountain ranges Qinling, Minshan, Qionglai, Liangshan, Daxiangling and Xiaoxiangling, which are on the edge of the Tibetian Pleateau in China (Zhang et al., 2007; Zhu et al., 2010). Figure 1 shows the historical distribution and the 2003 distribution of giant pandas. Figure 2 shows the giant panda population distribution in 2008. Their habitat choice is mainly influenced by environmental factors such as bamboo coverage, land cover type, elevation, slope and aspect. Although the giant pandas are said to be solitary, their territories would overlap, the animals are probably aware of each other and recognise individuals, but males show no evidence of territorial behaviour (Dungl et al., 2008).
Figure 1 (Wan et al., 2003) 2003 giant panda distribution is represented by black areas, white circles represent fossile records in Early Pleistocene and black circles represent fossil recods in Mid to late Pleistocene.
Climate change, human activities and natural catastrophes are believed to accelerate habitat fragmentation, and lead the giant pandas further towards their inevitable extinction in the near future (Zhang et al., 2007). Shen et al., 2008 suggested that if habitat fragmentation continues, the Minshan population will be separated into more-isolated populations.
Several incidences may have accelerated habitat fragmentation. A major earthquake in Sichuan in 2008 may have further affected the giant pandas. With 53% of the giant panda habitat, 67% population affected and thirty two damaged enclosures in Wolong’s giant panda breeding centre. There are now eight disconnected ranges with two large giant panda populations and sixteen small isolated groups (Wang et al., 2008).
Figure 2 (Wang et al., 2008) Giant panda in 8
(labelled I to VIII), with 2 larger and 16
small, isolated groups. Habitat connections
Large rivers may act as a barrier between habitats, the results from Zhang et al., 2007 suggested that this was not the case and human population expansion was the primary reason. The extensive human population growth in the giant panda habitats have led to habitat destruction for agricultural uses and may have led to the extinction of giant panda populations at the eastern edge of the Sichuan basin (Zhang et al., 2007).
A study by Zhang et al., 2011, suggested that it is more important to protect any existing old growth forest habitats than to protect a secondary growth forest of the equivalent area. It is possible that bamboo that grows underneath old growth is more nutritious, and the trees may be large enough to form cavities suitable for maternity dens (Zhang et al., 2011).
In a study by Shen et al., 2008, it was estimated that the giant panda core habitat has a 30.4% to 44.5% decrease, after anthropogenic information was incorporated.
To restore degraded giant panda habitats, Shen et al., 2008 suggested two approaches: 1. Reforestation with pioneer species to create a canopy cover, and then recolonizing the habitat with species that cannot tolerate open planting but representing a range of life forms and successional stages; 2. Reforesting with species representatives of a more mature successional stage, completely bypassing the natural succession sequence.
It is important to establish green corridors between the subpopulations in each mountain range to connect the mosaic habitat cover (Shen et al., 2008), even though the giant pandas might not migrate between mountains due to their sensitivity to human activity, the green corridors allows gene flow and genetic variation between the larger populations and smaller populations (for example the Daxiangling and Xiaoxiangling population of less than 40 individuals) to ensure long-term survival (Wan et al., 2003).
2. Digestive System
The giant panda can be described as a “facultative carnivore”. They have a digestive system (including dentition and jaw musculature) of a carnivore, but they have a vegetarian diet that mainly consists of bamboo and fruits. Fossil data suggested that the giant panda had started its bamboo diet as early as 7 Ma and completed the dietary change in by 2 Ma, which as accompanied by the functional relaxation of the Tas1r1 gene (Zhao et al., 2010). There are a total of 60 species of bamboo that is available for consumption, the giant panda is able to consume 16 species and only 11 species are consumed regularly (CBRGPB).
They are also very selective about the specific plant portions they consume (Diernfeld et al., 1982), although there was no evidence that the selectiveness is due to increased energy assimilation (Finley et al., 2011). The giant pandas
have preferred to move onto gentle slopes and consume old shoots as their primary food source in the winter (Zhang et al., 2009).
Despite the low digestible energy values in the normal giant panda diet, the high levels of intake (up to 6% of body weight in dry matter and spending 50% of the day foraging (Zhang et al., 2009)) accompanied by a fast rate of passage (passage complete in 8±3 hours), the giant panda meets its nutritional needs by utilising cellular contents rather than through the digestion of plant structural carbohydrates (Diernfeld et al., 1982). The giant panda compromises its high energy needs, despite the low protein, high cellulose diet, with maximised ingestion rates and avoiding movement to another forage patch to minimise energy expenditure (Zhang et al., 2009).
Although the wild giant pandas survive on a diet consisting almost entirely of bamboo (Diernfeld et al., 1982), it has been reported that wild giant pandas may consume dead animal material (www.bbc.co.uk, 2011). Diernfeld et al., 1982 also observed that their male subjects consistently consumed his gruel and feline diet.
By relying on bamboo as their primary diet, the bamboo die off during 1975 to 1985 has caused deaths of several hundred giant pandas from six mountain ranges (Wan et al.,2003).
Dentition and Jaw Musculature
Figure 3 A model of giant panda skull in CRBGPB
Figure 3 above shows a modelled giant panda skull in CRBGPB.
A comparative study by P Christiansen, 2008 (Figure 4) found that the giant panda had the smallest canines and the shortest canines relative to skull length,
while the diameter of the crown is the largest of any ursid compared. The author suggested that the short canines were not rounder than other species but were much sturdier, and only the giant panda has derived canines that are morphologically different from other species. The results of the study has demonstrated that the giant panda’s canine adaptations are probable derived from a shift from an omnivorous diet to a herbivorous diet (P Christiansen, 2008).
Figure 4 (P Christiansen, 2008) Comparative morphology of the upper canines of ursids viewed in direct lateral and anterior perspective. A: Giant panda, B: Spectacled bear, C: American black bear, D: Brown bear, E: Malaysian sun bear, F: Polar bear, G: Asiatic bear and H: Sloth bear.
A study by pastor et al., 2011 on the giant panda’s tongue suggested that it resembles animals with a herbivorous diet.
The giant panda’s skull is composed of dense compact bone with enlarged musculature (Diernfeld et al., 1982), although this does not produce a stronger bite force than other ursid species (P Christiansen, 2008). The dentition resembles a typical herbivore which is characterised by large, flat cheek teeth with elaborate crown patterns (Diernfeld et al., 1982).
The giant panda possess a digestive tract that is similar to the dog, cat and racoon, which consist of a simple stomach with a muscular pyloric region for mixing digesta before it enters the small intestine (Finley et al., 2011), a short straight colon about 10cm in length and no caecum (Diernfeld et al., 1982) (Figure 5).
Figure 5 (Zhang & Wei, 2006) A drawing of the giant panda digestive tract.
To adapt to the bamboo diet, the oesophagus has a horny lining to provide protection against shards of bamboo (Finley et al., 2011). The simplicity of the digestive tract along with high musous levels (Zhu et al., 2010) of the giant panda means that they have an extremely rapid transit time, and therefore limited capacity for microbial digestion (Diernfeld et al., 1982). Diernfeld et al., 1982 suggested that “hemicellulose was the only structural digester to any significant extent”. It is true that giant panda faeces is one of the better smelling faeces, as 80% of their diet is not digested (CRBGPB, personal communication).
Previous studies have found that the presominant bacterial species in the giant panda faeces was Escherichia coli (E. coli), other faecal bacteria include Streptococcus (including Enterococcus), Enterobacteriaceae and Clostridia (Wei et al., 2007). The proximity of the individualsm and diet may influence the type of bacteria detected (Wei et al., 2007). Although E.coli may also be detected in other herbivorous mammals, the E. coli detected in the giant panda did not appear to favour the utilisation of a fibre based diet, which made the giant pandas highly vulnerable to perturbations (Wei et al., 2007).
Other Physiological Features
The giant panda has elongated radial sesamoid (wrist bone of forepaw) with muscles attached to the “false thumb” (Figure 6), which give the bone mobility and allows the giant panda to have a “forceps-like grasp” (Diernfeld et al., 1982).
model in CRBGPB, showing the radial sesamoid elongation.
As described by Diernfeld et al., 1982 and also observed in the CRBGPB in August, 2011, the giant panda holds its food with the forepaws. By inserting the bamboo clums sideways into the mouth, the leaves are easily stripped and eaten with just a twist of the head. The larger clums are cracked with the molars and peeled with front teeth to expose the internal pith. In the case of bamboo shoots, the outer casing is easily removed by using the same method.
The large surface area of the skull that allows the attachment of temporal muscles to maximise biting force during mastication (Jin et al., 2007) may also assist the giant pandas in peeling and chewing bamboo.
Most of the information on reproduction of giant pandas are found using subjects from the captive population.
The female reproductive organ consists of a pair of ovaries, oviducts, uterus, vagina and external vagina. Figure 7 shows the anatomy of the female giant panda reproductive organ.
Figure 7 (Zhang & Wei, 2006) The female giant organ Uterine panda (dorsal tube; (2)
reproductive view). (1)
Ovary; (3) Fimbria tuabe; (4) Mesovarium; (5) Uterine broad ligament; (6) Corpus uteri; (7) Uterine vaginal cervical orifice of canal; cervix; (8) (9)
Hymen; (10) External urethral orifice; (11) Bulbus vestibule; (12) Vestibulum vaginae; (13) Labia vulva; (14) Clitoris; (15) Ostium gl. Vestivulares minores; (16) Vagina; (17) vaginal portion of cervix; (18) Uterine horn.
The size of the ovaries differ with age and body size, with an average ovary size of 30-32mm in length, 20-25mm in width, 15-17mm in thickness and 4-5g in weight. According to previous studies, it was found that there are ridges present in the ovaries, and they disappear with age (Zhang & Wei, 2006).
The oviduct is the site of fertilisation, and the ovarian capsule connects the oviduct with the ovaries. The ovarian capsule is usually 40mm×30mm (Zhang & Wei, 2006).
The giant panda uterus consists of a pair of uterine horns, a uterine body and a cervix. The uterus structure is very similar to that of a pig uterus with a pair of uterine horns leading to a cylindrical uterus. During oestrus, the cervix relaxes and becomes a petal shape structure (Zhang & Wei, 2006).
The vagina is an ovular structure that is in general 60-95mm long (Zhang & Wei, 2006). A hymen is usually present in the vagina and may serve as a pseudocervix.
3.1.5. External Genitalia
The external genitalia consist of the vestibule bulb, vulva and clitoris(Zhang & Wei, 2006).
The follicles in the ovaries develop from primordial follicles, primary follicles, secondary follicles, tertiary follicles to mature follicles. A corpus luteum of a size of 125mm×90mm×65mm would form if an oocyte was not released by the end of the follicular maturation phase. An average oocyte is 141.1 um in diameter (Figure 8), which is similar to that of a cow oocyte. In a normal ovulatory phase, 1-3 oocytes would be released. However, it was found that some follicles would become a corpus luteum before a mature oocyte was even relased. This may contribute to the reproductive difficulties observed in giant pandas (Zhang & Wei, 2006).
Figure 8 (Zhang & Wei, 2006) Giant panda ovum. Left: naked ovum. Right: intact ovum.
3.1.7. Oestrus Behaviour
The giant pandas are seasonally monoestrus (Chaudhuri et al., 1988) usually with a short period of oestrus during the springtime between March and May
(Bonney et al., 1982), with a receptive period of 2-3 days (Platz et al., 1983), devoting less than 1% of their annual lifespan on sexual activity (Aiken-Palmer et al., 2012). A female giant panda in the wild typically give birth every 2-3 years (Snyder et al., 2003). By removing the cubs before 6 months of age in captivity, they have the potential to breed every year. The oestrus periods of the giant pandas can be easily related to urinary hormone metabolite levels and sexual behvaiours.
There are extensive records on giant panda oestrus behaviour. Female oestrus behaviour can usually be observed for 10-20 days, and the length of the oestrus period is dependent on age and health, a younger, healthier female would have a longer oestrus period. The whole oestrus period can be divided into three periods: pro-oestrus, oestrus and post-oestrus.
Bonney et al., 1982 observed a rise in oestrogen levels from 10 to 68 ng/mg creatinine during the pro-oestrus phase, and a dramatic decline to basal concentrations when the female was in oestrus. This late spike in oestrogen metabolite probably lead to ovulation as the overproduction of oestrogen beyond the threshold stimulatory levels may be required (Montfort et al., 1989).
Other than oestrogen, follicular stimulating hormone (FSH) may also be sampled. Monfort et al., 1989 sampled a female giant panda during the periovulatory and pregnancy interval. The results from the study showed that early morning urinary bioactive FSH levels may reflect serum FSH
concentrations, which may serve as the index for estimating FSH secretion (Monfort et al., 1989). The researchers suggested that monoestrus species (giant pandas in this case), may not require a period of sustained FSH release or multiple FSH peaks for the maturation of follicles after prolonged ovarian inactivity (Monfort et al., 1989).
The female giant pandas have been observed to produce pro-oestrus behaviours such as scent-marking, bleating and increased activity (Bonney et al., 1982). A female giant panda would produce a high-pitched tonal chirp with higher jitter and harshness to solicit male attention when it is in oestrus, and increase in frequency as ovulation approaches (Charlton et al., 2010). These pro-oestrus behaviours are considered to be directed towards successful male contact establishment and successful sexual encounters (Bonney et al., 1982).
During oestrus, the female would bleat at a frequency of 0.18-5.4kHz. The female bleats are usually accompanied by male bleating to signal oestrus. The female would have a swollen vulva and would masturbate on most vertical structures. The behaviours is usually accompanied by a lordosis posture and with the female becoming more docile and receptive towards other giant pandas regardless of their gender (Zhang & Wei, 2006).
During the post-oestrus phase, the female decreases its bleating frequency and resumes food consumption. Although the female may still exhibit lordosis, the female is not receptive for copulation.
The female oestrus behaviour is summarised in the table 1 below.
Table 1 Summary of female giant panda oestrous behaviour, with the first 4 days indicating no signs of oestrous.
The male reproductive organ consists of a pair of testes, epididymis, vas deferens, accessory sexual gland, penis and scrotum. Figure 9 shows the ventral view of the male reproductive organ.
Figure 9 (Zhang & Wei, 2006) The male giant panda reproductive
organ (ventral view). (1) A.V. et. N. spermatica; (2) Anulus
vaginalis; (3) Urinary bladder; (4) Ampulla Ductus prostate; ductus deferentis; (6) (5)
spermaticus; (8) Tunica vaginalis communis; (9) Cavum vaginale; (10) Cauda M. ischiocavernosus; (12) (11) area
epididymidis; (13) (14)
Caput epididymis; (16) Tail; (17) Glans penis; (18) Anus; (19)
Corpus penis; (20) Scrotum; (21) M. cremaster externus.
The giant panda possess a pair of testes, with one on each side of the lower abdominal area. Under normal circumstances, the testis would descend into the scrotum during puberty. The giant panda testis is oval or pear shaped, with the testicular size varying with age and body size. A normal testis would be 75-135g in weight, 70-88mm in length, 45-66mm in width and 33-41mm in thickness (Zhang & Wei, 2006).
It was reported that testis volume during the female anoestrus period was reduced compared to the volume during oestrus period, and semen volume, sperm motility and plasma androgen concentrations were also decreased (Tsutsui et al., 2006; Aiken-Palmer et al., 2012). The authors inferred that spermatogenesis occurred from 3 months before oestrus to 2 months after oestrus in the presence of a mature female giant panda (Tsutsui et al., 2006).
During oestrus periods, sperm would collect at the epididymis. When at anoestrus periods, no sperm would be found at the epididymis (Zhang & Wei, 2006). This may imply that spermatogenesis does not occur during anoestrus periods.
3.2.3. Vas Deferens
The vas deferens extends from the epididymis to enter the urethra. The size of the ampulla vas deferens is dependent on age and body size (Zhang & Wei, 2006).
3.2.4. Accessory Sexual Gland
The giant panda only possess the prostate gland and none other assessor sexual glands. The prostate gland is divided into two leaves on each side, and the glandular secretion mixes with the semen during ejaculation (Zhang & Wei, 2006).
The penis in giant pandas is relatively small, and has a cylindrical L (or S) shape (Figure 10). The average penis length is 80-95mm (Zhang & Wei, 2006).
Figure 10 (Morris & Morris, 1966) Showing an anatomical drawing of the L or S shaped giant panda penis.
The scrotum is important for testes temperature regulation. The scrotum in giant pandas is located between the thighs and is only slightly protruding from the body. During oestrus periods, the scrotum becomes softer, has a higher elasticity and protrudes more from the body (Zhang & Wei, 2006).
Figure 11 (Spindler et al., 2004) (a) Acrosome intact sperm appear blue, (b) mottled or patchy staining indicate damaged acrosome, or (c) detached membrane. (d) Sperm with missing acrosome have a white or pink cap with no evident blue staining.
The acrosome reaction at the zona pellucida is essential for successful sperm penetration and fusion with the oolemma, which involves the imagination of outer acrosomal memberane into the swollen acrosomal matrix and the decondensation of acrosomal matrix followed by the vesiculation of the outer acrosomal membrane (Sun et al., 1996). The distribution of Ca2+ and Ca2+ATPase in the giant panda sperm was different to other mammals, and the acrosomal reaction is dependent on the uptake of extracellular Ca2+ (Sun et al., 1996). It was shown that Ca2+ ionophore can accelerate acrosome reaction and may be used to induce sperm capacitation in giant pandas, and the capacitation was dependent on Ca2+ (Sun et al., 1996; Spindler et al., 2004). The appearance of various acrosome damage is shown in Figure 11. The capacitation observed in giant panda sperm is demonstrated in Figure 12.
Figure 12 (Spindler et al., 2006) (a) “Normal” giant panda sperm before decondensation was opaque. (b) “Partially decondensed” spem was
comparatively more transparent. (c) “Fully decondensed” sperm was transparent and enlarged. (d) Sperm with only the calyx remaining was categorised as diffuse headed sperm.
3.2.8. Sexual behaviour
In normal circumstances, the giant panda matures at 6-7 years old and 4.5-5.5 years old in some extreme cases in captivity (Zhang & Wei, 2006).
Male giant pandas would bleat to signal sexual activity, and may contain information on androgen levels and body structure (Charlton et al., 2011). This allows the female pandas to choose high quality males for copulation and allows other male pandas to avoid competition (Charlton et al., 2011) which may lead to increased energy costs.
Male giant pandas would compete for mating opportunities with females and follow the female for up to a month before copulation, but shows no male guarding behaviour (Charlton et al., 2010).
It is no surprise that the pair of giant pandas in Edinburgh Zoo had failed to mate in 2012 (www.bbc.co.uk, 2012).
In general, the inability to conceive has been considered to be behavioural. The statement is true to a certain extent as the giant pandas are in heat for 2-3 days on average (Platz et al., 1983), and some giant pandas are known to be aggressive or lack sexual interest towards the opposite gender whilst they are in the same cage (CBRGBP, personal communication; Snyder et al., 2003; Spindler et al., 2004). There are several factors that may affect the exhibition of sexual behaviours in giant pandas (Zhang & Wei, 2006): 1. Age can affect the activity level of the individual, the older giant pandas would bleat and walk less than younger giant pandas; 2. Space available can affect successful copulation, as sexual behaviours require space; 3. Body weight and health can affect hormonal balance, which extreme weight loss can affect when the female is in oestrus.
A study by Snyder et al., 2003 suggested that early social deprivation have negative effects on sexual and maternal behaviour, and social play in species such as rhesus macaques, chimpanzees, orang-utans, gorillas, domestic dogs and domestic cats. It was believed that the behaviours are detrimentally influenced by the lack of natural maternal exposure. The authors also suggested that by separating giant panda cubs from their mothers permanently before the age of 6 months may cause behavioural changes (more so in males than in females), as observed in rhesus macaques and rats (Snyder et al., 2003).
However, it cannot be ruled out that the some of the reasons can also be physiological.
The study by Hori et al., 2006 suggested that in natural mating a large amount of sperm is lost before it reaches the external uttering orifice as ejaculation may occur in the vaginal vestibule before the pseudocervix. The curved structure of the pseudocervix also means that direct advancement of insemination equipment may be difficult (Hori et al., 2006).
A study by Chaudhuri et al., 1988 have shown that the giant panda exhibits delayed implantation with a late increase in progesterone metabolite in urine about 3 months after successful artificial insemination (AI) (Figure 13). The black bear, which is known to delay implantation, would show a similar pattern of increase in progesterone concentrations near the occurrence of implantation (Chaudhuri et al., 1988). The exhibition of delayed implantation may be due to the phylogenetic similarities between the bears and giant pandas (Chaudhuri et al., 1988).
Figure 13 (Chaudhuri et al., 1988) Urinary concentrations of conjugated oestrogen and pregnanediol-3-glucuronide (PdG) in the urine of a female giant panda during oestrus and pregnancy in 1984. The arrows indicate the day of mating (♂), and the day of parturition (P).
Along with delayed implantation, the giant pandas give birth to extremely underdeveloped foetuses (1/10 of mother’s body weight) which imposed difficulties in ultrasound detection and increased the probability for
pseudopregnancies (Zhang et al., 2009).
Previous studies suggested that low genetic variability in the wild population has been detected and population decline is partly due to genetic factors (Zhang et al., 2007). The results from Zhang et al., 2007 implied that the giant panda has a good level of genetic variability and the diminished genetic diversity must not be accounted for its endangered status. The wild population still possess a
significantly rich gene pool, and would be able to survive proving their habitat is protected and demographically stable (Zhang et al., 2007).
4. Recognition of Conspecifics
Since giant pandas are solitary animals, it is very important for them to recognise their conspecifics in encounters. The signals for recognition can be categorised by distance, with vocalisations travelling the furthest, then smell, then visual recognition when the individuals are within close proximity.
The giant panda produces at least 11 types of sound (Dungl et al., 2008). Vocalisations have been shown to contain information for vocal recognition, and it would be expected to be selected to maximise differences between individual vocalisation and for receiver to extract information from the callers (Charlton et al., 2009). Such information may include genetic and physical features such as sex, age, body size, larynx and vocal tract length, sex hormone levels (Charlton et al., 2011). The extensive use of vocalisations in the giant panda may be due to the difficulties in identifying visual cues, and vocalisations usually travel for a longer distance in a dense forest.
Giant pandas are particularly vocal during the breeding season. Vocal recognition of familiar and unfamiliar individuals is also important in the sexual context. The primary vocalisation is the bleat, which signals non-aggressive intent and is produced at high frequencies during mating rituals (Charlton et al.,2009). The bleat was also thought to be the easiest to identify and contains more information. Vocalisations are thought to be important for maintaining contact between two individuals and may also be used by males to familiarise females with their presence during mating season, which have a role in initiating and controlling the other animal prior to mating. Males with higher androgen levels would generally produce a longer and higher frequency bleat (Charlton et al., 2011). It was found that females may be more receptive to vocally familiar males (Charlton et al., 2009). Charlton et al., 2010 also found that female chirps (Figure 14) before and after ovulation is significantly different. It was suggested that female chirps that advertise fertility can facilitate mating, and provide dominate males with cues to copulate the females at the optimum time for
insemination (Charlton et al., 2010). It therefore suggests a very important role of vocalisations in reproduction. Figure (Charlton 2010) (a) Waveform and (b) spectrogram et 14 al.,
of a female giant panda high-
pitched tonal chirp characterised by a rapid shift downward in fundamental frequency towards the end.
Vocalisations may potentially be used in tracking individuals given the high rates of vocal activity during the breeding season. This may allow the estimation of population size and dispersal of the species (Charlton et al., 2009).
Giant pandas have been observed to communicate with conspecifics using olfactory signals, and chemical-rich scent marks on environmentally prominent features have been found (Hagey & McDonald, 2003). Males would scent mark throughout the year, and females would scent mark within two weeks prior to their oestrus period. A major component of scent marks are volatile short chain fatty acids directly transferred to the mark from the scent gland region, other components would include chemicals from urine and vagina (in females). Scent
marks are believed to contain information on sexual receptivity, dominance, territory ownership, individuality and kinship (Hagey & McDonald, 2003). Although females would use urine to mark their territories, giant pandas seemed unable to react to olfactory cues more than one metre away and urine is easily washed away (Dungl et al., 2008). Males seem to have a higher preference for the urine of oestrus females, but it was suggested that the animals can only discriminate the scent of the anogenital secretion and not the urine itself (Dungl et al., 2008).
By chemical profiling scent marks, it may be possible to identify and track individuals and measure dispersal with the lowest human influence (Hagey & McDonald, 2003).
Dungl et al., 2008 have conducted an experiment on facial recognition in giant pandas. To account for the poor olfactory recognition, the giant pandas have been found to claw at trees or peel bark from trunks to guide conspecifics along with olfactory deposits (Dungl et al., 2008). In fact, 93% of trees that have been scent marked also had claw marks. It was concluded that visual markings are necessary for olfactory communication. The authors also identified that the fur patterning in the giant panda face can provide information on individual identification and communication, and the giant pandas are able to remember the patterns for up to a year (Dungl et al., 2008).
An individual’s ability to find and remember the location of resources, such as food, potential mates, and threats (e.g. predators), is essential for survival (Perdue et al., 2009). The results from Perdue et al., 2009 suggested that giant pandas are able to remember spatial information in the absence of external cues with a memory threshold of 10-15 seconds delay in which remembering becomes difficult. The results also suggested that the animals are able to remember general information but not specific locations, and motivation, distraction and preservation may affect individual’s performance (Perdue et al., 2009). By remembering locational information such as forage patches and potential mates, the giant panda would be more adapted to the environment and have a higher reproductive rate.
5. Giant Pandas in Captivities/Reserves
The captive giant panda population is highly valued by the Chinese Government (Aitken-Palmer et al., 2012). They act as an asset for research, education, foreign relations and as insurance against any catastrophe that might affect the wild population. The collective ex-situ population of giant pandas has nearly tripled from around 120 to more than 330 individuals in the last 11 years (Aitken-Palmer et al., 2012).
Table 2 below summarises the nature reserves in China where Giant Pandas might be found (Zhang & Wei, 2006).
Reserve Jianshan Baishuijiang Yuhe
District Wenxian Wenxian, Wudu Wudu Songpan Jiuzhaigou Jiuzhaigou Songpan Jiuzhaigou Qingchuan Pingwu Pingwu Pingwu Beichuan Beichuan Anxian Maoxian Shifang, Mianzhu Pengzhou Wenchuan Baoxing Tianquan Congzhou Dayi
Area/km2 100.40 2137.50 749.44 551.00 643.00 162.00 767.00 271.00 400.00 322.97 260.00 138.00 77.00 82.93 177.00 196.00 637.00 303.86 2000.00 390.00 234.00 101.00 308.00
Category Provincial National Provincial Provincial National Provincial Provincial Provincial National National Provincial Provincial Provincial Provincial Provincial Provincial Provincial National National National Provincial Provincial Provincial
Huanglong Jiuzhaigou Baihe Baiyang Wujiao Tanjiahe Wanglang Si’er Xiaohegou Xiaozhaizigou Piankou Qianfoshan Baodinggou Jiudingshan Baishuihe Wolong Fengtongzhai Labahe Anzihe Heishuihe
Caopo Wawushan Yele Hongba Shenguozhuang Ma’anshan Mamize Liziping Mabian Dafengding Meigu Dafengding Shanxi Foping Changqing Laoxiancheng Ningshan Majiashan Guanyinshan Tianhuashan Sangyuan Motianling Niuweihe
Wenchuan Hongya Mianning Jiulong Yuexi Ganluo Leibo Shimian Mabian Maigu Foping Yangxian Zhouzi Ningshan Ningqiang Foping Ningshan Liuba Mianxian Taibai
1224.00 166.00 180.00 360.00 337.00 408.26 388.00 409.03 345.00 160.00 292.40 299.86 126.11 267.32 102.00 135.00 254.85 138.00 117 134.92
Provincial Provincial Provincial Municipal Provincial Provincial Provincial Provincial National National National National Provincial County County County County County County Provincial
Table 2 (Zhang & Wei, 2006) List of giant panda nature reserves in China with their area and protection status.
Since the setting up of Wolong China Research and Conservation Centre for the Giant Panda in 1980, a lot has been done to improve captive breeding of giant pandas. The four biggest captivities in China are the Chengdu Research Base of Giant Panda Breeding, Wolong China Research and Conservation Centre for the Giant Panda, Beijing Zoo and Shaanxi (Louguantai) Rescue and Breeding Cenre for Rare Wildlife (Zhang & Wei, 2006).
The giant pandas are generally chosen and sent to other zoos while they are in their sub-adult stage. The main aim for loaning out giant pandas is to allow foreign research to take place and therefore increase the breeding success of the giant pandas and eventually genetic success.
Below is a list of zoos outside China with giant pandas on loan:
• • • • • • • • • • • • • •
Schoenbrunner Tiergarten, Vienna, Austria Zoo Aquarium Madrid, Madrid, Spain Zoologischer Garten Berlin, Berlin, Germany San Diego Zoological Garden, San Diego, California, USA National Zoological Park, Washington, District of Columbia, USA Zoo Atlanta, Atlanta, Georgia, USA Memphis Zoological Garden & Aquarium, Memphis, Tennessee, USA Chapultepec Zoological Park, Mexico City, Federal District, Mexico Chiangmai Zoological Garden, Chiangmai, Thailand Kobe Oji Zoo, Kobe-Shi, Hyogo, Japan Adventure World, Nishimuho-Gun, Wakayama, Japan Adelaide Zoological Gardens, Adelaide, South Australia, Australia Edinburgh Zoo, Edinburgh, UK Zoo Bois de Vincennes, Paris, France
5.2.1. Main Aims for Captive Breeding
One of the aims for captive breeding is to improve the quality and quantity of giant panda population. The international workshop in 1996 on ‘Giant Pand Captive Management Planning’ has recognised the need for more research to improve assisted breeding of giant pandas (Spindler et al., 2006). Since the setting up of various giant panda captivities, the number of giant pandas has now increased significantly (from about 30 individuals to more than 316 alive animals in 2010). Therefore the quality of breeding has now become priority for all existing captivities.
Another aim is to study the behavioural development of captive giant pandas. There are evidence suggesting that male captive giant pandas are less capable of natural mating behaviour, possibly due to the intensive breeding process that was carried out to increase giant panda numbers and also being separated from mother giant pandas at an earlier age than wild giant pandas. Furthermore, there is some strange behaviour exhibited by captive giant pandas, such as head shaking and bobbing, which is also observed in other captive species as stereotypic behaviours.
The third aim is to study the nutrition of giant pandas. As giant pandas have a very different diet to the common bears, it becomes important to study their nutrition so as to provide them enough energy and nutrients. Moreover, the
giant pandas change their diet according to the seasons as described in (Finley et al., 2011).
All captive giant pandas are now listed in the stud book to avoid inbreeding and to maintain genetic diversity. The ultimate goal of captive breeding is to create a sustainable captive population that sustains 90% of the original population’s genetic variability for 100 years, which may serve as a source for sustaining wild populations in the future (Shen et al., 2009).
5.3.1. Natural Mating
Natural mating is encouraged in captivities; the detailed mating procedure is described in the book by Zhang and Wei, 2006 (Figure 15). Natural mating usually occurs in early mornings (5.00-9.00). When the giant pandas are in oestrus, the male would appear unsettled and would bleat and sniff. The female would produce lordosis posture, and also bleat to signal oestrus. When the giant pandas are ready to copulate, the male would hug or bridge on top of the female. When penetration is successful, both the male and the female would bleat together, while the female backs into the male, the male would sit down on the floor. The laverage copulation lasts for 1-4 minutes with the longest lasting more than 17 minutes. After ejaculation, the female might threaten the male to leave while the male refuses until the female escapes its grasp. After copulation, the female would attack and threaten the male; some males would escape while some others would remain near the female until the female produces a lordosis posture and copulates with the female again. It has been observed that one female giant panda is capable of copulating with one male several times or with several males in the same day when it is in oestrus (Zhang & Wei, 2006).
Due to the lack of competition and mate selection, the average pregnancy rate of natural mating in captivity is only 60%. Also, most pregnancies are now achieved by assisted breeding. This further depletes the sexual behaviour in captive giant pandas, with only 10% males classed as sexually active (i.e. successful copulation with females) and the other 90% as sexually inactive (i.e. exhibits sexual behaviour but unable to copulate successfully). No significant difference in semen quality has been found between sexually active and inactive males. The classification for females are different to males, as sexually inactive
females (around 30%) are described as the lack of lordosis postures, and sexually active females (around 70%) exhibits lordosis. Both sexually active males and females generally vocalise more to signal oestrus. The reason why there is such a high percentage of sexually inactive individuals could be because of the separation from mothers at a young age (less than 6 months), lack of interaction between both genders and the lack of sufficient management (Zhang & Wei, 2006).
Figure 15 (Zhang & Wei, 2006) Natural mating of a pair of giant pandas in 2005. (A) The male and female were exhibiting lordosis postures prior to copulation.
(B) Mounting attempted by the male. (C) Successful mounting, and copulation began. (D) The male restrained the female while sitting down. (E) The female attempted to escape the restrain from the male while the male continues to hold on to the female. Copulation was complete at this point.
5.3.2. Assisted Breeding
The use of assisted breeding is now well recognised in China to ensure that every individual is represented in the captive population (Spindler et al., 2006).
Artificial Insemination (AI)
AI requires sperm collection from the male giant pandas. The CRBGPB uses electroejaculation procedure for the collection of sperm and is believed to be consistently effective in collecting good quality sperm without endangering the health of the animal (Platz et al., 1983; Spindler et al., 2006). The results from Platz et al., 1983 indicated that the ejaculate volume and sperm motility tend to be depressed during the female anoestrus period, and that the electrical stimuli appears to have no effect. However, giant panda semen is easily contaminated by urine with one out of five samples contaminated in the study by Hori et al., 2006.
Fresh semen have played an important role in captive breeding to give the most AI successes and to overcome the problem of sexual incompatibility (Spindler et all., 2004). Fresh semen was able to capacitate in-vitro with or without the use of accelerators over a 6-hour interval of incubation.
AI can also be carried out using cryopreserved sperm, and have been used to produce healthy offspring in the giant panda (Spindler et al., 2004, 2006). The use of cryopreserved sperm would facilitate gene flow between different captivities without compromising political and legal restrictions (Spindler et al., 2004). Cryopreserved sperm can potentially be obtained from a wild animal to introduce new genetic material into the captive population without the extraction of more giant pandas from its habitat (Spindler et al., 2004). By following the cryopreservation and thawing protocols described in Spindler et al., 2004, the authors concluded their technique was ‘field-friendly’ that it imposed little to no detrimental effect on fertilisation or decondensation, and can maintain the functionality of the giant panda sperm. Despite the discovery of high proportion
of motile and acrosome-intact sperm, the authors suggested that may not be sufficient for normal fertilisation and found that giant pandas sperm undergo capacitation by 5.5-6.5 hours of incubation, and that Ca2+ ionophore and zonae emulsions are capable of eliciting acrosome reaction (Spindler et al., 2004). Although the cryopreserving and thawing sperm may accelerate capacitation and producing hyperactive sperm by inducing calcium flux and changes in the lipid bilayer structure along with tyrosine phosphorylation, it may contribute to reduced sperm longevity post-thawing as the changes are usually destabilising (Spindler et al., 2004).
AI in giant pandas have been carried out by intravaginal insemination using bovine insemination equipment (Hori et al., 2006). Huang et al., 2011 have found that the optimal time for AI would be before the notable 50% or more drop in urinary oestrogen. Magnitude of urinary oestrogen, number of AI attempts, male identity and male semen quality appeared to have no influence on the probability of AI success using cryopreserved semen (Huang et al., 2011).
In the wild, giant panda cubs remain with their mothers for at least 1.5 years (Snyder et al., 2003). When twinning occurs in the wild (approximately 50% of all litters), mothers would abandon one neonate and wild giant pandas generally would not be raised with a littermate (Snyder et al., 2003). In captivity, twin births are kept with the greatest effort. The keepers would swap one twin with another by wrapping the other twin a towel that was scented with the previous twin. This process is done regularly to alternate care of the cubs between their mothers and humans for periods throughout the day (Snyder et al., 2003). The keepers would separate the cubs from the mothers before 6 months of age but after the mother has finished producing essential colostrum which contains high concentrations of free arginines and secretory immunoglobulin A (Wei et al., 2009).
Chengdu Research Base of Giant Panda Breeding (CRBGPB)
CRBGPB was founded in 1987, with 100 acres of land and still planning to expand to about 200 acres. The main aim for CRBGPB is to promote educational tourism, which is to educate the public about habitat conservation, and breeding of giant pandas. At the CRBGPB, the giant pandas are fed three times a day with
fresh bamboo, bamboo shoots, panda cakes and apples. The panda cakes are special cakes that were produced in the CRBGPB for the improved nutrition of the giant pandas, the ingredients include corn, soya, rice, oats, salt, CaHPO4, CaCO3 and vegetable oil. All the ingredients are washed and dried before being moulded into the panda cake that is shaped similar to the Chinese moon cake.
Other than the panda cake, the CRBGPB primarily feeds the giant pandas with four species of fresh bamboo: Bashania fargesii, Chimonobambusa
pachystachys, Sinobambusa tootsik and Indocalamus longiauritus; and three species of bamboo shoots: Bambusa quadrangularis, Praecox prevernalis, Bambusa stenostachy.
The CRBGPB is located at the Northern Suburbs in Chengu City, Sichuan. CRBGPB has its own veterinary hospital and research centre on site, and also has a separate education department for public education.
Chengdu is situated between the Longmen Mountain and Qionglai Mountains which was one of the historical habitats of the giant pandas. Due to the geography of Chengdu City, the climate is humid, subtropical and monsoon influenced, with humid summers and mild winters. The temperatures in Chengdu range from -5.9oC in the winter to 40.0oC in the summer at the extremes. Despite the cold temperatures in winter, snow is rare in Chengdu, and temperatures can never be as low as it is in the mountains where the giant pandas originated from.
Behaviour of Giant Pandas ex-situ
The behaviour of giant pandas ex-situ is well documented and an ethogram have been produced for the purpose (Appendix 2). A table from Snyder et al., 2003 (Figure 16) was also used as a reference.
Figure 16 (Snyder et al., 2003) A detailed description of captive giant panda behaviours.
In CRBGPB, the cubs are separated from their mothers before the age of 6 months and are reared by keepers and would remain with the fellow cubs that were born in the same year in the same nursery until they reach maturity. To learn more about the giant pandas, the CRBGPB kindly allowed behavioural observations in their No. 2 giant panda enclosure in August and December, 2011. No. 2 giant panda enclosure has six three-year-old sub-adult giant pandas (1:1 male to female ratio) in the same pen and two mature males in separate pens. There were some slight changes to the number of subjects during the winter observations, which will be explained below.
5.5.1. Summer Time
The behaviour of the giant pandas in the summer was observed during the hottest week in Chengdu in August, 2011, with an average temperature range from 21.1oC to 29.1oC. All giant pandas were housed indoors except for two occasional days where they were housed outdoors when the temperature dropped below 27oC. The giant pandas all followed a similar feeding pattern every day, spending an average of 46.3% of the day active (Zhang & Wei, 2006), and the rest of the day sleeping or making small walks to urinate or
defecate. Males generally demonstrated a higher level of play fighting than females, consistent with the conclusion from Snyder et al., 2003. While the giant pandas engage in play fights, they would make noise and utilise some skills that may be useful for self-defence or foraging (e.g. scratching). An apparent social hierarchy was also observed during the visit, with the male with the fastest food consumption rate being on top of the hierarchy and generally bigger than the others and the initiator of play fights. However, the social hierarchy constantly changes and there were no fearful behaviour displayed by the lower ranking individuals.
5.5.2. Winter Time
The behaviour of the giant pandas in the winter was observed during the coldest week in Chengdu in December, 2011, with an average temperature range from 3.8oC to 10.4oC. There were some slight changes to the animals kept in No. 2 enclosure; four out of the original six sub-adult giant pandas were moved to other enclosures for quarantine as they were to be translocated to other zoos and one additional mature male was added to the enclosure in a separate pen. Despite the decrease in number of subjects, the giant pandas in general were more active during the week and spent more time engaged in activities such as walking, climbing and play fighting (only for the two sub-adult giant pandas in the same pen), and less time sleeping.
Also, a new pair of giant pandas was moved to No. 2 enclosure on the last day of the observation period. The pair of giant pandas that have been in No. 2 enclosure shall be referred to as the “old pair”. There were some interesting behaviour observed in the new pair. The new pair was behaving normally when they were first introduced into the indoor cage adjacent to the outdoors enclosure where the old pair was present. However, as the keepers called the old pair nearer to the indoor cage, the new pair began to exhibit fearful behaviour such as whining, uncontrolled urination and defecation, and attempts to stay as far away as the old pair as possible. In fact, the new pair quickly climbed the rails as the old pair approached and many attempts by the keepers to lure the new pair back to ground were unsuccessful (Figure 17 shows an attempt by the keeper). However, the old pair only exhibited behaviour that resembles a curious canine. This may have implications on the reintroduction of the species.
Figure 17 The new giant panda pair showing fearful behaviour
towards the old pair by climbing the bars to high places. A keeper attempts to lure the pair down but unsuccessful.
5.5.3. Stereotypic Behaviour
There were several stereotypic behaviours observed in the giant pandas in No. 2 enclosure, namely stereotypic pacing, head tossing and paw sucking. There have been disputes on why stereotypic behaviours occur in captive animals. One view is that the behaviour arises from stress and anxiety. However, recent studies have concluded that stress and anxiety may not be the primary reason for the occurrence of stereotypic behaviours (Zhang & Wei, 2006).
To reduce the occurrence of stereotypic behaviour due to stress and anxiety, the CBRGPB have put in various different types of environmental enrichment (such as plastic tubes, plastic balls, wooden sticks, ice cubes and iced apples) around the captive areas. The use of environmental enrichment is believed to increase the active forage time of giant pandas and to reduce time spent on stereotypic behaviours (Zhang & Wei, 2006).
All graphs and statistics in this section was done using Microsoft Excel.
5.6.1. Rising Numbers of Captive Giant Pandas
The numbers in CBRGBP have increased significantly, with six giant pandas rescued from the wild to start with; there are now 104 births in total. Figure 18 and Figure 19 shows the number of births and the cumulative number of births each year respectively.
Number of births each year
10 Births 8 6 4 2 0 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Year Figure 18 Number of births in CBRGPB each year from 1987 to 2011.
120 100 Number of births 80 60 40 20 0 1987 1992 1997 Year Figure 19 The cumulative number of births each year from 1987 to 2011. Simple regression analysis shows an increasing trend in number of alive animals in CBRGPB. 2002 2007 y = 0.2331x2 - 927.92x + 923565 R² = 0.988
From the figures above, it can be clearly seen that the numbers in CBRGPB is increasing, largely due to the increase success in breeding and raising cubs.
5.6.2. Male to Female Ratio
Sex determination of young cubs is usually quite difficult because of their small size and underdeveloped nature. One recent study by Xu et al., 2010 has discovered a new method (a single-reaction test) for sex identification of giant pandas whose sexual dimorphisms are not obvious (Figure 20).
Figure 20 (Xu et al., 2010) Agarose gel displaying results of Ursidae sex identification with GTf/GTr. Genders, species and resources of samples are exhibited at the bottom. M: GeneRulerTM 100bp DNA Ladder.
Figure 21 and Figure 22 shows the number of males to females born and the cumulative number of each gender born every year respectively. The figures above show that the male to female ratio at the CBRGPB is diverging from 1:1 and there are generally more females born than males.
Males and Females Born Every Year
12 10 8 Number 6 4 2 0 1987 -2 M y = 0.1952x - 388.18 R² = 0.5413 F Linear (M) Linear (F) y = 0.2326x - 462.99 R² = 0.3407 1992 1997 Year 2002 2007
Figure 21 Number of males and females born every year from 1987 to 2011. A simple regression analysis shows that the male and female ratio at the CBRGPB is diverging from 1:1.
Cumulative Male and Females Born Every Year
60 50 Cumulativ Frequency 40 Cumumulative M 30 20 10 y= 0 1987 1992 1997 Year Figure 22 The cumulative number of each gender born every year from 1987 to 2011. Simple regression analysis show that there are more females born in CRBGPB, but as numbers of females increase, the number of males also increase. 2002 0.1213x2 2007 Cumulative F Poly. (Cumumulative M) Poly. (Cumulative F) - 483.35x + 481318 R² = 0.9833 y = 0.1003x2 - 399x + 396765 R² = 0.9889
5.6.3. Genetic Variability
The whole ex-situ population was founded by 45 individuals from 288 wild caught animals, which the 45 individuals became the primary breeders for the current population (Shen et al., 2009). Also, the capturing of wild giant pandas was banned and introduction was terminated. This had resulted in a significant loss of genetic variability in the ex-situ population (Shen et al., 2009). As the breeding management plans for the ex-situ giant panda population set up by the Conservation Breeding Specialist Group (CBSG) and IUCN is porrly followed (Shen et al., 2009), fewer captive males than females breed (Snyder et al., 2003), which resulted in a few males being over-represented in the population (Shen et al., 2009).
Although a high level of heterozygosity in ex-situ population was found (Spindler et al., 2004), the level of heterozygosity and allelic diversity is still lower than that in the wild population (Shen et al., 2009).
6. Future of Giant Pandas
There have been public debates on whether or not the endangered species should be saved (www.bbc.co.uk, 2011). The argument put forward by the side for not saving the giant pandas was because they have to be maintained in captivity. Therefore they cost too much to maintain and their chances of returning to the wild are slim. The argument against is that by conserving the species, their natural habitats are more likely to become a nature reserve which may ultimately benefit the survival of the species. Taking views from both sides, it can be said that if the giant pandas can successfully be reintroduced, they may prove to have more ecological value than being in captivity.
Releasing into the Wild
In 1989, the ex-situ population made a transition from a population that relies on the capture of wild individuals to a source for potential reintroduction of captive individuals in to the wild (Shen et al., 2009).
Since the 1980s, there have been eight attempts to reintroduce the giant panda back into its natural habitat, while the release was successful, the survival of
reintroduced giant pandas have yet to be improved. Zhang & Wei, 2006 have summarised four points that affect the successful reintroductions: 1. Giant pandas seem to have memory/homing behaviour, which allows them to return to where it was raised. However the successful return would be highly dependent on the distance and geography of the site of release; 2. Captive giant pandas rely on the help from keepers, they are conditioned with the calls of the keepers therefore pre-reintroduction training is needed; 3. The captive environment can affect the success rate of reintroduction, the amount of human contact they receive in captivity would affect how well the giant pandas can adapt to the wild; 4. There is limited tracking of the reintroduced individuals, most of them are not tracked and therefore makes record collecting extremely hard.
The captive ex-situ population will serve as a reservoir for individuals to be reintroduced into appropriate habitats. The CRBGPB has set up a new Chengdu Field Research Centre for Giant Pandas in December 2011 in Majiagou, Yutang Town in Dujianyan City. The institution aims to release 40-50 sub-adult giant pandas into the wild for training. The individuals will be trained to forage, live outdoors, mate, etc. The environment at the Research Centre is fully consistent with the UN description of the giant panda habitat (Yang et al., 2011)
6.1.1. Rules for Successful Reintroduction
There are eight rules outlined by D Kleiman, 1989: 1. A self-sustaining captive population with enough breeding stock to provide a surplus and genetic variation; 2. A sustainable amount of adequate and protected habitat with protection for the species in the future; 3. Effective techniques to prepare animals for reintroduction, animals must be trained to survive, interact with conspecifics and shelter construction; 4. Post-release monitoring and evaluation to ensure that the program is suitable; 5. Professional and public education to create local support to sustain reintroduction efforts; 6. Sufficient long term funding potential to avoid suspension of
projects/programs; 7. Side benefits which may serve as basis for continued habitat protection;
8. Reference to examples allows project participants to learn from successful and unsuccessful stories.
In the book by Zhang & Wei, 2006, they have produced some guidelines for the preparation for giant panda reintroduction:
a. Biology of the giant panda
Research must be conducted on the dispersal and den selection, social structure and space requirements, reproductive physiology and behaviour, genetic heritability and variability, diseases, interaction with other species in the habitat, and captive behaviours. By conducting research on the above sectors, it is possible to condition the giant pandas back to their natural behaviours and increase survival. The health, gender and genetics of the individual to be reintroduced must also be considered.
A suitable site of reintroduction must be chosen carefully to ensure habitat fragmentation is minimal. One good example for a site of reintroduction is Emeishan as giant pandas became extinct in the mountain mainly due to poaching and is between Qionglaishan and Daxiangling.
b. Social, economical and legal guarantee
It is important to gain local support before reintroducing, as the giant panda habitats overlaps with human social-economical activities. It is more important to gain government and legal support to set up nature reserves at the site of reintroduction so as to minimise potential habitat destruction.
c. Planning and preparing
reintroduction should be set up to ensure there is enough experts and money to facilitate the whole process. canine distemper virus (CDV), canine parvovirus (CPV), canine coronavirus (CCV), canine prarinfluenze virus (CPIV) and rabies virus have been the primary viruses that causes casualties in captive environments (Qin et al., 2010). Currently captive giant pandas are exposed to the common canine viruses and are not vaccinated. The giant pandas due for reintroduction should be vaccinated against common diseases to increase
survival. To prepare the giant pandas for reintroduction, training (such as increasing food choice, increasing movement, avoiding predators, social
behaviours and important conspecific signal recognition) must be given to allow them to adapt to the natural environment. There should also be an emergency plan in place such that any unsuccessful cases or injured individuals can be quickly brought back into captivity.
After successful reintroduction, the individuals must be tracked constantly to allow research with minimal human influence. Any deaths should be collected for any further research on the reason of death. Emergency human interference should be carried out in case of disease, injuries, forage or den assistance. A review should be carried out to assess the reintroduction programme.
Examples in other species
Since the giant panda is a native species in China and international organisations have been involved in the possibility of reintroducing the species, it may be possible to take some inspiration from other species that have been reintroduced by the joint effort of the Chinese government and international organisations.
6.2.1. Père David’s Deer
The first deer was reintroduced in 1985 by the Chinese Government and 1986 by the World Wildlife Fund (WWF) with 26 individuals (wwf.panda.org, 2005). Père David’s Deer is a significant example in the successful reintroduction of native Chinese species which had been extinct from its natural habitat. The
reintroduced population gave birth to the first wild offspring in 2003 (Zhang & Wei, 2006) and had been shown to have a healthy net growth rate (Yang et al., 2007). However, salinization of soil and human influence is still a threat to the deer’s natural habitat which may endanger the deer population once again (Yang et al., 2007).
6.2.2. Chinese Alligator
The work to revive the species began in 1999, with a shrinking population of 130 (www.wcs.org, 2009). With efforts to restore their natural habitat since 2001 (H
Jiang, unpublished), the first reintroduced Chinese Alligator was release in Honghing reserve of Xuancheng County in Anhui in 2003 (www.wcs.org, 2009). To ensure genetic diversity, the project imported another twelve individuals from the US and three individuals from Changxing in 2007. The reintroduced individuals had produced fifteen hatchings in 2008 (www.wcs.org, 2009). A review by H Jiang (unpublished) concluded that the captive Chinese Alligator is able to adapt to restored habitats and recover breeding capacity in the wild very smoothly. In a study conducted by Wang et al., 2011, they have also found that most reintroduced alligators have not moved away from the reintroduction site. This may implicate that the reintroduction habitat is prime for the alligators, or the alligators have lost the ability to explore other suitable habitats.
6.2.3. Black Rhinoes
First rhino introduced back to Kenya in 2008 (news.bbc.co.uk, 2008). The Department for Environment Food and Rural Affairs (defra) has organised the Darwin Initiative to reintroduce the black rhino into its former range in Uganda from 2010 to 2011 (darwin.defra.org.uk, 2011).
Like the giant panda, the black rhino is also large in body size and has reproductive difficulties (although not as many as the giant panda). The first eastern black rhino was imported from Tanzania to Zoo Dvur Kralove in 19711974 with the first calf born in 1977 (www.waza.org). It took the black rhino more than 30 years to be reintroduced back to its natural habitat.
This research project was set out to investigate the possibilities to reintroduce the giant pandas back into its natural habitat.
With the basic biology of giant pandas being researched since they entered captivities, their diet and reproductive physiology has almost been thoroughly researched on. With the current knowledge on the giant panda physiology, it was found that the giant panda is closely related to the bears and has adapted to a vegetarian diet despite the low energy content and the carnivores stomach. The giant panda, therefore, can plausibly be called a “facultative carnivore”. Their special bamboo based diet is one of the reasons why giant pandas are endangered.
The giant panda’s difficulty in reproduction is also one of the problems that cause its endangered status, with the males lacking sexual drive and the females just being in oestrus for 2-3 days a year along with delayed implantation and extremely underdeveloped new born. It is clear that the reproductive success is relatively low in the wild.
With the rising number of births in captivity, it is possible to say that the captive population have transformed to a sustainable population. However, the increase in birth rates is a result of human intervention. The possibility of captive giant pandas being able to revert to wild behaviours is unconvincing, as the captive giant pandas are raised by humans from a very young age and therefore may associate human presence with food. They are also raise with other giant pandas of similar age. The wild giant pandas however, are very solitary and would avoid human contact and even conspecifics except during the breeding season, the wild population uses a lot of visual, vocal and olfactory cues to communicate with each other. This adds to the low birth rates in the wild.
The giant panda’s endangered status cannot be blamed only on its physiology and behaviour. Human activities have a huge impact on the giant panda habitats and population. With poaching banned, at least the giant pandas would be dying of natural causes. However, habitat fragmentation is still a process that greatly affects the giant pandas. With the urban areas expanding, and agricultural activities close to the nature reserves, the giant panda populations in isolated mountain ranges will become more disconnected. This in turn affects the genetic diversity of the wild population. However, the reintroduction of captive animals may introduce new genes into the gene pool. The introduction of new genes may enhance the overall survival of the species.
To ensure successful reintroduction, a detailed plan for the reintroduction programme must be in place. This makes sure all the problems that might occur during reintroduction attempts are addressed. For example, the biology of the animal must be well prepared to ensure that they are able to survive, forage and reproduce without human influence; habitat management plans must be agreed on to ensure minimal habitat destruction or fragmentation occurs to further isolate the giant panda population; genetic variance of the giant panda population must be well monitored to avoid problems that may arise from inbreeding. For the reintroduction plan to run smoothly, it will require efforts from the local residents to the state government.
Taking examples from other species of similar sizes and are successfully reintroduced (e.g. the Chinese alligator, Père David’s deer and Black rhinos). They all took at least 10 years for the Chinese alligator to have wild hatches, 18 years for Père David’s deer to give its first wild birth and 30 years for the black rhinos to return to its natural habitat. Considering the biology and behaviour of the giant pandas, it is plausible to say that it would take the giant pandas at least 30 years, if not more, to be successfully reintroduced.
There were debates on whether or not the giant panda should be saved. Both sides have put sound arguments together, although no conclusions were made from the debate. It is clear that if the reintroduction programmes are successful, the giant pandas may prove to have more ecological value and therefore worth saving.
In conclusion, the successful reintroduction of the giant pandas will require: 1. Knowledge on the biology of the giant pandas (wild and captive) mainly on diet, reproduction and behaviour; 2. A sustainable ex-situ population and environment; 3. A detailed plan for the reintroduction programme; 4. An estimated time frame for reintroduction; and 5. Support from residents, local and state government.
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Appendix 1 – Cryopreservation and Thawing Methods (Spindler et al., 2006)
The semen is diluted immediately within 15 minutes of collection with commercially available TEST egg yolk buffer modified to contain 5% glycerol. The semen is then diluted in a 15ml conical plastic tube slowly over a 3-minute period to give a final concentration of 400×106 motile sperms per ml.
Each tube is then placed in a water jacket (37oC water, 400ml) and placed a refrigerator to cool slowly to 4oC over a 4-hour period.
Cooled semen is then pipetted into 0.25ml sterile plastic straws, and each straw is sealed using a Nyclave impulse heat sealer. Each straw is placed 7.5cm above liquid nitrogen for 1 minute, then 2.5cm above for another minute to achieve a rapid cryopreservation rate of -40oC per minute and -100oC per minute respectively. Frozen samples are plunged into the liquid nitrogen and stored for at least 24 hours before thawing.
The straws are exposed to air for 10 seconds for thawing, followed by plunging the straw into a 37oC waterbath for 30 seconds. Both ends of the straw are cut off to allow the thawed sample to flow into a 5ml sterile plastic tube and it diluted slowly over a 3 minute period with 2ml HF10 at 37oC.
microcentrifuge tube at 200g for 8 minutes. The supernatant is discarded and the sperm pallet is resuspended in HF10 at 37oC to achieve a final concentration of 50×106 cells per ml.
Appendix 2 – Giant Panda Behaviour Ethogram (CBRGPB)
1. Normal behaviours
The processing and consumption of food. The type of food can be categorised into the categories below: • • • • • • • • • Biscuits (b) Bread (br) Whole provisioned bamboo (wb) Bamboo leaves (l) Stem or clum of bamboo (s) Gruel (g) Fruit (f) Vegetables (v) Other vegetation growing in the enclosure (ov)
The consumption of water.
The liking of area that the animal had been sniffing. If the animal licks or bites another animal, score as LIag.
The elimination of metabolic wastes from the body which include defecation (DE) and urination (UR). If the animal urinates over the edge, place an “e” behind the code.
Vertical ascent or descent into trees or other structures with all four limbs above ground. Only includes active climbing, if the animal is resting on the structure or tree, “rest” is scored.
Short directional travelling between two points, or sustained travel in a nonstereotypic manner.
Scratching body with paws, licking and/or biting fur.
Lying or sitting, the animal can be either awake or asleep.
Olfactory investigation (OI)
Placing nose close to a substrate or an object and sniffs, and/or appear attentive for more than one second.
1.10. Stationary alert (SA)
Alert, standing on four limbs, sitting or lying quietly, remaining attentive, moving head from side to side and/or sniffing air, attention to external stimuli.
1.11. Scent marking (SM)
Scent marking can be categorised into four categories by the way the animal is rubbing its anogental region repetitively against a substrate or an object: • Squat mark (sm) The animal scent marks in a squatting position with its tail up. • Leg-cock mark (lm) The animal scent marks with its tail up against the object with one hindleg raised. • Reverse mark (rm) The animal backs up to a vertical surface before scent marking with its tail up and standing with all four limbs. • Handstand mark (hm)
The animal elevates its hind quarters with weight on front legs before sent marking, both hind legs must be above ground.
1.12. Locomotive play (LP)
Solitary, superfluous, apparently purposeless activities such as gambolling, frisking, somersaulting, rolling and leaping.
1.13. Object play (OP)
Dragging, batting or tossing objects with excessive force. The animal appears to be using the object for entertainment rather than investigating the object.
1.14. Social play (SP)
Social play can be categorised into different categories: • • • Reciprocal fighting (rf) Non-reciprocal fighting (nrp) Approaching (ap) The animal appears to be orientated towards another animal and moves from a distance of more than one adult body length from the other animal to within one adult body length of the animal. • Leaving (lv) The animal that is within one adult body length proximity of another animal travels to more than one adult body length away from the animal. • Contacting (ct) The animal touches any part of another animal for more than one second, usually occurs after approach. It is more important to score when an interaction begins. • Breaking contact (bc) The animal ceases to touch any part of the other animal for more than 1 second, usually as part of leaving behaviour. It is important to score after an interaction has occurred. • Charging (chg) The animal approaches another animal at a speed faster than a walk and makes contact with the other animal. The animal is usually running head down and uses its head to hit the other animal. Score “c” when contact is made.
Lunging (lg) The animal rapidly projects its body towards another anima without travelling and from a stationary position, usually involves standing on hind legs and thrusting body forward or pouncing. Also includes leaping from higher elevation onto animal below. Score “c” if contact is made.
Following (fl) The animal travels behind another animal for a distance of at least 2 adult body lengths, with both animals moving no faster than a walk.
Chase (chs) The animal follows another animal for a distance of at least 2 adult body lengths moving at a pace faster than a walk, usually as part of a play bout.
1.15. Vocal (VOC)
Vocal behaviours are scored using the 1-0 method. A vocalisation is only recorded once during each minute when it occurs, regardless of how many times it has occurred. Vocalisations can be categorised into the following categories: • Bleat (bl) Twittering, goat-like call of around 1-3 seconds. • Chirp (ch) Short, tonal high-pitched call with descending pitch towards the end. • Bark (ba) Short, fairly loud and similar to a dog bark. • Moan (mo) Low pitched, low to medium amplitude call of variable duration. Often has several short starting elements and usually grades with barks, chirps and bleats. • Growl (gr) Long, loud, low pitched and similar to a dog growl. • Honk (hk) Short, tonal, low pitched, nasal, falling in pitch and produced repetitively in a series. • Roar (ro) Very loud, intense and harsh vocalisation. • Chomp (cm) Rapid opening and closing of jaw with teeth clicking together audibly. • Huff (hu)
Audible expulsion of air through an open mouth. • Snort (sn) Intense audible expulsion of air through the nose. • Squeal (sq) Short, high pitched, open mouth call. • Whine (wh) High pitched, medium intensity, often repetitive vocalisation. • Groan (grn) Low to medium pitched, often repetitive, medium length vocalisation. • Other (vo) Any vocalisations the animals makes that is not defined above.
2. Stereotypic behaviours (STE)
Stereotypic pacing (PC)
Back and forth or perimeter travel in a repetitive, sustained, stereotyped pattern. The animal must travel at least 3 times on the same route before scoring.
The animal stands on its hind legs and spins for more than 90o.
Sit pirouette (SPI)
The animal sits on the ground with an erect torso and spins for more than 90o.
Head toss (HT)
The animal abruptly lifts its head upward and/or to the side in a swinging motion. Individual head tosses are scored accordingly.
Head bob (HB)
The animal grips cage bars and bobs its head up and down in a stereotypic manner.
Suck chest (SUC)
The animal uses its mouth to suck on its own chest area, with sucking vocalisations. The separation of hair on chest to reveal the nipple is sometimes observed.
Suck paw (SP)
The animal sucks on its paw with its mouth, with sucking vocalisations.
Suck other (SO)
The animal sucks on other parts of the body, or the body of another animal.
The animal shifts its weight from side to side while remaining stationary.
2.10. Regurgitate and reingest (RR)
The animal vomits then reingests the vomit.
2.11. Teeth click (TC)
The animal opens and closes its jaw repetitively and produces vocalisations.
2.12. Coprophagia (CP)
Consumption of faeces.
3. Other (OT)
Any other behaviour that does not fit into any of the definition on the ethogram.
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