A technique for identifying the sex of a bird embryo in the egg was requested by managers of endangered species conservation programs, to choose the sex of offspring to meet demographic and genetic goals, and to place single sex eggs in wild bird nests as a tool for reintroduction programs. A study was initiated at the International Crane Foundation (ICF) to determine if small blood samples could be safely collected from developing crane eggs and then used to determine the embryo’s sex using a DNA probe/restriction fragment-length polymorphism technique.
Thirty-five fertile eggs from seven crane species (brolga [Grus rubicundus], demoiselle [Anthropoides virgo], hooded [Grus monachus], red-crowned [Grus japonensis], sandhill [Grus canadensis], Siberian [Grus leucogeranus], white-naped [Grus vipio]) that were identified as surplus to the Species Survival Plan’s (SSP) breeding and reintroduction program goals were used in the study. The 12 control and 23 “sexed” eggs were artificially incubated using standard ICF protocols.1 Embryonic development was monitored by weighing and candling. Between the 16th and 25th day of incubation, a 1-cm circular area of the shell above the equator of the egg was removed using a belt sander. A drop of sterile mineral oil was applied to the exposed shell membrane and, with candler transillumination, underlying chorioallantoic vessels were visualized. A vessel was cannulated using a 27–30-ga needle and 1-cc syringe, and 0.01–0.05 cc of blood was collected and then transferred to a tube containing 2 cc of 70% ethanol. The egg shell hole was covered with a piece of sterile adhesive semi-occlusive wound/IV dressing (Tegaderm, 3M Animal Care Products, St. Paul, MN) that extended 2–4 mm beyond the hole. The eggs were then incubated until they hatched or until the normal incubation period was exceeded. Chicks were euthanatized on the day of hatch. All the chicks and unhatched eggs were necropsied, and gonadal sex was determined grossly and histopathologically. Fixed blood samples from four species were submitted to AviGene Services, Inc., Madison, WI, where the DNA was isolated and subjected to RFLP analysis using a commercial turkey DNA probe that marks both Z and W chromosomes.
There were no differences in the egg weight loss rates between control and “sexed” eggs. Nine of 12 (75%) of control eggs successfully hatched; 15 of 23 (65%) of “sexed” eggs hatched. However, hatching success of “sexed” eggs improved markedly during the second half of the breeding/research season (83% versus 45% for the first half), as the blood collection technique was refined. Of the eight “sexed” embryos that died, six died within a few days of the blood collection procedure, and the remaining two died close to the time of hatch. Infection was a possible factor in only one of these eggs; a coagulase negative Staphylococcus sp. was cultured. Blood was successfully collected from 20 of the 23 eggs (87%). There was 100% (11/11) concurrence between the DNA probe and gonadal sex determination results.
The sex of avian embryos can be determined in the egg by collecting blood for DNA probe analysis, with a good chance for continued normal development and successful hatching of the egg. Factors that seem to increase the success of this technique include using it on species whose eggs are thin-shelled and lightly pigmented so chorioallantoic vessels can be identified by candling, collecting the blood during the second half of the incubation period, and careful management of the eggs after the shell membrane has been exposed, especially during hatching. This technique for in ovo avian sex determination has the potential to be a useful tool for the management of commercial, zoo, and endangered species breeding flocks.
1. Gabel R, Mahan T. Incubation and hatching. In: Ellis DH, Gee GF, Mirande CM, eds. Cranes: Their Biology, Husbandry, and Conservation. Blaine, WA: Hancock House Publishers; 1996:59–76.