Biomedical Survey Expertise And General Methods

The Biomedical Survey was successful because of three key elements: effective communication; multi-skilled participants; and adequate funding. Constant translation was provided by two team members who were fluent in both languages, had lived in both cultures and were comfortable with biological terms and laboratory/animal procedures -these people probably were the most valuable of all the participants. Diversity in skills was achieved by selecting team members representing a wide range of scientific disciplines, including veterinary medicine, animal behaviour, reproductive physiology, genetics, nutrition and pathology. Each person had also demonstrated a history of successfully working in teams. Having people from different home organisations (Box 3.1) enriched the team with assorted philosophies while increasing overall problem-solving capacity. Numerous donors (Box 3.2) ensured

Box 3.1. Participating institutions and investigators

Beijing Zoo

Jinguo Zhang Zheng Xin Peng Cheng Lin Zhang Shi Quang Huang Yi Luo Yan Lu

Ming Hai Yang Tian Chun Pu Yan Ping Lu Wanmin Wang Jiang Jun Peng Fei Bing Zhu Qiming Hou

Chengdu Research Base of Giant Panda Breeding and Chengdu Zoo Anju Zhang Yuezhong Tian Guangxin He Guanghan Li Yunfang Song Jianqiu Yu Zhiyong Ye Qiang Wang Zhihe Zhang Zi Yang

Shunlong Zhong Hongwei Chen Xuebing Li Mingxi Li Xiangming Huang Jingchao Lan Meijia Zhang Shurong Yu Jishan Wang Rong Hou Wei Zhong

China Conservation and Research Centre for the Giant Panda, Wolong Nature Reserve Hemin Zhang Peng Yan Wang Guiquan Zhang Chun Xiang Tang Quan Chen Li Jian Yiang Ping Tan Xian Yan Wang Pong Rongping Wei Yan Huang Desheng Li Jun Du

Chinese Association of Zoological Gardens Zhong Xie Menghu Wang

Chongqing Zoo Ximu Zhou Youxin Xie Wei Guo Xiancum Zheng Aiping Wang Denfu Wu

Columbus Zoo

Ray Wack

Conservation Breeding Specialist Group, IUCN/SSC Susie Ellis (team leader)

St Louis Zoo R. Eric Miller

Smithsonian's National Zoological Park David Wildt (team leader) JoGayle Howard Richard Montali Rebecca Spindler

University of California at Davis

Lyndsay Philips

Zoo Atlanta Rebecca Snyder

Zoological Society of San Diego

Barbara Durrant Mark Edwards Donald Janssen Arlene Kumamoto Mabel Lam Mary Ann Olson Bruce Rideout Sandra Skrobot Meg Sutherland-

Smith Jeff Turnage Lee Young

Box 3.2. Donors to the Biomedical Survey

Air-Gas, Inc. British Airways Columbus Zoo

Giant Panda Conservation Foundation, American Zoo and Aquarium

Association Heska

InfoPet Identification Systems Smithsonian's National Zoological Park Nellcor Puritan Bennett Ohaus

Olympus America, Inc. St Louis Zoo Sensory Devices, Inc. Zoo Atlanta

Zoological Society of San Diego the final element - funding to support the Survey, including monies needed for travel, daily allowances, equipment, supplies and data analysis/distribution.

The Survey was conducted in February and March (the onset of the giant panda breeding season) in each of the years 1998, 1999 and 2000. During the first year of collaborating within a given institution, a memorandum of understanding (MoU) was signed between each Chinese breeding centre or zoo and the CBSG team. This is a common practice in China, which greatly facilitates understanding mutual expectations while avoiding misunderstandings (see the sample MoU Appendix 3.A).

Biomedical Survey methods were consistent throughout all three years to allow data comparisons over time. Anaesthesia was induced in all giant pandas through the intramuscular (i.m.) administration of ketamine hydrochloride (see Chapter 4). The primary advantage of this anaesthetic was ease of administration (usually via blow dart or pole syringe) and rapid onset and recovery. Once each animal was tractable, the following procedures were performed:

(1) Using a needle inserter, an electronic transponder chip (Trovan,

Eidap, Inc., Sherwood Park, Alberta, Canada) was inserted subcutaneously in the interscapular area at the dorsal midline at the cranial aspect of the black and white hair interface of each animal (Fig. 3.1). ATrovan transponder reader confirmed that each chip was working, and that the correct number was recorded.

(2) Each animal was tattooed in the mucosa of the upper left lip with its studbook (SB) number (Fig. 3.2).

(3) A0.5 x 0.5 cm skin biopsywas incised from the inner thigh of a hind limb, and 20 hairs and 10 ml ofheparinised blood were collected for preparing samples for genetic analysis (see Chapter 10). Each skin biopsywas minced, immersed in 1 ml of cryopreservation medium and placed in cryovials containing 1 ml of freeze medium. The medium consisted of alpha minimum essential medium (MEM, Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (Irvine Scientific), 1% glutamine (Sigma-Aldrich, St Louis, MO) and 1% penicillin-streptomycin with 10% dimethyl sulphoxide (Sigma-Aldrich). Cryovials were frozen by being placed directly into a primed liquid nitrogen vapour shipper. The blood samples were mixed 1:1 with storage buffer consisting of 0.2 M NaCl, 0.1 M EDTA (ethylenediaminetetra-acetic acid) and 2% sodium dodecyl suphate and frozen at —30°C. Hair samples were stored at ambient temperature in labelled plastic bags. All biosamples were maintained at each of the respective sites until later genetic analysis in China.

(4) Selected, healthy males were subjected to an approximately 20 minute electroejaculation procedure (see Chapter 7). Ejaculate volume was recorded, and fresh semen was evaluated for sperm motility, normal/abnormal morphology and sperm acrosomal integrity. Fresh semen was also mixed in various diluents used historically by the Chinese and USA team members (see Chapters 7 and 20). Sperm were then evaluated for the ability to retain viability or to survive cryopreservation in pellets or straw containers. Aliquots from each sample were thawed and evaluated by both teams together to determine optimal freezing and thawing methods, and to assess the ability of sperm from various males to survive a cryopreservation stress (see Chapter 7). All cryopreserved sperm samples were stored on site at the respective collection locations.

(5) A general physical examination was performed which included body weight, body measurements, oral/dental examinations, and assessment of limbs and external genitalia; 25 to 30 ml of blood

Figure 3.1. Using Trovan injection device (arrow) (photograph by S. Ellis).
Figure 3.2. Tattooed mucosa of the inside upper lip for identification purposes (photograph by D. Janssen).

(including that used for the genetic analysis) were collected from the jugular vein (Chengdu institutions and the Wolong facility) or from the cephalic vein (Beijing Zoo) of each panda. Portions were used for haematology and serum chemistry analysis. Testes were measured and palpated for tone and consistency. A vaginal smear for cytology was prepared from each female and a faecal smear for cytology and Gram stain from many individuals of both sexes. An ultrasound examination of the abdominal cavity was conducted. During anaesthesia, each animal was monitored by noninvasive blood pressure, transcutaneous pulse oximetry, body temperature and direct observation. Some blood parameters (total number of white blood cells and percentage values for neutrophils, band neutrophils, lymphocytes, monocytes, eosinophils and basophils) were assessed onsite using a portable analyser (I-Stat, I-STAT Corporation, East Windsor, NJ) or haemocytometer. Other blood traits (e.g. sodium, potassium, chloride, pH venous, partial carbon dioxide, blood urea nitrogen and total and serum protein) were evaluated at a local human hospital (see Chapter 4). Remaining blood products were stored on site at the respective locations.

(6) A nutrition survey form was distributed at each location and then evaluated (see Chapter 6). The focus here was on:

a. identifying and quantifying nutrient content of all offered food items;

b. evaluating offered diets, especially mass;

c. generating data on food intake, especially mass consumed;

d. measuring feeding frequency of various diet components;

e. characterising food distribution within enclosures and sites of food presentation;

f. determining seasonal variations in provided diets;

g. recording information on food storage and preparation facilities.

Diets were evaluated using Zoo Diet Analysis Program software (Allen & Baer Associates, Silver Spring, MD). Faecal volume and consistency traits also were described, including typical faecal output over 24 hours and mucous stool frequency, with and without blood.

(7) Historical and behavioural data were collected, specifically origin, date of birth, health and reproductive history, past reproductive success and opportunities to breed. Information included evaluations provided by 38 keepers on more than 20 behavioural characteristics (e.g. calmness, shyness or aggressiveness), which then were statistically analysed (see Chapter 5).

At each location, all results were discussed between the CBSG and various Chinese teams to reach a consensus on findings and interpretation. All data and resulting management recommendations were provided to all participants in electronic and hard-copy format.

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