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Full text of "Cryopreservation of peregrine falcon semen and post-thaw dialysis to remove glycerol"

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John E. Parks, Willard R. Heck and Victor Hardaswick 

ABSTRACT — Peregrine Falcon (Falcoperegrinus) semen was found to have a mean ejaculate volume, sperm concentra¬ 
tion and initial sperm motility of 95 /xl, 47 X 10 8 sperm/ml and 70%, respectively. When frozen in a medium containing 
0.3,0.9 or 1.48 M glycerol, post-thaw sperm motility was 29,47 and 54%. Because of the contraceptive effect of glycerol, a 
dialysis procedure was developed to remove the cryoprotectant from post-thaw semen. Percent motility during post-thaw 
incubation was greater in samples from which glycerol had been removed by dialysis than in controls (P< 0.05). Of 6 eggs 
from a single $ American Kestrel (Falco sparverius) obtained after insemination with frozen-thawed, dialyzed peregrine 
semen, 2 were fertile and survived to pip. One interspecific hybrid was hatched and raised successfully. 

Captive breeding has been used effectively for 
the conservation of birds of prey for many years. 
Artificial insemination has been a useful technique 
in captive breeding programs since the early 1970’s 
(Weaver 1983). However, there are many situations 
in which the efficiency of a breeding program may 
be reduced because semen is not available at the 
time or place where it is needed. The ability to 
freeze raptor semen would facilitate captive 
breeding under these and other circumstances. 
The ability to freeze semen also would permit 
banking germ plasm from rare or endangered 

At present, a limiting factor in the use of frozen 
semen in domestic avian species is the inhibition of 
fertility by the cryoprotectant in the medium 
(Brown and Graham 1971; Lake and Stewart 1978; 
Sexton 1979; Lake et al. 1980; Lake et al. 1981; 
Graham et al. 1982). In order to overcome this 
problem, cryoprotectants such as glycerol or di- 
methylsulfoxide (DMSO) must be removed post¬ 
thaw (Lake and Stewart 1978; Lake et al. 1981; 
Graham et al. 1982) or new, less problematic cryo¬ 
protectants must be identified. 

The purpose of this study was to develop proce¬ 
dures for processing and freezing raptor semen, 
using the Peregrine Falcon (Falco peregrinus) as a 
semen source; and to develop a procedure for re¬ 
moving the cryoprotectant glycerol from thawed 
semen without further loss of sperm viability. 

Materials and Methods 

Semen Collection and Handling. — Semen from 3 adult male 
Peregrine Falcons was collected up to 2 times/d (Boyd and 
Schwartz 1983) over a period of approximately 2 m. On each 
occasion, ejaculates from 1 - 3 J d* were pooled. Semen was diluted 
1:3 (v/v) at 20°C in 12 X 55 mm vials containing Lake’s freezing 
diluent (Lake and Stewart 1978), placed directly into an ice water 
bath (0 - 2°C) and transported to the laboratory (see Table 1). All 
remaining steps up to freezing and during the thawing process 
were carried out at 4°C. 

Semen Evaluation. — Sperm motility was assessed microscopi¬ 
cally by estimating the percentage of sperm moving progressively 
forward (percent motility). Unfixed smears prepared from sam¬ 
ples diluted in freezing diluent were placed on a slide warmer at 
37°C for 30 sec immediately prior to evaluation. Percent motility 
was estimated in several microscopic fields to the nearest 5% using 
a phase contrast microscope at a total magnification of 400x. 

Sperm concentration of the semen diluted 1:3 was determined 
with a hemacytometer after an additional dilution of 1:1 (v/v) in 
fixative (4% glutaraldehyde). Duplicate counts of each prepara¬ 
tion were averaged for use in calculating sperm concentration of 
the original ejaculate. 

Semen Freezing and Storage. — Aliquots of approximately 50 /al 
of cooled, diluted semen were placed into 0.25 ml French straws 
(IMV) for freezing. Diluent was aspirated into the straw ahead of 
the semen with a small air space separating the 2 liquids. Filling the 
straws in this way served to seal the polyvinylchloride (PVC) plug 
at the end of the straw without loss of semen and also prevented 
straws from floating when placed in liquid nitrogen. Straws were 
then sealed and loaded into a Planer R204 freezer (2°C). Semen 
was frozen in nitrogen vapor at 6°C/min to -180°C and then 
plunged into liquid nitrogen (Brock et al, 1984). One straw from 
each freezing procedure was then thawed in water (4°C) for evalu¬ 
ation of percent motility. Straws were wiped dry and the semen- 
containing portion was emptied into precooled tubes (6 X 50 mm). 
Remaining straws were stored for 1-2 months prior to thawing. 

Initially, semen was frozen in diluent containing 0.3, 0,9 or 1.48 
M glycerol. Percent motility was estimated on aliquots prior to 
freezing and immediately post-thaw. Based on these initial trials 
using different levels of glycerol in the freezing medium (Table 2), 
1.48 M glycerol was selected for routine use in subsequent ex¬ 

Method for the Dialysis of Diluent and Diluted Semen. — 

Dialysis to remove glycerol from the freezing medium and thawed 
semen was carried out using semi-micro dialysis tubing (2.55 mm 
diameter, molecular weight cutoff of 12,000 - 14,000, Spectra/ 
Por). Tubing was washed thoroughly in twice distilled water and 
stored wet at 4°C prior to use. All subsequent steps in the dialysis 
procedures were carried out at 4°C. Tubing was tied and cut into 
lengths of 8-10 mm from the tied end, filled with the freezing 
diluent (1.48 M glycerol) and equilibrated for 10-20 min in the 
same diluent. Diluent was then completely removed from the 
tubing and 50 fx 1 aliquots of fresh freezing diluent or of thawed 
semen were pipetted into the tubing using a fire-polished 200 /al 
capillary pipet and a capillary suction apparatus (Clay-Adams). 
The tubing was then closed with a Spectra/Por closure and 
dialyzed with stirring against 500 volumes of Lake’s thawing 
medium (Lake and Stewart 1978). 


Raptor Research 20 (1): 15-20 

Spring 1986 

Freezing Falcon Semen 


Table 1. Characteristics and pre-freezing treatment of Peregrine Falcon semen. 

Volume for 
Collection Period 3 
0 * 1 ) 





(x 10 6 /ml) 

Interval Between 
Collection and Cooling 0 



All b 
































a Semen was collected between approximately 0830 - 0930 H and 1630 - 1730 H. 

k Volumes for pooled ejaculates included 2 samples (partial or whole ejaculates) from 2 individuals and 1 sample from 3 individuals. 

Volumes are included for individual birds from 3 collection periods in which ejaculates were pooled prior to freezing. 
c Intervals were timed from collection of the last ejaculate for pooled samples. Freezing was begun within approximately 15 to 30 min 
after initial dilution and cooling. 

Estimation of the Efficiency of Glycerol Removal by Dialysis. — 

Removal of glycerol from the freezing medium was measured by 
supplementing the medium with [2- 3 H]-glycerol (New England 
Nuclear, 200 /zCi//zmol) at a level of 2 x 10 5 dpm/50 /xl. Appear¬ 
ance of radioactive glycerol in the dialysate relative to the initial 
amount placed in the dialysis tubing was used to calculate the rate 
and extent of glycerol removal. 

Preliminary trials (n = 2) indicated that > 99% of the glycerol in 
the freezing medium was removed after 30 min of dialysis. To 
establish the time-course relationship of glycerol removal, dialysis 
of freezing medium containing 1.48 M glycerol was carried out for 
2 h against thawing medium containing no glycerol (n = 5). Input 
samples; 0.2 ml aliquots of the dialysate taken at 0, 0.25, 0.5, 1.0, 
2.0, 5.0,15,30,60 and 120 min of dialysis; and residual material in 
the dialysis tubing were analyzed for 3 H content (glycerol) by 
liquid scintillation spectrometry. 

Evaluation of the Effect of Dialysis on Sperm Motility. — 

Because glycerol was removed so rapidly by the dialysis proce¬ 
dure, damage to sperm due to osmotic effects was considered a 
potential problem. Therefore, dialysis conditions were established 
to remove the glycerol more gradually. Material to be dialyzed was 
transferred at 15 min intervals to thawing solutions containing 
glycerol decreasing in equimolar increments (1.1,0.74 and 0.37M 
and no glycerol). It was assumed that the rate of glycerol equilib¬ 
ration (and thus removal of glycerol from the thawed semen) in 
these steps was approximately equivalent to that observed in the 
one-step procedure, and the extent of total glycerol removal was 
calculated on this basis. An experiment was designed to assess the 
effect of the step-wise dialysis procedure on falcon sperm motility 
during post-thaw, post-dialysis incubation. A split-ejaculate 
technique was used in which all treatments within each experi¬ 
ment were imposed on aliquots of the same ejaculate. At the 

Table 2. The effect of glycerol level on pre-freeze and post-thaw motility of falcon sperm. Values are percentages. 

Percent Motility 



Glycerol Level 

0.3 M 

0.9 M 

1.48 M 

0.3 M 

0.9 M 

1.48 M 






























Parks, et al. 

Vol. 20, No. 1 

Table 3. Post-thaw motility of falcon sperm after dialysis to remove glycerol (n = 4). a Values are percentages. 


Hours of Incubation After Dialysis 







1.48 M Glycerol 







Four-step Procedure 

(1.1 M to Glycerol-Free) 







a Post-thaw, pre-dialysis motility for these samples was 55±7%. 
* P < 0.05 

completion of dialysis, semen was emptied into tubes (6 X 50 mm) 
at 4°C. Percent sperm motility was determined immediately and 
after 30, 60, 90 and 240 min of post-dialysis incubation (38°). 

Artificial Insemination. — Female peregrines were not availa¬ 
ble for testing the fertility of post-thaw, dialyzed semen. One 
unpaired $ American Kestrel (Falco sparverius) was inseminated 
with approximately 40 to 50 /i\ of thawed semen, dialyzed by the 
step-wise procedure. Six single inseminations were made within 4 
h after oviposition and the first egg laid after each insemination 
was artifically incubated. Thawed samples were maintained at 4°C 
until the oviduct was everted for insemination. The semen was 
then transferred to an insemination syringe and deposited into the 
oviduct (Weaver 1983). Total time between thawing and insemi¬ 
nation including dialysis was approximately 90 min. 

Statistical Analysis. — Means and standard deviations were 
calculated for semen characteristics and for motility estimates on 
semen diluted and frozen in different levels of glycerol. The 
effects of dialysis on post-thaw motility were analyzed by analysis 
of variance after arcsin transformation of the percentage data. 


Semen characteristics and information related to 
initial handling of semen are presented in Table 1. 
Semen was not scored on appearance, but only a 
low to moderate level of contamination by extrane¬ 
ous cell types and other debris was observed in the 
ejaculates used for freezing. Initial experiments in 
which glycerol was the only variable tested (Table 2) 
indicated that 1.48 M glycerol provided greater 
protection during freezing than 0.9 M or 0.3 M 
glycerol based on post-thaw sperm motility. How¬ 
ever, glycerol levels were tested on separate semen 
collections so comparisons were not made on a 
statistical basis. The response to freezing, when us¬ 
ing 0.3 M glycerol, was consistently poor; but the 
difference in post-thaw motility between 0.9 M and 
1.48 M diluents was small, especially when ex¬ 

pressed as the difference between pre-freeze and 
post-thaw motility (13 vs 16%). 

Rate and extent of glycerol removal during a 
single step dialysis of samples are presented graphi¬ 
cally in Fig. 1. Approximately 90% of the glycerol 
was removed by 15 min of dialysis, and after 30 min 
glycerol had been completely removed. Based on 
this rate of equilibration, a sample frozen in 1.48 M 
glycerol and dialyzed by the step-wise procedure 
was considered to contain less than 30 mM glycerol 

containing 1.48 M glycerol and the appearance 
of radioactivity in the dialysate was measured. 
Bars represent standard deviation for each 
time point (n = 5). 

Spring 1986 

Freezing Falcon Semen 


A comparison of post-thaw motility for sperm 
dialyzed by the step-wise procedure or directly 
against the freezing medium (1.48 M glycerol) is 
presented in Table 3. It is apparent that sperm 
survived the dialysis procedure with fair to good 
motility. During post-dialysis incubation motility 
declined with both treatments (P< 0.01). Although 
no significant time X treatment interaction was 
found, percent motility remained sufficiently 
higher after glycerol removal to demonstrate an 
advantage over the control treatment (P < 0.05). A 
consistent difference in the motility pattern was also 
observed between the dialysis treatments. Sperm 
from which glycerol had been removed exhibited a 
greater velocity, and motility was more progressive 
with less amplitude in the flagellar motion than with 
sperm in glycerol. This difference was not qualified 
but was readily apparent to other observers. Sperm 
in suspensions after glycerol removal also seemed 
more resistant to dessication on slides prepared for 
microscopic examination than those remaining in 
high glycerol medium, based on maintenance of 

Of 6 American Kestrel eggs potentially fertilized 
by frozen, thawed and dialyzed Peregrine Falcon 
semen, 2 eggs were fertile and developed to pip. 
One of these young died at pip while the other 
interspecific hybrid hatched and was raised success¬ 


At present, there is very little detailed informa¬ 
tion on semen characteristics of raptorial species. 
This study provides such information on ejaculate 
volume, sperm concentration and percent motile 
sperm for semen from the Peregrine Falcon. Val¬ 
ues for these characteristics have also been reported 
for the American Kestrel (Bird and Lague 1977). A 
comparison of the semen characteristics for these 2 
species indicates an 8-fold greater ejaculate volume 
for the peregrine which approximates the differ¬ 
ence in body weight between it and the kestrel. 
Sperm concentration is over 30% greater for the 
peregrine. The much greater total sperm/ejaculate 
for the peregrine may be a necessary adaptation for 
ensuring adequate sperm numbers at the site of 
fertilization in this larger species. Percent motile 
sperm appears to be slightly higher for the pere¬ 
grine than the kestrel, but this may be due to dif¬ 
ferences in conditions under which sperm were 
examined. This type of information on semen 

parameters is necessary for making the most effec¬ 
tive use of artificial insemination in the species of 
interest, and for effective processing of semen for 
cryopreservation. A knowledge of semen charac¬ 
teristics may also serve as a basis for comparison 
when examining the effects of environment or en¬ 
vironmental contamination on reproduction (Bird 
and Lague 1977). 

In the present study, we found that peregrine 
semen freezes well in Lake’s diluent. It appears that 
a broad range in glycerol level might be acceptable, 
but more definitive work is required to establish the 
optimum glycerol concentration. Brock et al. (1984) 
reported excellent post-thaw motility for kestrel 
semen frozen in Lake’s diluent, but fertility of the 
semen was < 5%. The requirement to remove 
glycerol and other cryoprotectants from post-thaw 
semen in order to obtain acceptable fertility has 
been established in domestic avian species (Brown 
and Graham 1971; Lake and Stewart op . cit .; Lake et 
al. 1980; Lake et al. 1981; Graham et al. op. cit.). 
This also may be true for falcon semen. Removal of 
glycerol from post-thaw cock semen by dilution and 
centrifugation greatly improves fertility (Lake and 
Stewart, op. cit), but this approach is not practical 
when working with microliter quantities of falcon 
semen. Graham et al. (op.cit) reported that the level 
of cryoprotectant (DMSO and ethylene glycol used 
in combination) necessary to maintain vigorous 
post-thaw motility of turkey semen depressed fer¬ 
tility. Use of dialysis to remove the cryoprotectant 
significantly improved fertility; although dialysis 
time, dialysate composition and pH, and se- 
men-to-dialysate ratio all influenced the level of 
fertility observed. Dialysis can be adapted for use 
with the small semen volumes associated with rap¬ 
torial species and is a milder approach for removing 

Lake et al. (1980) demonstrated that in order to 
minimize its inhibitory effect on fertility in the 
Domestic Chicken (Gallus spp.), glycerol must be 
reduced to a level below 1% (0.11 M) in diluted 
semen. It is apparent from the present study that 
under the appropriate conditions glycerol can be 
reduced below the level of 1% within 30 min by 
dialysis. By controlling the sample volume/dialysate 
ratio or by adjusting the level of glycerol in the 
dialysate, rate of glycerol removal can be regulated 
to minimize the post-thaw to insemination interval 
necessary to remove cryoprotectant while main¬ 
taining optimal sperm viability. In this study, man- 


Parks et al. 

Vol. 20, No. 1 

ipulations required to transfer and dialyze the mi¬ 
croliter volumes of frozen semen were carried out 
with only a small reduction in motility. Maintenance 
of post-thaw motility was slightly, but significantly 
improved with glycerol removal. The relevance of 
greater velocity in these samples is not readily appa¬ 
rent. However, differences observed in motility of 
sperm after glycerol removal may translate into 
enhanced sperm survival in the more favorable en¬ 
vironment of the female reproductive tract. 

The results of this study are based on a limited 
number of observations, leaving many questions 
regarding cryopreservation of falcon semen un¬ 
answered. However, several important points can 
be drawn from these results. Peregrine semen can 
be frozen using glycerol as a cryoprotectant with 
good post-thaw sperm motility, and the glycerol can 
be rapidly removed from post-thaw semen by 
dialysis without substantial loss of sperm motility. 
The techniques used in these procedures are sim¬ 
ple, relatively inexpensive, and can be adapted for 
practical application. Finally, the development of 2 
kestrel eggs in a clutch of 6 suggests that post-thaw 
dialysis is potentially useful for successful breeding 
with frozen falcon semen. Use of homologous 
species for insemination may provide a more useful 
measure of fertility. Refinement of these proce¬ 
dures and use of additional females to test fertility 
will help to establish whether post-thaw glycerol 
removal will make the use of frozen semen a practi¬ 
cal approach to captive breeding of falcons and 
other birds of prey. 


This project was supported in part by The Peregrine Fund, Inc., 
and the American Wildlife Research Foundation, Inc. 

Literature Cited 

Bird, D.M. and P.C. Lague. 1977. Semen production in 
the American Kestrel. Canadian J. Zool. 55:1351-1358. 
Boyd, L.L. and C.H. Schwartz. 1983. Training im¬ 
printed semen donors. In: Falcon Propagation - A 
Manual on Captive Breeding (J.D. Weaver and T.J. 
Cade, Eds.) pp. 24-31, The Peregrine Fund, Inc., 
Ithaca, New York. 

Brock, K., D.M. Bird and G. Ansah. 1983. Cryogenic 
preservation of spermatozoa of the American Kestrel 
Falco sparverius. Internat. Zoo Yearbook 12:67-71. 
Brown, K.I. and E.F. Graham. 1971. Effect of some 
cryophylactic agents on turkey spermatozoa. Poultry 
Sci. 50:832-835. 

Graham, E.F., D.S. Nelson and M.K.L. 
Schmehl. 1982. Development of extender and 
techniques for frozen turkey semen. 1. Development. 
Poultry Sci. 61:550-557. 

Lake, P.E., R.B. Buckland and O. Ravie. 1980. Effect 
of glycerol on the viability of fowl spermatozoa - impli¬ 
cations for its use in freezing semen. Cryoletters 1:299- 

Lake, P.E., O. Ravie and J. McAdam. 1981. Preserva¬ 
tion of fowl semen in liquid nitrogen: application to 
breeding programs. Brit. Poult. Sci. 22:71-77. 

Lake, P.E. and J.M. Stewart. 1978. Preservation of 
fowl semen in liquid nitrogen - an improved method. 
Brit. Poult. Sci. 19:187-194. 

Sexton, T.J. 1979. Cytotoxicity of DMSO as related to 
components of a turkey semen extender. Poultry 
Sci. 58:1024-1030. 

Weaver, J.D. 1983. Artificial insemination. In: Falcon 
Propagation - A Manual on Captive Breeding, (J.D. 
Weaver and T.J. Cade, Eds.) pp. 19-23, The Peregrine 
Fund, Inc., Ithaca, New York. 

Department of Animal Science, 201 Morrison Hall, Cornell 
Univ., Ithaca, NY 14853. Address of second and third au¬ 
thors: The Peregrine Fund, Inc., Sapsucker Woods Road, 
Ithaca, NY 14853. 

Received 25 March 1985; Accepted 15 July 1985