CRYOPRESERVATION OF PEREGRINE FALCON SEMEN AND
POST-THAW DIALYSIS TO REMOVE GLYCEROL
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
species.
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¬
periments.
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).
16
Raptor Research 20 (1): 15-20
Spring 1986
Freezing Falcon Semen
17
Table 1. Characteristics and pre-freezing treatment of Peregrine Falcon semen.
Volume for
Collection Period 3
0 * 1 )
Initial
Motility
(%)
Sperm
Concentration
(x 10 6 /ml)
Interval Between
Collection and Cooling 0
(min)
Individual
Pooled
All b
Ejaculates
Ejaculates
Ejaculates
X
95.4
89.0
94.6
70.1
47.4
18.4
S.D.
51.7
38.5
49.7
6.9
16.1
6.8
Range
27-208
50-127
27-208
60-85
29-81
12-45
n
23
3
26
25
15
24
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
Pre-Freeze
Post-Thaw
Glycerol Level
0.3 M
0.9 M
1.48 M
0.3 M
0.9 M
1.48 M
X
75
66
70
29
47
54
S.D.
4.1
4.9
7.4
11.0
2.6
5.8
Range
70-80
60-75
60-85
20-45
45-50
45-65
n
4
6
15
4
6
15
18
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.
Dialysate
Hours of Incubation After Dialysis
0
0.5
1.0
1.5
4.0
X*
1.48 M Glycerol
41
26
24
25
13
26
Four-step Procedure
(1.1 M to Glycerol-Free)
43
33
35
31
20
32
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.
Results
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
post-dialysis.
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
19
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
motility.
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¬
fully.
Discussion
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
cryoprotectant.
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-
20
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.
Acknowledgments
This project was supported in part by The Peregrine Fund, Inc.,
and the American Wildlife Research Foundation, Inc.
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Received 25 March 1985; Accepted 15 July 1985
