Progeny from cryopreserved Golden eagle spermatozoa
A Knowles-Brown 1 and GJ Wishart 2
1 Crookedstane, Elvanfoot, South Lanarkshire
2 University of Abertay Dundee, Bell Street , Dundee DD1 1HG
Successful production of progeny of non-domestic avian species from cryopreserved spermatozoa has been limited to a few species (see Wishart, 2000), based on interest in their conservation, the availability of captive birds and the expertise required for semen collection and artificial insemination (AI). Falconers have developed techniques for semen collection and AI in several raptors and progeny have been produced from cryopreserved semen of American kestrels (Brock and Bird, 1991) and peregrine falcons (Parkes et al, 1996; Samour, 1987; J. Blanco, personal communications).
One of the problems of cryopreservation of semen from non-domestic birds has been the need to use sophisticated laboratory equipment under non-laboratory conditions , so we have sought to utilise ‘field methods', the simplest of which involves rapidly freezing sperm in pellets formed by dropping diluted semen directly into liquid nitrogen (Tselutin et al., 1995). Whilst this has been succesful for some species (Hartley et al., 1999), we had been unable to obtain progeny from raptors with this fast-cooling method. Recently, it was shown that spermatozoa from eagles and falcons are particularly resistant to the hyperosmotic conditions that occur during cryopreservation (Blanco et al., 2000). We therefore investigated cryopreservation of golden eagle spermatozoa at slow cooling rates, using a simple freezing apparatus - ‘Mr Frosty' (Nalgene) – placed in a domestic freezer.
The sperm donor was a 5-year-old golden eagle. Approximately 40 µl s emen was collected, twice-daily, by voluntary deposition onto a polythene-glove and then transferred to a capillary tube. After cooling to 5 ° C in a refrigerator, the semen was diluted with the same volume of a glutamate-based solution (Lake and Stewart, 1978) in a small cryovial (Nalgene). Samples were equilibrated at 5 ° C for 15 min and then mixed with the same volume of diluent, containing 18% dimethylacetamide. These were inserted into a ‘Mr Frosty', also equilibrated at 5 ° C, and then placed inside a domestic freezer for 1.5-2h - after which the cryovials were removed from ‘Mr Frosty' and plunged into liquid nitrogen. Alternatively, samples were dropped into liquid nitrogen to form pellets (Tselutin et al., 1995), which were then placed into cryovials. All samples were stored in liquid nitrogen and were thawed by dipping cryovials into a bath of warm (~37 ° C) water. The proportion of motile spermatozoa was estimated by manual assessment of individual spermatozoa in replayed video-recordings and the proportion of live spermatozoa were estimated after staining with nigrosin/eosin (Hartley et al., 1999). Samples frozen in Mr Frosty were inseminated into a 8-year-old Steppe eagle by transferring the thawed semen into a 100 µl capillary tube and blowing the contents directly into the cloaca, after stimulating the eagle for copulation. Inseminations were performed twice daily for 5 days before lay and 3 times daily for 2 days after the laying of each egg - which occurred at 3-day intervals. Eggs were artificially incubated in a forced air incubator at 37 ° C, with a calculated average weight loss of 13%.
The cooling rate for a sample contained within Mr Frosty is shown in Figure 1. This was logarithmic, with rates ranging from 0.3 ° C/min between 5 and -5 ° C to 0.08 ° C/min between 12 and 14 ° C. Nucleation occurred at –9.6 ± 2.5 ° C, 70 ± 11min after placing in freezer (n=4) . Sample volume and sperm concentration were typically 24 ± 5 µl and 183 ± 112 million sperm/ml, respectively (n=4). Fresh samples showed 39 ± 4% motile spermatozoa and 87 ± 3% ‘live' spermatozoa (n=3), whilst in cryopreserved samples, these parameters were reduced to 11 ± 8% and 35 ± 14%, respectively (n=4). Samples frozen as pellets had 3.1% motile and 4,1% live spermatozoa (mean of two estimations). A total of 5 eggs were laid by the female eagle during a period of 14 days. All were fertile. Four eggs hatched at an average weight of 68gms after 39 days incubation; one embryo died at 36 days due to malposition at the time of pipping.
This is the first published account of progeny from cryopreserved eagle spermatozoa.
We thank Maurice Lindsay and Don Downie for skilled assistance and are very grateful to Nalge ( Europe ) Ltd for the gift of ‘Mr Frosty' and to DCP Microdevelopments Ltd for fabricating the temperature sensors.
Blanco, J.M., Gee, G., Wildt, D.E. and Donoghue, A.M. 2000 Species variation in osmotic, cryoprotectant and cooling rate tolerance in poultry, eagle and peregrine falcon semen. Biology of Reproduction 63: 1164-1171.
Samour, J. 1987. Studies of male reproductive function, semen preservation and artificial
insemination of the budgerigar (melopsittacus undulatus) with some observations on birds of prey. PhD Thesis, University of London .
For all other references, see:
Wishart, G.J. 2000. The cryopreservation of germplasm in domestic and non-domestic birds. Pages 179-200 in Cryobanking the Genetic Resource. Eds P.F. Watson and W.V. Holt. Taylor and Francis, London and New York .