Karyoty,pe and Identification of Sex Intwo Endangered Crane Species

A laboratory procedure for sex identification of monomorphic birds was developed using modern cytological methods of detecting chromosome abnormalities in human amniotic fluid samples. A pin feather is taken from a pre-fledging bird for tissue culture and karyotype analysis. Through this method, the sex was identified and the karyotype described of the whooping crane (Grus americana) and the Mississippi sandhill crane (G. canadensis pulla). Giemsa-stained karyotypes of these species showed an identical chromosome constitution with 2n = 78 ± 2. However, differences in the amount of centromeric heterochromatin were observed in the Mississippi sandhill crane when compared to the whooping crane Cbanded karyotype. Analysis of sex chromosomes has been used to determine the sex of many sexually monomorphic avian species (Hungerford et. 1966; Jovanovic & Atkins 1969; Mengden & Stock 1976; Rasch & Kurtin 1986; Prus & Schmutz 1987). The karyotype also provides information of taxonomic utility (Ray-Chandhuri et a1. 1969; Takagi & Sasaki 1974; DeBoer 1976; Shields 1982). Karyotype analysis as a sexing method offers a safe alternative to surgical sexing. Feather pulp has been used as a source of mitotic figures suitable for karyotype analysis in a variety of avian species (see Rasch & Kurtin 1986). The techniques for cell culture of feather pulp were first developed by Mengden & Stock (1976) who also suggested using the karyotype to sex avian species. Although tissue culture techniques were taught to others in the avicultural community (Training Seminar on Sexing and Artificial Insemination of Birds, Houston Zoological Society, 18-20 May 1977) its use was limited by frequent culture failures and the difficulty in finding metaphase spreads adequate for analysis. Although blood culture provides another possible source of mitotic figures, culture of avian blood was not as successful as in mammalian species until Biederman & Lin (1982) demonstrated an effective technique for the separation and culture of lymphocytes from whole blood. The technique has been successfully used to sex the California condor and the whooping crane (Biederman et a1. 1982). An evaluation of the use of the blood culture method of sexing birds is provided by Prus & 219 Proc. 1988 N.Am. Crane Workshop Schmu tz (1987). The present study was undertaken in order to identify the sex of whooping cranes and Mississippi sandhill cranes at the Patuxent Wildlife Research Center, Maryland. The crane samples provided an opportunity to test the method of genetic sexing as applied to an endangered species. The method combines the feather pulp technique of obtaining tissue for cell culture and the "in-situ" technique of chronlosome preparation as developed for psittacine species at Vivigen, Inc. (Seluja & Goodpasture unpubl. data). MATERIALS AND METHODS Tissue for cell culture and mitotic cell analysis was obtained from pin feathers taken from immature birds that had been hatched and reared at Patuxent. Pin feathers were plucked from birds, surface sterilized with alcohol, placed into tubes of transport media and express mailed to our laboratory. The transport media contained antibiotics (Gibeo 600-5140 and 600-5340, 1 ug/ml each solution, and fetal bovine serum (Gibeo 200-6140,5 ug/ ml) in Hank's balanced salt solution (Gibco 104020AJ). AlL samples were in transport media for approximately 28 hours prior to cell culture due to Federal Express transit time. Pin feather tissue was extracted using a technique similar to that previously described by Van Tuinen & Valentine (1982). Cells were grown on