Sixty-one animals were available for the Biomedical Survey. To allow age comparisons, we divided the population into three groups: juveniles (animals 1 to 18 months of age); subadults (19 months to 4.5 years); and adults (>4.5 years). The rationale for the age division between the juvenile and subadult category was largely based on knowledge that young appear to be weaned in nature at about 1.5 years of age (Zhu et al., 2001). For the other division, our Biomedical Survey indicated that some males and most females were pubertal (had reached sexual maturity) by 4.5 years of age (see Chapter 7). Of the surveyed population, nine individuals were juveniles, 12 were subadults and 40 were adults. Six adults were examined twice, so our data set includes a total of 67 examinations (from 61 individuals) over a three-year period.
Each of the 61 pandas was anaesthetised using a ketamine hydrochloride (HCl)-based protocol. Including the six animals evaluated twice, our findings were based on a total of 67 anaesthetic episodes. Each individual was fasted for 0 to 12 hours before anaesthetic administration by a remote delivery darting system (Daninject Pistol System; Wildlife Pharmaceuticals, Fort Collins, CO), usually in the animal's home enclosure. Induction time was defined as the time from drug administration to recumbency (head and body contact with the ground). Procedure length was considered the time from first physical contact with the animal to the point of last physical contact. Most entire examinations required less than 60 minutes.
In addition to using ketamine HCl, on 22 occasions sedative drugs, including chlorpromazine (mean 0.40 mg kg-1; range 0.26-0.59 mg kg-1; n = 12), xylazine HCl (mean 0.35 mg kg-1; range 0.21-0.48
mg kg-1; n = 7) and/or diazepam (mean 0.13 mg kg-1; range 0.09-0.21 mg kg-1; n = 4), were given in an attempt to smooth the body rigidity normally incurred with ketamine HCl alone. With the exception of one procedure, supplementary ketamine HCl (administered intramuscularly or intravenously) or the use of isoflurane gas was always required to provide the needed time to complete the medical examination. Supplemental ketamine HCl was given intramuscularly or intravenously.
In 12 cases where the procedure was longer than normal (e.g. to conduct abdominal laparoscopy), we used inhalation anaesthesia delivered by face-mask or endotracheal tube. To accomplish the latter, isoflurane and oxygen were administered via a face-mask using a precision isoflurane vaporiser set at 4-5% and a circle-system anaesthetic machine. The animal was placed in lateral recumbency. Once jaw relaxation was achieved, the animal was intubated using a 12-, 13- or 14-mm internal diameter endotracheal tube. The head was extended dorsally as much as possible, and the mouth was held open with ropes held by assistants. The larynx was directly visualised with a laryngoscope and then intubated, or the endotracheal tube was gently guided through the mouth and into the larynx without viewing. In adult animals, laryngoscopy required an extra-long (35-cm) blade to view the vocal folds. Once intubated, the animal was maintained on the appropriate concentration of isoflurane to maintain a surgical plane of anaesthesia.
All pandas were given supplementary oxygen by a face-mask covering the mouth and nares. Animals were initially placed in right lateral recumbency and were monitored at frequent intervals with one or more pulse oximeters (N-20 and N-40, Mallinckrodt, Hazelwood, MO) with clip-type sensors located on the ear tip, tongue, vulva or prepuce (Fig. 4.1; Plate III). Temperature, heart rate and respiratory rate were tracked regularly. Additionally, indirect blood-pressure was monitored (Criticon Dinamap, Mallinckrodt) using an appropriately sized blood-pressure cuff (width approximately 40% of limb diameter) placed on the left foreleg.
We chose to use a systematic approach to ensure complete and comparable data sets for all animals and all facilities. The goal of each anaesthetic procedure was always to safeguard the individual animal while
collecting information that could benefit the entire captive population. Multiple medical procedures were chosen for the purpose of most effectively collecting baseline biomedical data, providing unique permanent identification and conducting diagnostic evaluations for individuals with reproductive or medical problems. The medical procedures performed included a physical and reproductive examination, body measurements, body weight and blood sampling. A small (approximately 0.5 x 0.5 cm) skin tissue sample was collected from the medial tibial area for genetic evaluation (see Chapter 10). Biological samples were also obtained for vaginal cytology, urinalysis, faecal parasite examination and faecal cytology. Photographs were taken of dentition, general body condition and detectable lesions.
Both males and females were subjected to an ultrasound scan at the end of the medical evaluation while each animal was in dorsal recumbency. An Aloka SSD-500 portable ultrasound machine (Aloka Co., Ltd., Wallingford, CT) with 3.5-MHz transducer and printer (or similar portable units available locally) were used to obtain ultrasound images of the abdomen. In some cases, we used a 5-MHz rectal transducer to obtain more detailed views of the uterine body. To minimise hair removal, we clipped small windows ventral to the urinary bladder and in an area caudal to the xyphoid. We concentrated on examining the reproductive tract for normality but we also visualised other abdominal organs, generally obtaining good views of all, or portions, of the liver, gall bladder, spleen, urinary bladder and occasionally the kidneys.
Three adult females were also subjected to laparoscopy (Olympus America, Inc., Melville, NY). In brief, this involved clipping most of the abdominal region free of hair before placing each female in a head-down (35°) angle on a surgical table. This area was surgically prepared and draped, the abdominal cavity filled with room air via a Verres needle and then a sterilised trocar-cannula inserted in the umbilical area through a 3 cm-long skin incision. The trocar was replaced with a fibre optic telescope. Internal organs were manipulated through a laterally placed, secondary trocar-cannula using an accessory grasping forceps. This technique allowed clear viewing of most aspects of the reproductive tract, including both ovaries as well as the spleen and liver. No anomalies were observed in any of the females undergoing laparoscopy. These examinations demonstrated the feasibility of this more invasive approach for detailed assessment of the abdominal cavity content.
During each anaesthetic episode, one team member recorded all detailed findings on a comprehensive data form. We then compiled the data into a spreadsheet for later analysis. Results (and in fact all findings and analyses) were recorded on raw and summarised data sheets which were shared with collaborators before leaving each facility.
Individual permanent identification for animals is critical for cooperative breeding and management programmes, largely to avoid confusion and inaccuracy. In our study, each animal was given two forms of physical identification. Each was tattooed on the mucosal surface of the upper lip using the unique studbook (SB) number (Spaulding Electric Tattoo Marker Kit, Voorheesville, NY). Each animal received a microchip transponder placed subcutaneously along the dorsal mid-line between the scapulae (ID 100 transponder, Trovan, Eidap, Inc., Sherwood Park, Alberta, Canada). This identifier could be read through the skin with a hand-held reader (Trovan, Eidap, Inc.). All pandas tolerated both identification procedures well. For example, animals were seen eating bamboo later the same day after tattooing.
At the onset of each evaluation, a digital, load-bar weighing device (Model 500 with RL 15 load bars, Reliable Scale, Calgary, Alberta, Canada) with 0.1-kg resolution was used to determine individual animal body mass. A nine-point subjective scoring system, which relied on manual palpation to assess distribution of muscle mass and subcutaneous adipose tissue, was used to generate a body condition score (BCS). Individuals having a marginal or poor body condition were assigned a BCS of 1-3. Animals with moderate body condition were given a BCS of 4-6 (5 being ideal) whereas pandas with excessive body condition received a BCS of 7-9.
During each medical procedure, simple external measurements of defined anatomical points were also collected. The actual measures recorded were those modified from recommendations for the red panda, with additional metrics collected on the basis of earlier ursid studies (Roberts, 1994; Lundrigan, 1996). Unless specified otherwise, the following metrics were obtained using a standard, flexible measuring tape (data collected in 1999 and 2000, but not in 1998).
• Head length. From the tip of the animal's nose along the curves to the base of the skull's occipital ridge.
• Body length. From the base of the skull's occipital ridge along the curves to the root of the tail.
• Tail length. With the tail held at 30-45° above the dorsal aspect of the body, from the root of the tail to the tip of the tail (excluding hair extending beyond the tip).
• Nose-to-tail body length. From the tip of the animal's nose along the curves to the root of the tail.
• Neck girth. The circumference of the neck immediately behind the base of the skull.
• Axillary girth. The circumference of the body immediately behind the forelegs (at the chest).
• Abdominal girth. The circumference of the abdomen around the widest portion of the abdomen, across the ilium.
• Fore foot. With the right foot positioned with toes straightened: the length from the calcaneum (heel) to the tip of the longest toe, sine unguis (excluding the tip of the claw).
• Hind foot. With the right foot positioned with toes straightened: the length from the calcaneum (heel) to the tip of the longest toe, sine unguis (excluding the tip of the claw).
• Elbow width. The distance across the condyles of the right elbow.
• Knee width. The distance across the condyles of the right knee.
• Axillary skinfold. The thickness of the skin fold in the right axillary region (using callipers).
• Inguinal skin fold. The thickness of the skin fold in the right inguinal region (using callipers).
• Wrist skin fold. With the right fore foot perpendicular to the fore leg in a plantigrade position: the thickness of the lateral skin fold at the wrist (using callipers).
• Ankle skinfold. With the right hind foot perpendicular to the hind leg in a plantigrade position: the thickness of the lateral skin fold at the ankle (using callipers).
Animals were grouped by age based on dentition and onset of reproductive activity. The mean, standard deviation and range for each age class were calculated, and data for males and females were combined (because there were no gender differences; p > 0.05).
Blood was collected from the medial cephalic vein using a Vacutainer system (Vacutainer; Becton, Dickinson and Co., Franklin Lakes, NJ) within 15 minutes of handling an anaesthetized animal. Blood analyses included complete cell blood count (from EDTA-treated blood), blood-gas analyses (from whole heparinised blood) and selected chemistries (on whole heparinised blood and serum). After sample collection, blood-gas and chemistry analysis was performed within 15 and 30 minutes, respectively, using a portable analyser (iSTAT; Heska Corporation, Ft Collins, CO). A complete blood cell count was performed using manual methodology. A total white blood cell count was calculated using a Unopette (Becton, Dickinson & Co., Franklin Lakes, NJ) technique and a haemocytometer. A differential cell count was calculated using JorVet Dip Quick (Jorgensen Laboratories, Inc., Loveland, CO) stained blood smears. Total serum protein was measured using a hand-held temperature compensated refractometer (Leica, Inc., Buffalo, NY). Blood pH, partial carbon dioxide, total carbon dioxide, bicarbonate and base excess were included in the venous blood-gas analyses. Glucose, blood urea nitrogen, haematocrit and haemoglobin values were included in the chemistry analyses.
Semen collection and evaluation
Every adult male was subjected to a standardised electroejaculation procedure while under anaesthesia. The purpose was
1. to determine the quantity and quality of spermatozoa as an index of male fertility and
2. for research to understand male gamete biology and to enhance the ability to cool, cryopreserve or culture sperm in vitro.
All details associated with this process and related findings are presented by Howard et al. (in Chapter 7).
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