CHARACTERIZATION OF NATURAL EJACULATES AND SPERM CRYOPRESERVATION IN A GOLDEN EAGLE ( AQUILA CHRYSAETUS )by Silvia Villaverde-Morcillo, Rubén García-Sánchez, Cristina Castaño, Eduar Rodríguez, Fernando Gonzalez, Milagros Esteso, Julián Santiago-Moreno

Journal of Zoo and Wildlife Medicine


Veterinary (all) / Animal Science and Zoology


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Author(s): Silvia Villaverde-Morcillo, D.V.M., M.S., Rubén García-Sánchez,

R.V.T., Cristina Castaño, D.V.M., Eduar Rodríguez, Zootech, Fernando

Gonzalez, D.V.M., Milagros Esteso, D.V.M., Ph.D., and Julián Santiago-Moreno,

D.V.M., Ph.D.

Source: Journal of Zoo and Wildlife Medicine, 46(2):335-338.

Published By: American Association of Zoo Veterinarians



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Journal of Zoo and Wildlife Medicine 46(2): 335–338, 2015

Copyright 2015 by American Association of Zoo Veterinarians




Silvia Villaverde-Morcillo, D.V.M., M.S., Rube´n Garcı´a-Sa´nchez, R.V.T., Cristina Castan˜o, D.V.M.,

Eduar Rodrı´guez, Zootech, Fernando Gonzalez, D.V.M., Milagros Esteso, D.V.M., Ph.D., and Julia´n

Santiago-Moreno, D.V.M., Ph.D.

Abstract: This paper describes the sperm characteristics and response to cooling and freezing of naturally ejaculated semen from a captive, adult golden eagle (Aquila chrysaetus) trained to allow sperm recovery via cooperative copulation. A basic spermiogram was prepared, and sperm motility and morphometric variables recorded using a computer-aided system. For sperm storage, the effects of a polyvinylpyrrolidone-based extender were evaluated at 58C. The same extender was also used in freezing procedures in which glycerol (11%) and dimethylacetamide (6%) were compared as cryoprotectants. The extender preserved sperm viability over storage periods of up to 6 days. Although sperm motility and percentage live sperm values were poorer for frozen–thawed (5.8–14.6% and 44–42%, respectively) than for fresh samples (46.7 and 74.6%, respectively), no differences were seen between the effects of the two cryoprotectants. These results could be of use when attempting to store the sperm of golden eagles and other raptors.

Keywords: Aquila chrysaetus, cryopreservation, ejaculate, golden eagle, semen, spermatozoa.


The golden eagle (Aquila chrysaetus) is found in

Eurasia, North America, and some parts of Africa and overall is catalogued as of least concern by the

IUCN. Artificial breeding techniques have been used with rather variable success in some wild birds.3–6 Further development of these techniques is, however, required; knowledge of the biologic characteristics and physiology of wild bird sperm is still lacking, and sperm cryopreservation protocols need to be optimized. Studying the sperm variables of wild avian species is the best way to develop appropriate cryopreservation protocols.

To the best of our knowledge, the literature contains no reports on the baseline characteristics of golden eagle semen.

Over a period of 2 yr, an adult (more than 30 yr of age), male golden eagle kept at the GREFA

Wildlife Rehabilitation Centre in Madrid, Spain (recovered from injuries but unable to return to the wild) was trained to allow cooperative semen collection. Because the bird was already used to human presence, although not imprinted, successful matings were obtained after 4 mo of training. The bird was housed on its own in an outdoor pen and was fed 1-day-old chicks, quails, and rabbits.

Semen collection consisted of voluntary false copulation (over the bird’s trainer’s back) from the onset of the breeding season until regular azoospermic samples were obtained. The procedure was undertaken in the eagle’s normal pen and always by the same trainer.5,11 Ejaculated semen was collected in a gloved hand, placed in a 1.5-ml microcentrifuge tube (Eppendorft, Eppendorf Ibe´rica SLU, San Sebastia´n de los Reyes,

Madrid 28703, Spain) and diluted 1 : 1 (v/v) with a tempered (388C) medium composed of sodium glutamate (1.92 g), glucose (0.8 g), magnesium acetate 4H2O (0.08 g), potassium acetate (0.5 g), and polyvinylpyrrolidone (PVP; Mr 10,000; 0.3 g) dissolved in 100 ml H2O (final osmolarity 343 mOsm/kg) (Lake and Ravie 1984). The extender was prepared in our laboratory using reagentgrade chemicals purchased from Sigma Chemical

Co. (St. Louis, Missouri 63103, USA). The diluted semen was then immediately cooled to 58C, introducing the vial directly into the refrigerator, and the evaluation of sperm variables performed at 0, 24, and 72 hr (range 3–6 days) following incubation at 388C for 10 min at each sampling. Sperm motility and concentration were determined using an SCAt (Microptic SL, BarceFrom the Veterinary Clinical and Research Services of the Fieb Foundation, Tres Cantos 28760, Madrid, Spain (Villaverde-Morcillo); the Department of Animal Reproduction of the INIA, 24040 Madrid, Spain (Castan˜o,

Rodrı´guez, Esteso, Santiago-Moreno); and the Veterinary

Service of the Wildlife Rehabilitation Hospital GREFA,

Majadahonda, 28220 Madrid (Garcı´a-Sa´nchez, Gonzalez).

Correspondence should be directed to Dr. SantiagoMoreno ( 335 lona 08029, Spain) computer-aided image analysis system coupled to a phase contrast microscope, with settings adjusted to detect avian spermatozoa (A2 ¼ 5 lm2: VCL (10–100 lm/sg). The percentages of motile spermatozoa, and spermatozoa showing progressive motility, were recorded. We also analyzed sperm movement characteristics: curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF).