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Methods/Procedures
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METHODS/PROCEDURES

In the last remaining strongholds where "custom" is still practiced, Naravé pig owners vehemently protect their most prized possessions. Many could not grasp the scientific aspect of my requests, while others, in a very direct manner, refused to allow me to "wound" their pigs in order to "take away" blood. It was at this point that Vira Joseph, the highly respected chief of Avunatari Village on Malo Island, came to my aid. Part contemporary/part "custom" man, Chief Joseph explained in words, as I never could, the importance of my study in regards to the preservation of their rapidly disintegrating "customs". He explained to the men of his village that by my studying and then recording the history of the Naravé pig, future generations might know the importance these animals played in the lives of their forefathers. With all that I, thankfully, was granted the opportunity to draw blood and obtain my "hands-on" documentation of ten pigs, seven of which were intersexual.

Examining internal anatomies was another problem altogether. Some owners would part with the young (without tusks) or inferior Naravé pigs with broken or missing tusks. The price range was from 8,000 to 80,000 vatu ($60 to $600 U.S.). With all but perhaps fetal pigs beyond the boundaries of this project's budget allotment, I purchased one young pig for dissection. Additional dissections arose from those animals ritually killed at upcoming Nimangki ceremonies rather than sacrifice any more from this unique gene pool for this study. I was not fortunate enough to be invited to the ceremony itself but was allowed to perform 6 gross anatomical examinations on the deceased pigs.

The general body form, coat colorations, and natural histories from each morphotype were determined through field examinations, recent and past literature, personal interviews with Ni-Vanuatu villagers and actual specimens examined. All ages designated for each pig described in this study are approximate and originated from the owners account for each individual pig. The field weights were estimated by the author.

Personal documentation from actual sightings was my primary form of verification whenever possible. Distributional data were acquired through personal interviews, surveys, and service messages. Personal visits enabled verification of sometimes conflicting accounts of the existence of these animals and also allowed me to conduct field morphological and behavioral studies. One particular procedure available to me was blood examinations (complete blood counts at the local clinic in Luganville and hormonal assays from serum sent to New Zealand with the aid of the Vanuatu Dept. of Agriculture). Three normal pigs (two normal males and one female) were also selected, in addition to the seven intersexes, to serve as controls. Each individual animal was hand-caught and manually restrained. The eyes were masked to disorient the pig, providing an opportunity to fasten a protective cushion around the valuable curved tusks before any attempt was made to throw the animal to the ground. The animal was restrained in lateral recumbency on its right or left side. With the legs and head restrained, a 21 ml. blood sample was drawn from the radial artery of the down-side foreleg. The external genitalia were examined and photographed. A skin biopsy (3 cm. diam. X 2 cm. thick, preserved in EDTA/TRIS) was removed from the up-side ear with a leather punch and a commercial premedicated, numbered, ear tag was inserted in the void left by the skin punch. (Owner's cooperation by their eagerness agreed to receive the "earring" as a gift). The pig was then released with the total procedure requiring 10 minutes per pig.

Complete Blood Counts(CBC's) and hormonal assay interpretations were run on seven intersexual pigs, (D, E, F, J, K, L, and M) table(1)(2), two normal males, C, N) and one pregnant female (B). Their CBC's were compared against the normal blood values for swine used at the Western College of Veterinary Medicine, University of Saskatchewan, Canada. These blood values have been compiled from the clinical laboratory, Department of Pathology; from O W Schalm(18) et al (1975) and Kaneko(19) (1980). Normal values of laboratory data are offered as a guide and it must be noted that values may vary depending on the individual laboratory as well as the sex, age, and geographical habitat of the target research animal.

The quality controls for testosterone were at 2 ng-assayed 2.4 ng (120%), 5.00 ng-assay 4.49 ng (90%), 10 ng-assayed 9.54 (95%). The quality controls for estradiol were at 20 pg-assayed 19.37 pg (97%), 50 pg-assayed 53.22 pg (106%) and 100 pg-assayed 146.37 pg (146%).

For testosterone 200 ul of sample was extracted with diethyl ether for assay.

For estradiol 3 ml of sample was assayed using the method described by Webb et al(35). The intra-assay coefficients of variation were 8% for testosterone and 9% for estradiol.

The antiserum for DHT was the same as that used for testerone. It was used at a dilution of 1:300. Major cross-reacting steroids were 5alpha dihydrotestosterone 75%, 5beta dihydrotestosterone 75%, 5alpha androstan-3alpha, 17beta-diol 37.5%, 5beta androstan-3alpha, 17beta-diol 16.5%, androstenedione 0.1%. The antiserum used for the estradiol assays was raised in an ovariectomized ewe against oestradiol-6 (O-carboxymethyl) oximine bovine serum albumin conjugate used at a dilution of 1:16000. The major cross-reactions of other steroids were estrone 7.3%, estriol 1.4%, estradiol 17alpha 1.4% and androstenedione 0.015%.