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Reproduction in Nondomestic Birds: Physiology, Semen Collection, Artificial

Insemination and Cryopreservation

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 Avian and Poultry Biology Reviews 15 (2), 2004, 47 ± 101

Reproduction in Nondomestic Birds: Physiology, Semen Collection,

Arti®cial Insemination and Cryopreservation

George F. Geea, Henk Bertschingerb, Ann M. Donoghuec, Juan Blancod and John Soleyb
a
USGS, Patuxent wildlife Research Center, Laurel, MD 20708
b
University of Pretoria, Veterinary Wildlife Unit, p/Bay XO4, Onderstepoort 0110, South Africa
c
USDA, ARS, University of Arkansas, Fayetteville, Arkansas 72701
d
Junta de Comunidades de Castilla-La Mancha, Centro de Estudioa de Rapaces Ib'ericas, Sevellija de la Jara,
45671 Toledo, Spain

ABSTRACT

Pioneering work by Quinn and Burrows in the late 1930s led to successful artificial insemination (AI) programs in the domestic
poultry industry. A variety of species specific modifications to the Quinn and Burrows massage technique made AI possible in
nondomestic birds. Massage semen collection and insemination techniques span the entire range of species from sparrows to
ostriches. Also, cooperative semen collection and electroejaculation have found limited use in some nondomestic species.
Artificial insemination produces good fertility, often exceeding fertility levels in naturally copulating populations.
However, aviculturists should explore other ways to improve fertility before resorting to AI. Artificial insemination is
labor intensive and may pose risks to nondomestic birds as well as handlers associated with capture and insemination.
Semen collection and AI makes semen cryopreservation and germ plasma preservation possible. Yet, semen cryo-
preservation techniques need improvement before fertility with frozen-thawed semen will equal fertility from AI with
fresh semen.

Keywords: Reproduction, nondomestic birds, semen collection, arti®cial insemination, cryopreservation

INTRODUCTION Arti®cial insemination programs have become

This review brings together basic information on
arti®cial insemination (AI) of nondomestic birds.
Although AI procedures used with domestic fowl
apply to nondomestic birds, minor to major differences
in nondomestic reproductive physiology, behavior and
anatomy require accommodations. These differences
include extreme variations in size, reproductive strate-
gies, and morphological features of reproductive struc-
tures.
Much of what we discuss comes from literature and
personal experience. We hope that these discussions
will lead to a practical understanding of nondomestic
avian AI and enable others to use it successfully with
birds not mentioned here.
Major changes have occurred since the ®rst insemi-
nation by Ivanov in 1913 (Smyth, 1968) and the ®rst
practical application in poultry by Quinn and Burrows
(1936). Today, the commercial turkey industry
depends on an ef®cient arti®cial insemination (AI)
program to survive since modern turkey breeds
(Meleagris gallopavo) cannot copulate effectively.
*To whom correspondence should be addressed: Email:
important components of recovery and conservation
programs for some nondomestic birds. Yet, you may
®nd more permanent, less intrusive ways to overcome
infertility challenges. AI is only one component in a
successful, nondomestic birds breeding program. AI is
a labor-intensive process that requires bird capture,
restraint and occasionally special pen design.
Capturing and handling nondomestic birds pose a
risk of injury to bird and handler, disease transmission,
and reproductive failure.
AI is not something you should undertake without
adequate support. Many AI projects that start with the
best of intentions fail not from the lack of proper
technique but from the lack of persistence and dif®-
culties associated with capture and management of the
birds.
Conditions other than inadequate copulation or poor
semen quality may cause infertility and early embryo
death. Examples include improper handling of the
eggs, poor nest conditions, nutrition, and disease
(Hagan and Dziuk, 1981; Hurlet, 1995; Wilson,
1995; Wishart, 1995). One can discover and then
48 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
address problems by examining eggs for fertility and
checking females for conditions that interfere with
fertile egg production (Hulet, 1995). The fertile blas-
todisc is obvious in most freshly laid eggs and you can
detect the embryo after a few days of incubation (Bakst
and Gupta, 1997).
Reproductive success relies on multiple factors and
seasonal, environmental and behavioral effects impact
results. As suggested above, integrating knowledge of
the physiology of avian species and speci®c adapta-
tions unique to the bird is extremely important in
establishing AI programs. These include species sex
speci®c differences in reproductive anatomy and copu-
latory strategy. In addition, semen collection strate-
gies, evaluation of semen quality and function,
insemination schedules, cryopreservation methods,
assessment of egg fertility, and incubation conditions,
vary by species.
Season
Unlike chickens (Gallus domesticus), substantial
changes in the reproductive tract take place from one
season to the next in most nondomestic birds (Lofts
and Murton, 1973; Immelmann, 1971; de Juana, 1992).
For most species, the breeding season is short (Lofts
and Murton, 1973) with reproductive organs response
rapid and robust at the onset of the season. Detailed
descriptions of seasonal changes in the reproductive
system are beyond the scope of this report but avail-
able in the literature.
Changes in the environmental conditions (like
photoperiod, temperature, and humidity) can have
major impacts on reproductive condition (Marshall,
1961a; 1970). Breeding birds transported between
southern and northern hemispheres show inappropriate
breeding responses and may take years to adapt to new
environments (Lofts et al., 1971; Flieg, 1974). In
addition, other birds taken from northern and southern
extremes of their range may take several years in
captivity to obtain synchronous reproductive seasons
(Porter and Wiemeyer, 1972). Due to the necessity for
adjustment to their new environmental signals, asyn-
chronous pairs may produce infertile eggs and exhibit
inappropriate behavior.
Environmental factors in¯uence crane reproduction.
Whooping crane breeding seasons are limited to two
months in Wood Buffalo National Park, Northwest
Territories; to four months in the Patuxent Wildlife
Research Center, Laurel Maryland; and to ®ve months
in the Audubon Park and Zoological Garden, New
Orleans, Louisiana (Mirande et al., 1996). Florida
sandhill cranes (Grus canadensis pratensis) in their
native habitat reproduce from January through June; at
the Patuxent Wildlife Research Center from February
through June; and at the International Crane
Foundation, Baraboo Wisconsin from March through
August (Mirande et al., 1996). Cranes will lay one
clutch of two to three eggs and not lay others if they
successfully incubate the eggs. In extended seasons,
the birds relay if the eggs are removed as laid or the
nest fails (Mirande et al., 1996). Fecal steroid studies
of reproductively active sandhill crane pairs show the
typical increase in androgen and estrogen levels in late
winter and spring with rapid declines in the summer
months (S. Leffer, personal communication).
Extending photoperiod with metal halide lights can
lengthen the greater sandhill crane (Grus canadensis
tabida) reproductive season in captivity (Gee and
Pendleton, 1992). Yet, light is only one of several
factors in¯uencing the reproductive season.
Like the crane, the ostrich (Struthio camelus) is a
seasonal breeder (Duerden, 1908). Egg production
appears to be linked to increasing photoperiod
(Hallam, 1992; Hicks, 1992; Mellett, 1993). Peak
egg production occurs in the months with the longest
day (Table 1). Egg laying has been reported throughout
the year in various countries (Smit, 1963; Jarvis et al.,
1985; Shanawany, 1995) suggesting that ostriches may
be opportunistic breeders, too. The onset of breeding,
or peaks in reproduction, coincide with periods of good
rainfall and the consequent improvement of food
resources (Sauer, 1972; Leuthold, 1977). Yet in most
cases, the ostrich is a seasonal breeder (van
Schalkwyk, 1991; Verwoerd, 1997) and various indi-
cators have linked laying and reproductive perfor-
mance to photoperiod. In the USA, ostriches are
summer breeders but the season may extend from as
early as January to as late as October (Stewart, 1989).
Birds in the northern United States lay from May to
September, while in the southern birds may produce all
year round (Hicks, 1992).
Male ostriches from the Oudtshoorn district (n ˆ 32)
examined in May (pre-season), October (peak-season)
and February (end of season) showed the highest levels
of normal sperm morphology in October (Bertschinger
et al., 1992) (Figure 1). Plasma testosterone determi-
nations performed on the same group of birds showed
a different pattern with testosterone concentrations
peaking in May and decreasing sequentially in
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 49

Table 1 Ostrich (Struthio camelus) breeding season

Region Production Months Ref.
Northern Hemisphere March ± September 7 Leuthold, 1977
Northern USA January ± October 10 Stewart, 1989
May ± September 5 Hicks, 1992
Southern USA January ± December 12 Smit, 1963; Jarvis et al., 1985; Hicks, 1992
Israel March ± September 7 Degen et al., 1994
August ± January 6 Degen et al., 1994
South Africa June ± February 8 Van Schalkwyk, 1991; Verwoerd, 1997
August ± February 7 Jarvis et al., 1985
August ± January 6 Bertschinger, personal communication
Zimbabwe (wild) July ± January 7 Jarvis et al., 1985
July ± December 6 Jarvis et al., 1985
Australia July ± March 9 More, 1996

October and February. This observation may indicate exposed to additional lighting. Additional lighting (to
that male ostriches are ``switched on'' as the longest give a lightydark cycle of 16y8 hours) was provided to
nights approach. A pre-season peak in plasma LH the experimental pairs for 4 weeks from 26 June until
production, and subsequent increase in plasma testos- 21 July. Egg laying was recorded from 1 August to 18
terone levels, has also been reported in male birds in November when the sexes were separated again. As
Israel (Degen et al., 1994). can be seen in Table 2, the treatment group outper-
Hens start to lay in August and continue until the formed the Control group in 3 of the 4 months
end of January in South Africa (van Schalkwyk, 1991; (September ± November) monitored. Although this
Verwoerd, 1997) or longer (More, 1996). Although was only a pilot trial, it may indicate that, similar to
immature, active ovaries are also found in sub-adult domestic chickens, the egg production of ostriches
ostriches sampled during periods of long daylight may be improved by controlling the light ± dark
(Madekurozwa, 2002). Like the males, plasma LH cycle. The fact that ostriches, where eggs are
levels in females show an increase a month before removed regularly, stop laying after about 6 months,
the onset of the breeding season and
then decline steadily for the & jj % M e a n m o t i l i t y j % M a l e s p e a ki n g ( m o t i l e s p e r m )
remainder of the season (Degen & == M e a n % n o r m a l sp e r m &} Males peaking (normal sperm)
et al., 1994). In contrast, estradiol
levels, which in¯uence egg forma-
% motile / normal / birds peaking

tion, are elevated from the ®rst

month of the laying season until a
month before the end of the season.
There is a peak in estradiol levels
during the third month of the season,
which corresponds to the peak in egg
production (Degen et al., 1994) and
with the longest day.
Additional evidence supporting the
role of photoperiodicity was obtained
from a pilot trial carried out in the
Oudtshoorn district to establish the
effects of additional lighting on egg May October January
production of ostrich pairs Mean % motile sperm and mean % normal sperm of ostrich collected in May, October and
(Bertschinger, Soley and Burger, January, respectively. The figure also shows the % of birds that showed peak semen quality
unpublished data). Six pairs of birds in terms of motility and morphology with the same collections.

served as controls and ®ve pairs were Fig. 1 Seasonal effects on ostrich (Struthio camelus) semen.
50 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Table 2 Lightaand ostrich (Struthio camelus) production.
August September October November
Gp Eggs Laid Eggs Laid Eggs Laid Eggs Laid
Gp Ave/f Gp Ave/f Gp Ave/f Gp
Control 20 3.3 18 3.0 25 5.0 17
(6)
Lighting 17 3.4 49 9.8 45 9.0 20
(5)
Gp ˆ group, ( ) ˆ number of pairs, Ave ˆ average, f ˆ female.
a
Effects of 4 weeks of lighting (16 lighty8 dark) on egg production
of ostrich pairs in the ensuing months from 1 August to 18
November.
indicates that once ``switched on'' birds will only lay
for a certain period of time from the initial cue.
In their native habitat, females ostriches typically
produce one clutch per season, a clutch consisting of
about 20 chicks. Once the female lays the last egg, she
incubates during the day and the male during the night.
Males develop huge edematous skin folds on the
wings, which may be important for effective incuba-
tion (Bertschinger et al., 1992; Irons et al., 1996).
In South Africa, highly productive captive females
will lay eggs every second to third day for a period of
about 2 months if eggs are removed when laid.
Removing the male after the ®rst laying period gives
the female a rest. A second laying period results if you
reintroduce the males and it lasts for another two
months. The females molt in February and stop laying
eggs. Productive birds lay from 50 to 80 eggs in a season.
Environment
Environmental extremes cause stress and interfere with
reproduction (Marshall, 1961a; Mirande et al., 1997;
Gee, 1995). In nondomestic birds, stress causes birds
to stop laying eggs, to break eggs, and to kill chicks.
Heat stress environments cause reduced shell quality,
reduced egg size, and increased cracked and broken
eggs (Draper and Lake, 1967; Payne, 1967). Prolonged
exposure of eggs to the sun can start embryo develop-
ment without incubation or even kill the embryo
(Landauer, 1967). Temperature ¯uctuations increase
bacterial infection susceptibility in eggs (Bean and
MacLaury, 1959). Hot spells can reduce semen
production and reduce fertility of the eggs laid
(Clark and Sarakoon, 1967; Kundu and Panda, 1990).
Heat and cold have behavioral effects that can reduce
copulation in both sexes (Bluhm, 1985; Marshall,
1970). Cold can freeze unattended eggs in captivity,
especially in birds out-of-sync with seasonal cycles. At
the Patuxent Wildlife Research Center, eggs laid in
January by the Hawaiian nene goose (Branta sandvi-
censis) would often freeze if not rescued from the nest.
Ave/f
2.8 Reproductive history
4.0
The reproductive history of the species and of the
individuals within the species provides information
basic to an assisted reproduction program. This infor-
mation includes behavioral and physiological
measures, and a production history.
The age at ®rst egg, date of the ®rst egg each season,
egg production, fertility of eggs laid, eggs broken,
hatch of fertile eggs, physiological condition and
similar measures provide data needed to formulate a
reproductive strategy (Ellis et al., 1996; Swengle et al.,
1996; Gee and Mirande, 1996). Change of mate,
change of pen location etc. may correct poorly fertile
or infertile pairs and avoid the need for an AI program.
One needs to observe reproductive related behavior
and record it from year to year. Some pairs may not be
good candidates for AI if capture causes excessive
disturbance. Such pairs require special conditioning
before starting an AI program (Gee and Mirande,
1996). Often providing a special food item or other
positive reinforcement as a reward after or during
capture sequences will reduce stress.
Some pairs exhibit speci®c behaviors that signal the
start of the reproductive season. In cranes these include
nest building, stereotypic rituals like increased unison
calls, moving of nesting materials, nest bill-down
display, increased copulation frequency and strut
(Swengle et al., 1996). Date of ®rst egg each season,
cloaca size, pubic bone expansion and behavior before
lay help us decide when to start AI.
In the ostrich, overt changes in behaviour occur
during courtship and mating (Pocock, 1909; Sauer
and Sauer, 1966; Bolwig, 1973; Berens von
Rautenfeld, 1977; Osterhoff, 1979; Hicks, 1992;
Sambraus, 1994). In the female these include head
held low, wings spread with the tips ¯uttering, and
snapping of her beak open and shut. In the male these
include the conspicuous red shins (tarsal scales) and
beak, a ritual courtship dance, head movements from
side to side and head thumping on the back, and a
booming sound (Hicks, 1992; von Rautenfeld, 1977).
For more detailed explanations of the complex repro-
ductive rituals in both wild and domestic ostrich see
Hurxthal (1979) and Bertram (1992).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
In many nondomestic birds the date of the ®rst egg
each season is remarkably consistent from year to year
(Gee and Mirande, 1996; Ellis et al., 1996). In some
years the season is later or earlier than in other years.
The behavioral cues and activity of others in the ¯ock
can lead you to adjust the AI schedule. A male in some
pairs may come into season later than a female in that
pair. Frozen semen from the previous year can be used
to increase fertility of the early eggs.
These examples point out the need for and use of
reproductive history in an assisted reproduction
program. The history should include a variety of
factors tailored to those critical elements in the
species under study. To evaluate reproductive perfor-
mance in cranes, we maintain 30 different records
(Ellis et al., 1996).
FEMALE
Reproductive tract
The female reproductive tract (ovary and oviduct) is
the site of egg formation, sperm storage, and ferti-
lization. The reproductive tract in nondomestic birds,
although similar to the domestic chicken, may vary
in size, length and relative ratios of the various
segments (Johnson, 1986a; Gee and Russman,
1996; Bezuidenhout, 1986). The right oviduct is
vestigial and left oviduct functional in most species
(Marshall, 1961b; Lofts and Murton, 1973; Johnson,
1986a). Some falcons and the kiwi may have two
functional oviducts (Johnson, 1986a; de Juana,
1992).
The oviduct contains ®ve morphological segments in
a breeding nondomestic bird: the infundibulum,
magnum, isthmus, shell gland and vagina (Figure 2).
In the nonbreeding bird, only the much-reduced ovary
may be visible (de Juana, 1992). The most anterior
segment, the infundibulum, is associated with the
ovary and engulfs the ovum upon ovulation (Sturkie,
1954; Gee and Russman, 1996). The infundibulum is
the site in the oviduct where sperm interact with the
ovum.
Within the magnum, albumen is secreted around the
yolk, shell membranes are added in the isthmus, and
¯uids, shell, and pigments in the uterus (shell gland).
The most distal segment, the vagina, opens into the
cloaca. The vagina adds the cuticle to the shell and the
utero-vaginal junction is the site of sperm storage
(Johnson, 1986a; Proudman, 1995).
51
Empty follicle
Immature Yolk
Mature Yolk
Infundibulum (13 cm)
Magnum (39 cm)
Isthmus (18 cm)
Uterus (Shell Gland) (17 cm)
Vagina (5 cm)
8
> Coprodeum
>
>
<
Cloaca Urodeum
>
>
>
:
Proctodeum
Art Kate Spencer
Fig. 2 Female reproductive anatomy of Sandhill crane (Grus
canadensis pratensis).
During the breeding season, many nondomestic
female birds will exhibit noticeable abdominal enlar-
gement (bagging down) from the expansion of the
oviduct and ovary, especially when an egg is present.
In the mature female ostrich the oviduct is a long
(approximately 1.2 m), convoluted, well vascularised
organ (MacAlister, 1864; Duerden, 1912; Fowler,
1991; Muwazi et al., 1982). In the crane, the cloaca
becomes swollen during this period and the ends of the
pubic bones may separate (Gee personal observations,
Figure 3). Separation of the pubic bones may also be
used for timing inseminations (DeMatteo et al., 1998).
These changes and nest building often signal the need
to start inseminations in nondomestic birds.
Cloaca
The cloaca, which is the common opening to the
digestive and reproductive tracks, contains three
regions, the coprodeum, urodeum, and the procto-
deum, the most posterior (King, 1981). In the ostrich
as in most birds the coprodeum and urodeum are partly
separated by a coprourodeal fold (Geoffroy-Saint-
Hilaire, 1822). The uroproctodeal fold separates the
urodeum from the proctodeum (Fowler, 1991). The
opening to the vagina is located in the upper left wall
of the urodeum (Lofts and Murton, 1973; Johnson,
1986a).
52 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Average distance between pubic bones
in Sandhill Cranes
3.30
3.17
3.04
centimetres
2.91
2.78
2.65
2.52
2.39
2.26
2.13
2.00
1±3 4±6 7±9 10±12 13±15 16±18
Days before first egg
Fig. 3 Pubic bone expansion and egg production in Sandhill crane
(Grus canadensis pratensis).
This is important to remember because deep vaginal
insemination (near the sperm storage tubules) produces
the best fertility. In some species, the cloacal region is
fully exposed and everted by pressuring the female's
abdomen and pericloacal region (see AI section).
Obviously, bacterial infections interfere with insemi-
nations and can be passed from bird to bird by
improper insemination technique.
Sperm storage
Unlike most mammals, a single insemination of sperm
in female birds can fertilize eggs from several ovula-
tory cycles without the aid of additional semen. Sperm
storage tubules (SST), located at the uterovaginal
juncture (UVJ) (Figure 4) store most of the sperm
and release them over time (Bakst, 1993; Bakst and
Bahr, 1993; Brillard, 1993; Proudman, 1995). Briskie
and Birkhead (1993) describe simple methods to count
and study SST in reproductively active nondomestic
birds. The SST may exist in all avian species (Bakst
and Bird, 1987; Gilbert, 1979; Hatch, 1983; Dixon and
Birkhead, 1997).
Sperm storage tubules differ in size and number
between species and may be related to the size of
sperm and semen volume (Birkhead and Mùller,
1992). The female reed bunting (Emberiza schoeni-
clus) SSTs are extremely long (mean length 865 mm) to
hold the males extremely long sperm (Dixon and
Birkhead, 1997). However, the relationship between
sperm and SST length is not always positive. Chicken
and turkey sperm are nearly identical in length
(Thurston et al., 1982; Marquez and Ogasawara,
1975) yet the SST in turkeys are pleomorphic and
often 900 mm in length (Bakst, 1994; King et al.,
2002). In contrast, chicken SST are relatively straight
and rarely exceed 600 mm. Interestingly, King et al.
(2002) observed that the larger more pleomorphic SST
in the turkey were more likely to contain sperm then
the smaller, straight SST.
The distribution of sperm in the SST varies from
species to species. In the zebra ®nch [Poephila gutta
(Estrildidae)], sperm in the SST are distributed
randomly at the distal end of the UVJ (Birkhead
et al., 1990). Some SST there contain many more
19±21
cells than others do, a nonrandom distribution. In
some species there is a discrete strati®cation of sperm
in the SST but not in others (Sheldon and Birkhead,
1994).
In the ostrich, a distinct difference in color exists
between the mucosa of the uterus and that of the
vagina. The uterus appears darker due to the presence
of large numbers of uterine glands in the lamina
propria. The transition between the two regions is
abrupt and well demarcated (Bezuidenhout et al.,
1995). In the ostrich, SST are present in the vagina
in a wide band immediately adjacent to the uterova-
ginal junction (Bezuidenhout et al., 1995; Groenewald
et al., 1996; Holm et al., 2000). The presence of SST
demarcates the utero-vaginal junction which is
reported to vary between 11.5 cm (Holm et al., 2000)
and 20 cm (Bezuidenhout et al., 1995) in length. The
tubules are situated in the lamina propria, are mostly
branched and slightly coiled, and lined by a non-
ciliated, simple columnar epithelium (Bezuidenhout
et al., 1995). Sperm seen within the tubules are
oriented along the length of the tubule and generally
positioned in the centre of the lumen. Recent informa-
tion indicates that SST are present at the uterovaginal
junction in sexually immature birds with active ovaries
(as judged by the presence of large yolk-®lled folli-
cles), but not in birds with inactive ovaries.
The duration of fertility is dependent on many
factors. Assuming adequate numbers of sperm are
transferred to the vagina and ®ll the SST, then the
duration of fertility is clearly dependent upon the rate
of sperm loss from the SST (Sax et al., 1998). Brillard
(1995) has shown this to be the case for the chicken
and turkey. Sperm release from the SST is more rapid
with the chicken, hence the shorter duration of fertility.
Furthermore, he has also shown that the aging chicken
has a shorter duration of fertility than the younger hen
because sperm release from the SST in older hens is
more rapid.
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 53

Fig. 4 Micrograph ostrich (Struthio camelus) sperm storage tubule. Light micrograph (6700) of a
vaginal fold near the utero ± vaginal junction. Note the secondary folds lined by ciliated, pseudo-
strati®ed columnar epithelium (arrowheads). An arrow points to a densely stained area consisting of
sperm heads situated in a sperm storage tubule, the lining of which is simple columnar non-ciliated
in nature.

In most nondomestic birds sperm are available in ¯ycatcher (Ficedula hypoleuca) and bearded tit
excess, therefore the rate at which sperm are released (Panurus biarmicus) have very short periods of
from the SST determines the duration of fertility sperm storage. This limits the time when copulation
(Birkhead and Fletcher, 1994). For example, the pied can occur that will give rise to fertile eggs (Birkhead
54 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
et al., 1997b; Birkhead and Hoi, 1994; Sax et al.,
1998).
Sperm transport
The SST provides long-term sperm storage yet fertili-
zation takes place in the infundibulum at the opposite
end of the oviduct. The mechanisms for the transfer of
sperm from the SST to the infundibulum remain
uncertain. Sperm do not need to pass through the
SST. Inseminations that occur just before ovulation
produce fertile eggs. Yet some studies report reduced
fertility from intramagnal and intraperitoneal insemi-
nations (Blanco, in press; Brown et al., 1963;
Wentworth and Mellen, 1964).
Sperm release from the SST is a slow but a
continuous limited process when the female is in egg
production (Bakst et al., 1994). Sperm ascend to the
infundibulum, the site of fertilization, in relatively
small numbers. Sperm are rarely observed and recently
Bakst (personal communication) questioned if the
infundibulum functions as a secondary sperm storage
site in the oviduct.
Sperm in the oviduct are swept from the reproduc-
tive tract by the developing egg. This results in an
epizootic delivery of sperm to the infundibulum.
Sperm stored in the crypts of the infundibulum
remain and play an important role in fertilizing eggs
(Bakst and Bahr, 1993).
Sperm competition and mating schemes
Sperm competition involves more than competition
between the sperm themselves. Sperm competition
involves anatomical, physiological and behavioral
processes in both sexes (Rodrigues, 1998). These
processes give the male and female genetic control
over the offspring produced. Therefore, each sex
constantly evolves ways to genetically out compete
the other sex and other individuals of the same sex
(Birkhead, 1998).
Birds may copulate on water, on land, or on a perch.
Males and females have evolved appropriate anato-
mical structures to accomplish successful copulation in
these situations. In some males, the phallus provides an
intromission organ to deliver semen into the female's
cloaca. This helps avoid harsh environmental chal-
lenges to the sperm like copulation on water or in dry
desert heat. Some passerines evolved with modi®ed,
often enlarged, cloacas to help collect sperm (Castro
et al., 1996; Birkhead and Biggins, 1998). Brightness
of male plumage and extra-pair paternity are asso-
ciated in many birds (Mùller and Birkhead, 1994).
Females may have success in controlling extra pair
copulations (Venier et al., 1993; Wagner, 1991a,b). In
some cases the female can solicit copulation during her
most fertile period effectively selecting the male
partner or extra-pair male (Mùller and Birkhead,
1994). The female pied ¯ycatcher controls mating in
the wild and may limit the extent of sperm competition
(Birkhead et al., 1997a).
Extra-pair copulation is only one behavior used to
get genetic control of offspring produced. Some birds
must complete extensive displays before making a
successful copulation (Hicks, 1993; Swengle et al.,
1996), limiting the number of acceptable partners.
Timing copulations with multiple partners in¯uences
paternity and maternity. In male passerines, birds that
deliver the largest number of sperm per ejaculate,
copulate frequently, produce sperm at the highest
rate, and produce larger sperm, have an advantage
(Birkhead, 1996). They have evolved with anatomical
structures to improve semen delivery (Birkhead and
Biggins, 1998).
Males improve their chances by protecting
(guarding) their mate from other males. Very attentive
males like the colonial breeding lesser kestrel (Falco
naumanni) avoid sperm competition from other males
by frequent copulation. These repeated copulations
may strengthen the pair bond and limit female avail-
ability to other potential mates (Negro et al., 1996;
Sheldon et al., 1997). Protracted copulation may be
another form of mate guarding to restrict potential
mates (Schulze-Hagen et al., 1995; Sheldon, 1994). In
mate guarded species, reduced sperm number and
semen volume for AI may require the collector of an
AI program to make more frequent inseminations (Gee
et al., 1985; Gee et al., 1993).
Males and females also control paternityymaternity
through song, alarm calls, and defense of territory. In
the sedge warbler (Acrocephalus schoenobaenus), the
female leaves her partner to mate with other males that
appear more vigorous as indicated by greater song
repertoires. Older males tended to have greater song
repertoires and larger testes (Birkhead et al., 1997b).
These older males may offer some selective advantage
to offspring. In monogamous birds, song may function
to stimulate the female to solicit copulations, espe-
cially during her most fertile period (Pinxten and Eens,
1998). A neighboring male even in monogamous pairs
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
can father many chicks (Sheldon and Burke, 1994;
Gavin and Bollinger, 1985).
Females in some species have the capacity to
differentially eject ejaculates and retain sperm of
preferred males (Pizzari and Birkhead, 2000). In
species where the females selectively retain ejaculates,
collectors using AI may need to condition the females
to retain the semen inseminated. In most birds, getting
good behavioral response to AI helps to improve
fertility (Gee and Mirande, 1996).
Female ostriches eject eggs from the nest which
were laid by competing females. In the wild, free-
ranging females typically lay one clutch per season.
Clutch sizes vary from 16 to 36 eggs (Bertram, 1992),
with many of the eggs being contributed by the
``major'' hen. Not all the eggs laid can be accommo-
dated beneath the adult birds during incubation. A
number of the eggs (reportedly those of the ``minor''
hens ± Bertram, 1979, 1992) are removed by the
``major'' hen. This results in an effective clutch size
of about 20 eggs. This breeding system is apparently
similar throughout the natural range of the ostrich
(Bertram, 1992; Jarvis et al., 1985).
Mating schemes in nondomestic species provide
clues to the best insemination strategies. Those that
copulate infrequently or have long fertile periods may
require fewer inseminations than other species. Some
mating schemes are elaborate. In tinamou and
Temmincks's stint, females mate with a different
male for each clutch of eggs laid (Bump, 1969;
Oring, 1982; Cabot, 1992). In all birds, AI sequence
is important in controlling pedigree of chicks
produced and may be especially ¯exible in birds
like the tinamou. Some species produce small
semen samples and copulate repeatedly (Gee et al.,
1993) and others may mate once and ¯y out to sea
for days before returning to the nest to lay (Hatch,
1983). In birds like the northern fulmar, only one or
two copulations with good quality semen produce
fertile eggs. Some copulate to maintain pair bonds
and to produce fertile eggs (Negro et al., 1996).
In concert with these anatomical and behavioral
adaptations, physiological mechanisms have evolved
that in¯uence paternal and maternal contributions to
the next generation. Some males meet competitive
challenges by producing more semen. In birds where
there are high levels of sperm competition, males
generally have developed larger testis size and
produce more sperm per ejaculate (Mùller, 1988;
Sheldon and Birkhead, 1994). The Stitchbird
55
(Notiomystis cincta) practices monogamy, polyandry,
polygamy and polygynydry. Males have large testes
(4.2% of body mass) and concentrated semen
(1:56109 spermyml). Both sexes have enlarged
cloacas to simplify sperm transfer (Castro et al., 1996).
The female stores the semen for long periods and
subsequently can lay several fertile eggs after separa-
tion from the male. The female also rejects most of the
sperm transferred to her cloaca (99%). However, small
changes in the number of sperm retained can have a
major in¯uence on the number of fertile eggs produced
(Birkhead, 1992; Brillard, 1995).
Generally, sperm from the last insemination or
copulation prior to ovulation fertilizes the egg
(Birkhead and Biggins, 1998; Cunningham and
Cheng, 1999) provided enough time remains for the
sperm to travel to the infundibulum. In the chicken,
sperm move through the oviduct in a few hours (Bakst
et al., 1994). Apparent sperm competition can often be
explained by the sequential deposition of semen. In the
sandhill crane, DNA analysis of chick genotypes
showed that the insemination by the male made
closest to but before ovulation produced most of the
chicks (Jones et al., in preparation).
Repeated copulations may not eliminate extra-pair
paternity. Sperm in the zebra ®nch are produced at a
constant rate of 1:96106 per day. Repeated ejacula-
tions decrease the sperm available when the rate
exceeds sperm production (Birkhead et al., 1995;
Birkhead and Fletcher, 1995). After the pair copulation
period, the male zebra ®nch builds sperm numbers and
consequently extra-pair copulations succeed because
of replenished sperm numbers (Birkhead et al., 1995).
In successful naturally fertile pairs of cranes, those
pairs with high fertility rates provide the collector with
less semen than pairs with low fertility rates (Chen
et al., 2001). In species like the crane and zebra ®nch,
separating a pair for a day before collecting semen
increases the number of successful collection attempts
and results in larger semen volumes.
Sperm competition between sperm from different
males in the reproductive tract is more dif®cult to
document but some reports indicate it can happen
(Birkhead, 1996). Sperm competition may play a role
in those nondomestic species that copulate frequently
with many different males and in AI when using
pooled semen. Sperm competition is a complex
process, one too lengthy for discussion in greater
detail here but is covered well by Bakst et al. (1994)
and Birkhead and Mùller (1998).
56 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Egg formation
The egg laying cycle (ovulation, egg formation, ovipo-
sition and repeat) differs between species. When
developing an AI program, establishing the ovulatory
cycle and using this interval to develop insemination
schedules is extremely important. Inseminating birds
with an egg mass in the oviduct generally results in
lower retention of semen (Smyth, 1968) (see quail for
exception). The laying cycle in the more common
birds used in AI (chickens and turkeys) form an egg
in 24 ± 26 h but there are many exceptions in nondo-
mestic birds. Cranes require 50 ± 60 h to form an egg
(Gee and Russman, 1996). The usual interval between
eggs in falcons is 52 h. Weaver (1985) recommends
inseminations of falcons within 6 h of laying to
fertilize the next egg and no later than 16 hours after
laying (about 36 hours to form an egg).
One can use the egg laying cycle from a closely
related species until the interval for a new species is
determined. Careful observations of oviposition
interval can be used to estimate the time needed to
form an egg. Generally, birds do not ovulate until after
oviposition. One can use a variety of other techniques
such as ultrasound to detect ovulation (Gee and
Russman, 1996).
In the ostrich, the follicles containing the ova range
in size from 1 to 9 cm in diameter (Fowler, 1991;
Bronneberg, 1997; Plooijer, 1998). The timing of
ovulation and the duration of egg passage in the
oviduct of the ostrich is not known (Irons, 1995).
Generally, a hen will lay an egg every second day
suggesting that the passage along the oviduct takes
about 48 hours. The activity of the ostrich ovary can be
monitored easily using ultrasound (transcutaneous
(trans-abdominal) ultrasonography) (Bronneberg,
1997; Plooijer, 1998; Wagner and Kirberger, personal
communication). More exact estimates of the time
needed to form an egg in the ostrich should be
available with ultrasound.
In nondomestic species, birds may hold a fully
formed egg in the shell gland for several days. We
have palpated a few cranes that held their eggs for
many days. This should not be confused with egg
binding where the bird is unable to expel the egg.
Such an egg bound bird requires immediate interven-
tion. Egg bound birds usually show signs of stress,
attempts to lay, and general aberrant behaviors. One
should determine the behavioral patterns associated
with egg laying for the species under study.
Behavioral consideration and acclimation
When initiating an AI program it is critical that the
female's reproductive cycle is not disrupted due to
stress. In some nondomestic species stress due to
handling, unfamiliar personnel, loud noise, etc. can
stop egg production (Mirande et al., 1997), and
increase the incidence of soft-shelled and broken
eggs (Mirande et al., 1996). Also, aberrant behavior
may result from AI disturbance. One member of the
pair may attack the other and these attacks can be
lethal.
A variety of techniques can be used to calm the
birds and increase their acceptance of the handling
needed to collect and inseminate semen. Starting
the handling procedures long before the reproduc-
tive season in new pairs often helps. Providing a
reward (usually a favorite food) after each
attempted semen collection or insemination serves
as positive reinforcement to AI (Gee and Mirande,
1996).
Birds that adapt to the AI program lay more
fertile eggs. The more detailed understanding of
the reproductive mechanisms in the nondomestic
species under study the more likely AI will
succeed. These details should include behavioral
considerations like those noted earlier (reproductive
history).
MALE
Reproductive tract
The male reproductive tract in nondomestic avian
species is similar to that found in the chicken and
turkey (Johnson, 1986b). It contains paired testes,
ductus deferens, and genital papillae. The papillae
empty into the urodeum, the central compartment of
the cloaca. The proctodeum, the most posterior
compartment of the cloaca is the origin of the
intromittent or non-intromittent phallus as well as
cloacal secretions (Quay, 1967; King, 1981).
The proctodeal foam gland in the Japanese quail
produces a mucoid secretion, a meringue-like froth.
One can remove the froth by gently squeezing on
either side of the vent and wiping it away with a
soft cloth. Remove the froth before collecting the
semen (Wentworth and Mellon, 1963). It has no
adverse effect on the sperm (Fujihara, 1992) but
can interfere with semen collection.
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 57

In ratites, some large raptors, and a few passer-
ines, a waxy, greasy or mucous material may be
present in the cloaca. Although conspicuous secre-
tions are not present in all birds, secretory anal
glands have been found in 72 species in 42 families.
None have been found in the domestic fowl (Quay,
1967). Excesses of this material should be wiped
away to avoid contamination of the semen. The
ostrich has a pouch-like ventral extension of the
coprodeum, which acts like a bladder and where
urine collects between voiding (Warui and
Skadhauge, 1998; Skadhauge and Dawson, 1999)
and can contaminate semen collected.
urodeum when the phallus is exteriorized
(Bertschinger et al., 1992).
As male ostriches approach their ®rst breeding
season at 3 ± 3.5 years of age, phallus length increases
from 26 to 31.1 cm. The development of the phallus is
testosterone dependent (Bertschinger and Soley,
unpublished data). The phallus returns to immature
size (lengths) when mature males are under prolonged
stress and when blood androgen levels decrease
(Bertschinger and Soley, unpublished data).

Phallus variations

The phallus in the domestic chicken is a rudimen-

tary organ useful in chick sexing but performs no
useful function in copulation. The phallus in many
nondomestic species including some galliformes like
cracids is well developed. These include ratites,
waterfowl, tinamous, storks, ¯amingos, thick-knees
(Charadriformes, Family Burhinidae) and one
passerine (genus Bubalornis, the red-billed and
white-billed buffalo-weaver ®nches) (Skinner,
1974; de Juana, 1992; Irons et al., 1996).
Sometimes the phallus contains rigid cartilage
similar to that of the ratites to aid in copulation
(King, 1981). The simple description above does
little to re¯ect the complex structure of the phallus.
A more comprehensive review of this subject is
found in King (1981).
In the ostrich, the coprodeum and urodeum are
partly separated by a coprourodeal fold (Geoffroy-
Saint-Hilaire, 1822; Fowler, 1991). When the phallus
is extruded the uroproctodeal fold, which is attached to
the base of the phallus on either side of the phallic
sulcus (groove), is pulled backwards creating a tube-
like entrance to the urodeum (Figure 5). The resting
phallus lies folded within the proctodeum.
The phallus is very large in the adult ostrich varying
from 30 to 39 cm in length with a deep phallic groove
(Figure 5). The phallus of the adult ostrich consists of
paired ®brous bodies, a deep phallic sulcus (groove),
an elastic vascular body and muscles. Most evidence Fig. 5 Semen collection from ostrich. The right hand of the
operator is seen reaching into the urodeum to massage the seminal
points to lymphatic engorgement as the mechanism of
papillae while the left hand is used to hold the phallus with a cloth
erection in the ostrich. This increases caliber but not (c) in the extruded position. An assistant (a) lifts the dorsal part of
length of the phallus. (Bertschinger et al., 1992). the cloacal sphincter to reduce the pressure on the operator's hand.
Semen ducts (ductus deferens) end in genital papillae, Semen running down the phallic groove (arrowhead) is caught by a
which are clearly palpable on lateral walls of the third person in a collecting tube.
58 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Cloacal protuberance

Sperm storage takes place in the ductus deferens

within the body of most male birds. Songbirds
provide the exception (Howell and Bartholomew,
1952; Salt, 1954; Wolfson, 1952 and 1960;
Middleton, 1974). Many passerines have an enlarge-
ment on the dorsal lip of the cloaca, (the cloacal
protuberance) that contains coils of the ductus deferens
(Figure 6). A gentle squeeze will yield semen.

SEMEN COLLECTION

The three basic techniques used to collect semen from

nondomestic birds include cooperative, massage, and
electroejaculation. Falconers with sexually imprinted
birds of prey (Hammerstrom, 1970; Temple, 1972;
Berry, 1972; Boyd and Boyd, 1976; Grier, 1973)
pioneered cooperative semen collection techniques.
The poultry industry has used the massage technique
for decades with poultry (Quinn and Burrows, 1936).
More recently aviculturists, have adapted this tech-
nique for use with nondomestic birds (Gee, 1983; Gee,
1996). To collect semen from geese, ducks, pigeons,
and psittacines, several investigators prefer electroeja-
culation (Watanabe, 1957; Chelmonska and Geborska- Fig. 6 American gold®nch (Spinus tristis) cloacal protuberance.
Dymkowska, 1980; Harrison and Wasmund, 1983; Note swollen vent due to the protuberance store of semen in this
Betzen, 1985; Samour et al., 1985). reproductively active male American gold®nch.

Massage semen collection

The massage method continues to be the most
common method for semen collection but as
described later, others have a place in the right
situations. Using the massage technique, handlers
can collect semen frequently from most reproduc-
tively active males. The massage method can be used
to collect semen from uncooperative, non-domestic
birds in the wild and from birds not imprinted on
humans. Gee (1972) collected semen from wild
greater sandhill cranes at the time of capture in
their native habitat. However, semen quality is the
best and most consistent when obtained from birds
conditioned for semen collection.
With many nondomestic birds, the most effective
semen collection technique uses two people. An
assistant holds and stimulates the bird by stroking
the inner shanks and the ventral abdominal region.
The collector stimulates the region around the tail,
abdomen and vent by stroking with the left hand,
from the post-dorsal region of the back to the
interpelvic tail region, and postlateral region below
the tail.
At the ®rst signs of response, the collector bends
the tail back with the left hand and with the right
hand, strokes the abdominal and sternal regions from
anterior to posterior. The bird may respond with a
partial eversion of the cloaca and occasionally an
ejaculation. The collector grasps the tissue behind the
dorsal lip of the cloaca with the thumb and index
®nger of the left hand (Figure 7). The collector holds
a collection device (cup, funnel, or capillary tube) in
the right hand.
To get the ®rst drop of semen, the collector applies
gentle pressure to the dorsal lip of the cloaca. The
collector repeats the cloacal stimulation until the
remaining semen is collected. Semen collection
takes ®ve to ten seconds. If one fails to get semen
from a reproductively active male, repeat the process
later in the day or week. Make slight variations in
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 59

Fig. 7 Semen collection from the Sandhill crane. Assistant stimulates the crane by stroking the inner thighs
while the operator collects the semen by gently squeezing the dorsal lip of the vent. The operator uses the glass
funnel to collect the semen.

behavioral conditioning, restraint and massage until Cranes and storks

an erectile tissue response is observed.
Minor variations in the massage technique may
be needed to collect semen from some nondomestic
birds. With modi®cations, this procedure has been
used to collect semen from ducks, peacocks,
®nches, canaries (Bonadonna, 1939; A.F. Leighton,
personal communication), pigeons and doves (Owen,
1941), waterfowl (Johnson, 1954; Lake, 1962;
Pingel, 1972; Skinner, 1974), pheasants (Smyth,
1968; Durrant and Burch, 1991; Durrant et al.,
1995; Rose, 1996), quail (Wentworth and Mellen,
1963), falcons (Bird and Buckland, 1976; Weaver
and Cade, 1985), hawks (Corten, l973), cranes
(Gee, 1969, unpublished; Archibald, 1974), curas-
sows and turkeys (G.A. Greenwell et al., personal
communication), condors (Gee, unpublished)
cassowaries (Pickett, personal communication) and
ostrich (Irons et al., 1996).
Modi®cations in semen collection techniques for
cranes and storks are the result of the need for
special handling or restraining methods to accom-
modate long legs, sharp talons, long beaks and
different body sizes. We hold cranes and storks in
the standing position (Gee and Temple, 1978; Gee,
1996). They can be aggressive during the reproduc-
tive season and technicians need to take precautions
during capture and handling. Cranes and other long-
legged birds can injure themselves when kicking
forward during capture and handling, cutting their
necks with toenails. A straw broom held between the
crane and the technician provides a place for the
bird to attack that prevents injury to bird and
handlers. With aggressive pairs, technicians may
bene®t from chaps or heavy pants to protect their
legs.
60 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
The assistant captures the male and guides him to a
corner or another comfortable location. The bird is
cradled between the assistant's legs with the head and
neck behind the assistant (bird facing to the rear) and
the bird's breast against but not between the assistant's
thighs (bird's body in front of the handler).
Outstretched wings allow the assistant to message the
thighs (Figure 6). Also, the assistant may need to
support the bird when he ejaculates or the collector
may lose the sample.
Eagles
Eagles can be held in the lap if the talons are taped
shut. We found placing the bald eagle with talons
extended on a thick wooden perch worked better and
allowed a more natural response. In both methods, the
eagle is hooded to avoid injury to bird and handlers.
The assistant holds and stimulates the bird by stroking
the thighs. The collector strokes the tail, abdomen, and
vent as described earlier. Condors and eagles have
strong cloacal muscles. Stimulation and semen collec-
tion takes a powerful massage to get a response and to
collect the semen. Also, properly conditioned eagles
respond well to cooperative semen collection
(described later).
Budgies
To collect semen, both sides of the abdomen are
massaged for a few seconds and a gentle squeeze is
applied to both sides of the cloaca. The 4 ± 6 ml semen
sample is collected in nonheparinized microhematocrit
capillary tubes. Interestingly, the eosin ± nigrosin live-
dead staining (described later) does not work with
budgie sperm (Hargrove, 1986). Something about
dead budgerigar sperm membranes does not allow
the stain to penetrate.
Waterfowl
By making minor modi®cations in the massage tech-
nique described by Skinner (1974), you can obtain
quality semen samples. Often, handlers hold water-
fowl, game birds and others in their lap (Skinner,
1974). The assistant holds the bird and may stimulate
the thighs. In most waterfowl, the collector can collect
semen without the assistant's help.
Many geese and ducks produce relatively large
semen samples (0.1 ± 0.3 ml). The semen can be
collected from the tip of the phallus in a small cup
or tube (Skinner, 1974). The phallus is everted during
the massage process and before ejaculation.
In some waterfowl, ratites and tinamous, the uneven
surface on the phallus can interfere with semen collec-
tion especially in birds producing small semen
volumes (Figure 8). With birds that produce small
semen samples, one can intercept the semen at the
base of the phallus with a suction device to avoid
loosing semen in crypts and crevices on the surface of
the phallus (Gee and Sexton, 1990).
Sparrow
Small passerines (canary, starling, blackbird, and
sparrow) yield semen with little dif®culty but the
semen requires special handling to collect intact
sperm. During the reproductive season, most songbirds
have a well-developed cloacal protuberance (Howell
and Bartholomew, 1952; Salt, 1954; Wolfson, 1952
and 1960; Middleton, 1974). The protuberance looks
like a small pea on the dorsal lip of the cloaca (Figure
6). Applying a steady but gentle pressure to the
protuberance (Gee and Sexton, 1983; Briskie and
Montgomerie, 1992) produces small semen samples.
Modi®cation of the message technique for small
birds yields the largest, most repeatable semen
samples. Depending on bird size, stimulation around
the base of the tail and the abdomen below the vent
with the thumb and index ®ngertip prepares the bird
for semen collection. In the ®nal stages of the collec-
tion effort, the collector grasps the dorsal-lateral
surface of the cloacal protuberance by the thumb and
fore®nger and squeezes in a downward motion. With
repeated pressure, the semen appears on the inner
surface of the dorsal lip of the vent. The squeezing
pressure spreads the lips of the cloaca (Gee and
Sexton, 1983).
Sparrow semen is extremely thick and collecting
neat semen usually causes sperm damage, especially
broken tails (Gee and Sexton, 1983). The small sample
also dries quickly. To keep the densely packed cells
alive, a small quantity of diluent can be added to the
semen on the cloaca (Figure 9) before aspirating the
sample.
Semen does not move well in a typical capillary tube
without suction. An appropriate-sized mechanical
pipette with disposable tip or a white cell diluting
pipette when attached to a propipette works well.
Propipette examples include devices like the rubber
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 61

Fig. 8 Semen collection from a Tule white-fronted goose. The operator everts the phallus before collecting the
semen. In geese with small semen volume, the operator aspirates the semen from the base of the phallus.

bulb-type pipet ®ller, Fisherbrand # 13 681 50 or
pipette ®ller Fisherbrand # 03 692 35. A measured
volume of extender is added to the tip of the pipette
and added to the semen in the cloaca before aspirating
the sample. The bulb of the white-cell pipette can be
used to collect several samples.
Kestrels
Kestrels when being handled to collect semen, can bite
an unprotected hand. The person who holds and
massages the bird should wear modi®ed protective,
¯exible gloves with the covering over the thumb and
index ®nger removed. To uncover the massage digits,
cut off the lower half of the thumb and index ®nger of
the massage glove. This uncovers the ®ngertips for the
sensitive massaging process (Bird et al., 1976; Bird
and Rehder, 1981). We prefer a two-person technique
with one person holding the bird and wearing the
protective gloves, and the other person collecting the
sample (Figure 10) (Gee et al., 1993).
The American kestrel (Falco sparverius) produces
small semen samples (10 ± 15 ml) with few cells
(Brock, 1986; Gee et al., 1993). Getting larger
volumes of semen is dif®cult since the semen sample
¯ows over the surface of the vent. A capillary tube is
useful in collecting the semen.
Adding diluent to the semen on the lip of the
cloaca increases ef®ciency of semen collection,
allows the sample to be collected without bubbles,
and reduces contamination. We added 10 ml of
Beltsville Poultry Semen Extender (BPSE) to the
semen on the ventral lip of the vent and removed it
with a small catheter attached to a 1-ml syringe. A
propipette, attached to the opposite end of the
syringe, provided suction. Most of the sperm in the
ejaculate were recovered. The semen was dilute
enough so that we could accumulate 0.1 ± 0.2 ml in
62 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Fig. 9 Semen collection from a seaside sparrow. The operator holds the legs of this seaside sparrow between the
®ngers. By applying a gentle pressure to the protuberance, the operator releases the semen into the cloaca. The
assistant adds a drop of diluent to the semen and aspirates the sample.

the syringe barrel without aspirating it into the
suction device (Gee et al., 1993).
Kestrel semen differs from that of other birds in
terms of sperm morphology, sperm motility, sperm
numbers and live-dead staining characteristics.
Although live-dead staining does not work with the
round kestrel sperm, motility can be used as an
indicator of viability. When properly diluted, most
sperm are motile (Gee et al., 1993).
Cracids
Working with a new nondomestic species can take
time because of the adjustments needed in technique
and the usually small number of birds available for
study. After considerable conditioning, DeMatteo et al.
(1998) collected semen from a cracid (common piping
guan, Pipile cumanensis) by the massage technique.
The guan is another example of a species where a
combination of massage technique and cooperative
training resulted in semen collection. The seasonal
effect was most pronounced and restricted the time
when good semen samples were available (DeMatteo
et al., 1998). In addition, birds tended to ejaculate
when the handlers entered the enclosures to collect
semen. Diluents extended the small sample to facilitate
insemination and semen preservation.
Collecting samples at dawn signi®cantly increased
the number of successful collection attempts and the
size of the sample collected (K. DeMatteo, personal
communication). Subsequent inseminations resulted in
the ®rst fertile egg from AI with fresh semen in the
spring of 2000 (DeMatteo et al., 1998).
Condors
Sexually imprinted birds like eagles and falcons will
deposit semen on the trainer or in a collecting device
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 63

Fig. 10 Semen collection from American kestrel (Falco sparverius). With gloved hands, the assistant can hold
the kestrel without injury to the bird or handlers. Note the position of the thumb and index ®ngers of the
collector's left hand in the ®nal stages of stimulation and semen collection.

(Grier, 1973). However, most large or otherwise Parrots

dangerous birds are not imprinted. With some birds,
restraining devices are used to protect the bird and
handler from harm or to enable one person to collect
samples unassisted.
Andean condors (Vultur gryphus) with their
strong necks and beaks can in¯ict serious
wounds if unrestrained. The loose skin around
the neck allows the bird to pull its head inside
the neck skin and makes holding the head
dif®cult. Gee built a guillotine like devise to
hold the head of the Andean condor and cradle
the body to allow a natural copulatory response
(Figure 11). The rubber collar in the device was
small enough to prevent the head from passing
through but loose enough to allow the bird to
breath and move its neck. Condors have strong
cloacal muscles. Stimulation and semen collection
takes a powerful massage to get a response and
to collect the semen.
Parrots (especially large parrots and macaws) can
in¯ict serious bites on the handler and themselves.
Psittacines can be sedated and electroejaculated.
However, any sedation calls for veterinary assistance
and is not practical for the average aviculturist
(Harrison and Wasmund, 1983; Betzen, 1985).
Massage semen collection is possible from parrots
held in the hand or held in a restraining device. When
held in the hand, the assistant holds the parrot's head
by cradling the back of the head in the space between
the thumb and index ®nger (palm down and heal of the
hand facing back). The assistant places the end of the
thumb and index ®nger directly under the parrot's
lower mandible. The assistant supports the parrot's
body in the other hand with the legs held between the
®ngers. In this way, the assistant exerts enough
pressure to extend the head and prevent the bird
from turning its head and biting. With the bird held
64 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Fig. 11 Semen collection from an Andean condor. The long wings of the Andean
condor can interfere with semen collection. The assistant in this position stimulates the
thighs (hand with watch) and controls the wings. The strong cloacal muscles in
condors and eagles require substantial force to stimulate and to collect semen.

in this position, the collector can collect the semen
using the standard massage technique (Figure 12).
Nevertheless, some parrots do not cooperate when
held in this way. Also, some aviculturists prefer a
massage method that uses only one person to hold and
collect the semen.
We created a successful restraining device for
parrots using a clear plastic tube to hold the birds
(Figure 13). The tube should be narrow enough to
prevent the parrot from turning around in the tube. The
tube should be long enough to prevent the parrot
reaching the end. The back of the tube is cut to
provide access to the tail and cloaca. The feet are
restrained with jesses through holes or slots in the
bottom of the tube. With the bird in this position, one
person can collect the semen using the standard
massage technique. Birds accustomed to being held
in the tube are comfortable because they can see. You
can release the parrot by removing the jesses and
slipping the parrot out (head ®rst) through the open
end of the tube.
Quail
Quail contain a cloacal gland that produces a white
frothy substance. Though not detrimental to sperm,
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 65

Fig. 12 Semen collection from Puerto Rican parrot (Amazona vittata). Jim and Beth Wiley prepare to collect
semen from a Puerto Rican parrot. Note Beth's hand around the neck behind the outstretched head of the parrot.
Parrots can turn and in¯ict serious injuries if not properly restrained.

Smyth (1968) recommends removing it before semen
collection. Wentworth and Mellen (1963) used small
metal cones to restrain the quail during semen collec-
tion. The cone resembled the one sometimes used to
hold turkeys during semen collection but much smaller
(Smyth, 1968).
Ratites: general observations
semen even from a wattled cassowary (Figure 14). The
ratites produce the typical sauropsid cell (von
Rautenfold, 1977; Soley, 1992; Pickett, personal com-
munication) and semen volumes may exceed 1 ml. In
ratites the phallus ®lls much of the cloaca and the
cloaca may contain mucous, waxy or greasy
substances. These substances should be wiped away
before collecting semen.

Larger ratites, like the ostrich, rhea, and cassowary, are
dif®cult to restrain during semen collection. Their feet
can in¯ict serious damage to the AI team. Because the
birds can kick more effectively forward than back-
ward, the team faces the greatest risk when in front of
the bird. Handlers should maneuver a larger unco-
operative ratite into a restraining device that prevents
the bird from turning around.
Before collecting semen, reach in the cloaca and
gently evert the phallus. If a ratite is properly condi-
tioned, one can use the massage technique to collect
Ostrich
The size and aggressiveness make the ostrich
dangerous, complicating semen collection. Handling
ostriches for semen collection requires an experienced
team. Mucous-like secretions on the phallus and urine
contamination also make collecting clean semen
samples dif®cult. Semen collection in the ostrich has
been described by von Rautenfeld (1977) and
Bertschinger et al. (1992). We provide a condensed
version below.
66 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Fig. 13 Semen collection Hispaniolian Amazon parrot (Amazona ventralis). Aviculturists can capture, restrain
and collect semen from parrots without the help of an assistant. This Hispaniolian Amazon parrot responded well
to semen collection when held in this clear plastic tube.

Handlers maneuver an uncooperative ostrich into a
squeeze cage, plucking crush, horse trailer, or similar
device to restrain the bird during semen collection
(von Rautenfold, 1977; Soley, 1992). The bird is
caught by the wings and is then lead with the head
held low into a plucking crush (Figure 15). Catching
poles, which look like a shepherd's crook, are some-
times used to catch the ostrich around the neck.
However, handlers must take care to avoid tearing
the delicate skin with the crook. When caught, the
ostrich is blindfolded to calm it down. Once in the
plucking crush, handlers insert a crossbar behind the
ostrich to prevent him from backing out. Although
ostriches mainly kick forwards, this also protects the
operator from ¯aying legs.
Due to the large size of the ostrich, semen
collection differs from the standard massage tech-
nique used in smaller species (von Rautenfeld, 1977;
Bertschinger et al., 1992; Irons et al., 1996). The
phallus must ®rst be extruded in order to locate the
seminal papillae. They are to be massaged to
stimulate semen ¯ow.
The dorsal border of the vent is pulled up with one
hand (by the operator or assistant) and the phallus
scooped out with the other hand. The tip is gripped
with a soft cloth and pulled down to fully extend the
phallus. The use of a soft cloth is essential as the
mucous-like secretions from glands situated at the base
of the phallus make it very slippery and dif®cult to
extrude and hold for semen collection (Bertschninger
et al., 1992). The secretions, however, do not easily
contaminate the semen sample. Feces can be rinsed off
the phallus using a physiological saline solution in a
squeeze-bottle and excess mucous present in the vent
can be wiped away. The pouch-like ventral extension
of the coprodeum, however, can be more problematic
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 67

Fig. 14 Semen collection from Southern cassowary (Casuarius casuarius). Charles Picket and Bill Mullens
developed a technique to collect semen from a Southern cassowary at the National Zoo in Washington, DC. Note
Bill faces forward with the cassowary to properly control the bird and avoid the bird's feet when released.

due to the collection of urine in this structure between
voiding (see below).
The ®ngers of the free hand are then inserted under
the uroproctodeal fold into the urodeum by following
the phallic groove. The seminal papillae are located
on the lateral walls of the urodeum. Then the
papillae are gently massaged alternately with the
®nger tip while periodically withdrawing the hand
to allow the semen to ¯ow down the phallic groove.
The collector catches semen in a semen collection
tube (Figure 16).
One problem experienced by the semen collector
during the procedure is the contraction of the very
strong anal sphincter muscles around the armywrist.
The pressure causes the collector to tire rapidly,
making it dif®cult to reach the papillae.
The fact that the ostrich has a ``bladder'' often leads
to urine contamination of the semen sample. Samples
collected after any sudden release of a large volume of
¯uid usually contain urine. The extruded phallus
results in the urodeum being stretched (Figure 16),
which, together with the intrusion of the ®ngers during
massage of the seminal papillae, depresses the ¯oor of
the urodeum. This allows the urine to ¯ow out of the
ventral extension of the coprodeum. This situation is
aggravated when the bladder is full. The color and
consistency of the urine are similar to semen, which
makes it dif®cult to distinguish and separate from the
ejaculate.
The mean semen volume is 0.6 ml with a range of
0.1 ± 1.5 ml. One can facilitate semen collection by an
intravenous injection of oxytocin (5 IU via the basilic
vein of the wing) 2 ± 4 minutes before collection.
Usually mean semen volume increases (mean 0.6 ml
without and 1.4 ml with oxytocin). If semen collec-
tion is unsuccessful, particularly in excited, unco-
operative or fractious birds, you can repeat the
injection after 10 minutes (Bertschinger et al.,
68 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Fig. 15 Male ostrich in plucking crush (Struthio camelus). A
lightweight material bag (large arrowhead) has been placed over
the head in order to cover the eyes to calm him and a crossbar
(small arrowheads) has been placed behind the femurs and below
the abdomen to prevent him from backing out.
1992). Oxytocin may also be useful stimulating
semen ¯ow in other birds.
Most ostrich copulations take place during the
earlier part of the morning (Sambraus, 1994). This
behavior has been recorded in wild birds, ostriches
housed in zoological gardens (Pocock, 1909) and in
commercial birds. Early morning semen collections
may increase semen samples from mated pairs.
Homosexual behaviour has been recorded in wild
male ostriches (Sauer, 1972) and involved cocks in
full breeding plumage performing the courtship
display in front of other males. Male courtship
display towards human handlers has also been
widely observed, a phenomenon that has been nega-
tively correlated with fertility of eggs (Bubier et al.,
1996 and 1998).
Cooperative semen collection
Falconers pioneered the use of sexually imprinted
birds of prey to collect semen (cooperative semen
collection) from males and later conditioned females
to accept insemination. The handling techniques of
falconry encouraged these raptors to regard human
beings as social and later, sexual partners.
Cooperative semen collection depends on a male
voluntarily ejaculating semen that the human handler
can collect and use to inseminate a female.
Even when not encouraged, some captive birds will
exhibit copulatory-like movements with perches or
other objects in the pen. This semen can be collected
when observed (Weaver, 1985). However, this beha-
vior can reduce fertility in naturally fertile pairs.
Interestingly, one famous zoological case was Topa
Topa, the captive California condor (Gymnogyps cali-
fornianus). Frank Todd rescued him from the wild as a
chick and raised him in captivity at the Los Angeles
Zoo. Topa Topa would mount Frank's shoe and
deposit semen. Fortunately, this aberrant behavior
was corrected when Topa Topa paired with a female
condor. The pairing process took avicultural ingenuity
and time to correct the behavior.
Human imprinting in birds is a complex phenom-
enon that will not be covered in detail here. Brie¯y, if
kept in close association with humans and isolated
from conspeci®cs, it is common to have hand-reared
birds socialize and express sexual behavior towards
their handlers. Male birds will perform courtship
behaviors with the aviculturist. If encouraged, the
birds will attempt to mount and copulate on the
handler or a surrogate device. If repeated frequently,
this type of behavior leads to full copulatory activities
with the bird ejaculating semen.
Cooperative semen collection can be quite
successful. Hammerstrom (1970) attempted coopera-
tive semen collection with golden eagles, (Aquila
chrysaetos). Temple (1972) collected semen from a
sexually imprinted red-tailed hawk, (Buteo jamai-
censis) that voluntarily ejaculated while mounting his
gloved hand. Berry (1972) did the same with
goshawks, (Accipiter gentilis), and Grier (1973)
succeeded with golden eagles. Boyd and Boyd
(1976) achieved similar results working with sexually
imprinted prairie falcons, (Falco mexicanus), and
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 69

p r o ct o d e u m w i t h f o l d e d p h a l l u s

terminal rectum

ureter
seminel papilla
urodeum

copro-urodeal fold
ductus deferens

bladder-like
extension of
coprodeum
c o n ta i n i n g
urine

c = coprodeum

Fig. 16 Male ostrich (Struthio camelus) cloaca.

J.H. Enderson (personal communication) with peregrine
falcons, (Falco peregrinus). If interested, Weaver and
Cade (1985) provide a detailed discussion of the
techniques.
In all these cases, the unrestrained males voluntarily
ejaculated semen that the handler could collect in or on
a suitable receptacle. Although the massage method
requires less cooperation by the bird than that neces-
sary for the cooperative collection method, both
require training and cooperation from the birds to
obtain good semen samples.
Electroejaculation
Since many wild birds like large psittacines are
dif®cult to handle, electroejaculation could be a
useful way to collect the semen quickly and reduce
the stress associated with handling (Harrison and
Wasmund, 1983). Electroejaculation does not require
the bird to cooperate. Electroejaculation is not a
common way to collect avian semen but is used
more frequently in domestic mammals (Almquist,
1968).
Some investigators claim advantages with
domestic ducks and geese (Serebrovski and
Sokolovskaja, 1934; Watanbe, 1957,) pigeons
(Betzen, 1985) and large parrots (Harrison and
Wasmund, 1983). Watanabe (1957), using electro-
ejaculation without prior training, collected duck
semen. It contained a greater cell concentration and
was in larger volume than that collected by the
conventional massage technique. The semen
collected produced fertile eggs comparable to
semen collected by the massage method, and no
adverse effects were observed in the drakes.
The cloacal probe (commonly used with small
mammals) contains both electrodes on the same
probe, is effective at lower amperage, and causes less
tissue damage. If using a cloacal probe, the semen
spreads out over the probe and through the vent. At the
Patuxent Wildlife Research Center, we observed the
same with Pekin ducks (Anas platyrhynchos) using an
70 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
internal probe and a Lane electroejaculator (Pulsator
II, Lane Manufacturing Inc. Denver, Colorado 80222)
For us in the Pekin duck, the massage method proved
simpler and quicker.
Betzen (1985) reported a pigeon electroejaculation
technique that overcomes some dif®culties associated
with massage semen collection. He used ketamine
hydrochloride as the anesthetic, a hypodermic needle
and alligator clips as terminals, and a cloacal retractor
to keep the vent open during stimulation. In the
ejaculation process, the cloacal muscles contract
closing the vent.
The external probes avoid the loss of semen on the
surfaces of the cloacal probe and associated collection
problems like drying of semen on the probe. If using
external probes, the semen spreads through the vent
and onto the exterior surfaces below the vent. The
retractor used by Betzen (1985) prevents the vent from
closing, facilitates semen collection and allows the
semen to pool. The collector collects small samples
in the urodeum.
Electroejaculation can cause serious burns if the
electrode is too small or the current to high (Harrison
and Wasmund, 1983; Betzen, 1985). Convulsions and
death have been reported in early studies (Serebrovskii
and Soklovskaja, 1934; Bonadonna, 1939; Burrows
and Quinn, 1935). Because of the need for a veter-
inarian and the possible adverse effects, we do not
recommend electroejaculation except in the most
special circumstances.
INSEMINATION
Cooperative insemination
One can adapt cooperative semen collection techni-
ques for use with females for insemination. Sexually
imprinted females respond to their handlers by
assuming copulation postures and by everting their
cloacas exposing the oviduct. No restraint is required
to inseminate. In some birds, a light pressure on the
lower back may facilitate the copulatory re¯ex. The
handler merely deposits semen in the cloaca or
oviduct.
As with collection of semen from imprinted males,
most of the work with inseminating imprinted females
has been done with birds of prey (Temple, 1972;
Berry, 1972; Grier, 1973; Grier et al., 1973; Boyd
et al., 1977; J.H. Enderson (personal communication),
and Weaver, 1985). Unlike massage insemination,
cooperative insemination causes little stress on the
birds.
Massage insemination
Using the massage insemination technique, we must
restrain the females during the insemination procedure.
The procedure used to inseminate females of nondo-
mestic species is essentially the same as the procedures
used routinely with poultry (Quinn and Burrows,
1936). Females are restrained and held so that the
cloacal region is fully exposed and accessible. Held in
this way, you can deposit semen into the cloaca or
directly into the vagina.
The aviculturist can locate the oviduct for insemina-
tion in one of two ways, palpation or eversion of the
cloaca. Placing ®rm pressure on the female's abdomen
and the walls of the cloaca can expose the vagina. With
some practice you can evert oviducts from birds as
large as cranes (Figure 17).
However, eversion of the cloaca may be dif®cult in
nondomestic birds. The stress may result in reduction
or cessation of egg production and possibly in breaking
a developing egg. In these cases, palpate the oviduct
(probe with the inseminating device or ®nger) to ®nd
the vagina. Guide the inseminating device into the
vagina to deposit the semen (Watanabe, 1957; Gee,
1983; Putnam, 1982; Johnson, 1954; Watanabe, 1957;
Olver, 1971). Speculums of appropriate size and shape
provide another way to locate the vagina. They help
one to visually guide the inseminating device without
everting the cloaca. Although sperm can fertilize the
egg without passing through the SST, we do not
recommend inseminations beyond the UVJ as a
regular practice.
Quail
Inseminating quail poses a bit more dif®culty than with
other birds. Smyth (1968) recommended the
McFarquhar and Lake (1964) technique. They start
by forcing the hard-shelled egg from the shell gland.
Once the eggshell is visible, the tip of the inseminating
device is placed against the shell. Then, maintaining
contact with the eggshell, the uterus is allowed to
return to its normal position. At that time, they
release the semen into the uterus. We like others
avoid inseminations in birds when a hard-shelled egg
is present in the oviduct, however, inseminating quail
is the exception (Smyth, 1968).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 71

Fig. 17 Insemination of Sandhill crane (Grus canadensis pratensis). Pressure on the abdomen can evert the
oviduct in the Sandhill crane and many other large birds. Generally, the operator can avoid cloacal eversion by
palpation, reducing the stress on nondomestic birds.

Eagle visualized the oviduct using a vaginal speculum

Good fertility results from vaginal insemination in
eagles (Grier, 1973; Gee and Temple, 1978).
Although massage and cooperative insemination
works, there are times when too little semen is
collected or is otherwise compromised to use typical
vaginal insemination. Blanco et al. (in press) devel-
oped a minimally invasive intramagnal insemination
technique for the eagle. He obtained fertile eggs with
very small semen samples after removing the contami-
nants.
Intramagnal inseminations were via an endo-
scopic procedure that required a plastic catheter
to by-pass the vagina, UVJ, and shell gland. He
followed the physical restraint with anesthesia
using iso¯uorane. Blanco placed the females in
the ventro-dorsal recumbency position and exposed
the cloaca by gently pulling the tail feathers up,
which in turn, allowed abdominal distention. He
adapted to the species size. A rapiscope, which is a
¯exible 2 mm-diameter endoscope, was used to visua-
lize the uterus lumen. He inserted either a 0.8 or
0.2 mm tomcat, non-commercial, sterile catheter with
stopper into the magnum lumen, 2 cm above the
uterus in golden eagles. Once the catheter was in
place, he removed the endoscope and pulled the
bird's tail further up so that the body was at a 75
angle. Contaminated semen was diluted with
Beltsville Poultry Semen Extender (Sexton, 1977),
centrifuged, the supernatant discarded and the
semen resuspended in extender. This was then put
into the magnum, using a 1-ml sterile non-toxic
syringe. After a 2-min wait, Blanco removed the
catheter and kept the bird in the same position for
an additional 5 min, allowing recovery from
anesthesia. Each bird was inseminated twice, 4 ± 5
days before oviposition of the ®rst egg of the clutch
with 66105 viable cells per insemination.
72 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Crane
Like other birds, a technician can evert the crane
oviduct (Figure 17) but should avoid it because of
the stress associated with the restraint needed. The
female response to the stimulation of the massage
technique results in visual exposure of the vagina if
the dorsal wall of the vent separating the urodeum
from the proctodeum (uroproctodael fold) is pushed
upward. With practice the technician can insert the
inseminating syringe into the vagina during the few
seconds it is observed. Even when not observed, one
can locate the opening to the vagina by palpation.
Archibald (1974) has successfully inseminated
imprinted female sandhill cranes. These females
readily assumed copulation posture and opened
their cloacas in response to massage stimulation
and handling.
Fertility levels usually exceed 90% if an adequate
amount of good semen is inseminated. Semen should
contain 16 million or more live cells per insemina-
tion. The bird should be inseminated one to two
weeks before oviposition, two or three times each
week during the breeding season and an additional
insemination made after each oviposition.
Ostrich
The mechanics of the insemination of an ostrich are
straightforward. Two assistants catch the bird and
hold it by the wings. The collector reaches into the
urodeum with the right hand and locates the opening
of the vagina with a ®nger. A curved pipette follows
the ®nger about 5 cm into the vagina where the
semen is deposited. If using fresh semen, insemina-
tion twice a week should be suf®cient. It is reported
(Madekurozwa, personal communication) that ostrich
hens will lay fertilized eggs for up to two weeks
after removal of the males thus indicating a two-
week sperm-storage period. Although this observa-
tion has not been scienti®cally tested, other studies
have indicated that fertile eggs can be laid for at
least 5 to 8 days after copulation (Swan and Sicouri,
1999).
The ostrich may be a spontaneous or re¯ex
ovulator. It appears that mating increases the rate
of egg laying. Egg production slows down or ceases
when the males are removed during the mid-season.
Males mate from once a week to a few times a
day.
Inseminating devices
Unlike the turkey industry where semen is dispensed
mechanically into specially designed insemination
straws, most nondomestic devices are single use
catheters, plastic tubes, or small syringes. French
straws with the blunt end minimize the loss of semen
in larger birds. We recommend Tomcat catheters for
smaller birds and for some passerines, intravenous
plastic catheters. Glass should be avoided because it
can break or chip and injure the bird. If you reuse the
device, avoid leaving soap or water residue, as both
will destroy the sperm. Take care to use material which
is not cell toxic and made for the purpose of semen
collection or tissue culture.
Depth of insemination and frequency
Much of the semen deposited in the vagina leaks back
into the cloaca and is lost. Turkeys and chickens retain
about 1% of the insemination dose (Brillard, 1995). In
the Bengalese ®nch, Birkhead (1992) also estimates
that the SST retain about 1% of sperm transferred
during copulation.
Deep vaginal insemination usually results in the best
use of semen and the highest fertility rates (Wentworth
et al., 1975; Lake and Steward, 1978). Good fertility
has been reported for the American kestrel at 1 ± 2 cm
(Bird et al., 1976), most falcons at 2 cm (Boyd et al.,
1977), the Houbara bustard at 1 cm (Saint Jalme et al.,
1994), the budgerigar at 0.2 cm (Samour et al., 1988),
the sandhill crane at 2 cm (Gee, personal observation)
and the ostrich at 5 cm within the vagina (Bertschinger,
personal observation).
However, vaginal inseminations may be dif®cult in
some nondomestic birds for a variety of behavioral and
anatomical reasons. While semen deposited deep in the
vagina results in the highest fertility, cloacal insemina-
tions are effective if done properly and frequently
(Smyth, 1968; Temple, 1972; Berry, 1972; Grier,
1973; Archibald, 1974; Gee, 1983; Bird et al., 1976;
Boyd et al., 1977). Gee found that in the sandhill
crane, fertility rates above 80% were possible from
cloacal placement of semen. The inseminations were:
(1) frequent (at least twice each week during the
breeding season), (2) accomplished with adequate
numbers of sperm (more than 16 million), (3) were
begun two or three weeks before the ®rst egg, and (4)
were completed within a few hours after every ovipo-
sition (Gee and Mirande, 1996).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 73

Table 3 Semen volume, sperm concentration, and duration of fertility for a variety of birdsa
Species Volume Concentrationb Fertility Refs
(days)
Domestic
Canary ˆ 10 ml 10 ± 13 Grif®th, personal communication
Chicken 0.5 ± 0.8 ml 3.56109 8 Smyth, 1968
Duck 0.2 ± 0.4 ml 2 ± 96109 8 ± 16 Johnson, 1986b; Watanabe, 1957
Goose 10 ± 800 ml Gee and Temple, 1978; Olver, 1971; John-
son, 1954
Pheasant, 50 ± 250 ml 106109 11 Smyth, 1968; Cain, 1978
Ring-necked
Pigeon 10 ± 20 ml Owen, 1941
Quail, Japanese 10 ml 56109 4±5 Smyth, 1968
Turkey 0.2 ± 0.3 ml 86109 45 Smyth, 1968
Nondomestic
Blackbird, Brewer's ˆ 10 ml ‡ Wolfson, 1960
Blackbird, Rufous 18 ± 24 ml 6.5 ± 6.76109 Anderson, personal communication
Budgerigar 5.4 ‡ 1.1 ml 2.56107 Hargrove, 1986
Cassowary, Wattled 1 ± 5 ml Picket, peronal communication
Cockatiel l50 ml Harrison, personal communication
Condor, Andean 0.1 ± 0.2 ml Gee, unpublished
Crane, Sandhill 10 ± 200 ml 0.36109 10 Gee and Temple, 1978; Putnam, 1982
Dunnock 10606106 Castro et al., 1996
Eagle, Golden 0.2 ml 9 Grier, 1973; Grier, et al., 1973
Falcon, Prairie 50 ± 100 ml 0.026109 6±8 Boyd, 1978; Boyd et al., 1977
Finch, House 10 ml ‡ Grif®th, personal communication
Finch, Zebra (1.96106 Birkhead et al., 1995
sperm producedyday)
Goshawk 20 ± 30 ml Berry, 1972
Hawk, Red-tailed 0.1 ml 6 Gee and Temple, 1978
Longspur, sg, Smith's 2176106 Castro et al.,1996
Monal, Chinese 31 ml 1.96109 Durrant et al., 1995
Monal, Himalayan 18 ml 1.76109 Durrant et al., 1995
Ostrich 0.6 ml 36106 14 Madekurozwa, personal communication
Ostrich 5±8 Swan and Sicouri, 1999
Ostrich with Oxytocin 1.4 ml Irons et al., 1996
Parakeet, Quaker 1.02 ± 2.96 75 ± 5796106 Anderson, personal communication
Parrot, Eclectus 50 ± 100 ml Ð Gee and Beall, unpublished
Sparrow, House ˆ 10 ml ‡ Gee, unpublished
Sparrow, Seaside ˆ 10 ml ‡ Gee, unpublished
Sparrow, Swamp ˆ 10 ml ‡ Wolfson, 1952
Stitchbird 14606106 Castro et al., 1996
Thrush, Wood 10 ml ‡ Wolfson, 1960
Tit, sg Bearded 65.66106 Birkhead and Hoi, 1994
Tragopan, Temminck's 33 ml 1.36109 21 ± 26 Durrant et al., 1995
Warbler, sg, Aquatic 1986106 Castro et al., 1996
Warbler, sg, Sedge 12 ± 176106 Birkhead et al., 1997a
a
Modi®ed from Gee (1983).
b
Number of spermatozoayml of semen.
‡ Greater than the domestic chicken.
ÿ Less than the domestic chicken.
ˆ Approximate.
sg, Seminal glomera.
74 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Insemination frequency depends on species speci®c
characteristics such as cell concentration, duration of
fertility (Table 3), and insemination method. Birds
with a short duration of fertility require more frequent
inseminations (Brillard, 1993). Recommended insemi-
nation frequency varies from once every other day in
Japanese quail (Lepore and Marks, 1966) to once every
other week in turkeys (Smyth, 1968). Chinese phea-
sants inseminated with fresh semen two to three days
per week during the season produced excellent fertility
(Durant et al., 1995).
Observing breeding behavior before egg laying has
lead to the practice of inseminating the female a few
weeks or days before the onset of egg production. By
doing this, the bird has adequate numbers of live sperm
in the SST and at the site of fertilization for the ®rst
ovulation (Bakst and Bird, 1987; Bakst, 1993; Brillard,
1993). In the wild, the northern fulmar copulates with
its mate, leaves to feed at sea for about three weeks,
and then returns to lay her single fertile egg without
mating again (Hatch, 1983). Some wild birds copulate
on migration many weeks before the ®rst egg (Quay,
1989). To improve fertility, crane inseminations are
initiated 10 to 14 days before the ®rst egg (Gee and
Mirande, 1996). Brillard (1993) has suggested that the
ef®ciency of sperm retention by the SST before
oviposition is nearly two times greater than after the
start of egg production in poultry. With this knowl-
edge, inseminators improved fertility in commercial
turkey production. Turkey hens are routinely insemi-
nated twice before the onset of egg production.
As a rule when working with a new species, we
recommend starting inseminations two weeks before
the ®rst egg. We repeat the inseminations every three
to four days and 4 ± 5 hours after each oviposition
during egg production. We avoid inseminations when a
hard-shelled egg is present. Frequent copulations
during the laying season and after each oviposition
occurs in some natural mating systems too (Sheldon,
1994).
Brillard (1993) lists three conditions that affect
fertility in domestic fowl. These include the hen's
age, the timing of insemination before the onset of
the egg production, and the timing of inseminations
during oviposition and the ovulatory cycle. Our
general rule, developed by trial and error over the
years, adapts to these same conditions in nondomestic
birds.
Older nondomestic birds appear to continue with the
same fertility levels from year to year although fertility
levels may decline during the season (Gee and
Mirande, 1996). Aviculturists have not conducted the
studies in nondomestic birds necessary to detect the
affect of age. Disease or injury, more likely in older
birds, can affect egg production (Carpenter et al.,
1987).
SEMEN VOLUME
Semen volume can vary from a few microliters to
several milliliters (Table 3). As expected, larger semen
volumes come from larger birds but not all large birds
produce large semen samples. Although a large bird,
mean crane semen volumes vary by species and range
from 0.03 to 0.1 ml, when collected by the techniques
as described in this report (Mirande et al., 1996).
Semen volume also varies with semen collection
method and the amount of accessory ¯uids added to
the semen at ejaculation. For example, the volume of
chicken semen is greater than that of turkey semen.
Yet, the concentration of sperm is less because the
relatively large volume of ``transparent ¯uid'' dilutes
chicken semen. In some wild birds, semen volume
decreases markedly when collected more than once a
day. Semen volume and semen quality may vary
throughout the reproductive season (Birkhead and
Fletcher, 1995). However, this is not so with turkey
males. Turkey semen quality varies little during the
season. Unlike domestic birds, most nondomestic birds
have short reproductive seasons (30 ± 60 days)
restricting the time available for semen collection
(Gee and Sexton, 1990; Birkhead and Fletcher, 1995).
Although lower volumes generally occur early and
late in the breeding season, smaller volumes can occur
during the height of the breeding season. The males
copulate or attempt to copulate so frequently that
semen available for collection decreases (Birkhead
and Fletcher, 1995). Larger semen volume was
collected from naturally fertile crane pairs with lower
fertility records than from birds with high fertility
records. Presumably, less semen was available
because the birds copulated more frequently (Chen
et al., 2001). A researcher should ®nd (with their
method of collection) the normal semen volume for
the species under study. Often you can improve
collections (successful attempts and volume) by
collecting early in the morning before the active
period of the day (Gee and Sexton, 1990). This
proved critical in the collection of semen from the
piping guan noted earlier (DeMatteo et al., 1998).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
Peregrine falcons also copulate early in the day and
semen collected later in the day is characterized by
lower volume, fewer cells and less sperm motility
(Blanco personal observation). In the domestic
turkey, semen is collected twice weekly. If insemi-
nation doses are made by sperm number rather than
by semen volume per dose, then it may be advanta-
geous to collect semen three times per week in the
turkey. Semen volume per collection will be lower
but sperm number over the course of the week will
be higher.
In the ostrich the mean semen volume obtained
using the techniques described above is 0.6 ml with a
range of 0.1 ± 1.5 ml (Bertschinger et al., 1992).
Similar values have also been reported by other
authors (Irons, 1995; Irons et al., 1996; Hemberger,
1996; Hemberger et al., 2001). After injection of
oxytocin 2 ± 4 minutes before semen collection, the
volume increases to a mean of 1.4 ml (Bertschinger
et al., 1992). It should be emphasized that, due to
limitations imposed by the collection technique, semen
samples obtained by this method probably do not
represent a physiological ejaculate.
SEMEN EVALUATION
The following discussion provides a few observations
related to nondomestic birds. Additional information
of semen evaluation procedures and how to do them is
found in Bakst and Cecil (1997a).
Small semen volume and remote locations often
make semen evaluation from nondomestic birds more
dif®cult than with domestic poultry. Bird behavior and
training of both the bird and the semen collector have
profound effects on semen volume, semen quality and
the number of successful collection attempts. We delay
evaluating the semen until the bird responds to almost
every collection attempt and exhibits cooperative
behavior. These samples usually contain less urate
contamination. Once a responsive pattern is estab-
lished, most captive birds will provide samples of
repeatable volumes and quality (Gee and Temple,
1978; Gee and Mirande, 1996).
A variety of pipettes and syringes are available to
measure the small semen samples from nondomestic
birds. For measuring small (10 ± 500 ml) semen
volumes, we use calibrated disposable capillary tubes
(20 ± 100 ml) with suction control and microliter
syringes (50 ± 1000 ml) attached to disposable capillary
tubes (Gee and Sexton, 1983; Gee and Mirande, 1996).
75
We ®nd white and red cell diluting pipettes attached
to microliter syringes work well to measure the volume
of small semen samples (20 ± 100 ml), to dilute the
sample collected, and to hold pooled samples in the
bulb part of the pipette. A short plastic tube (the center
section from a 1 ml syringe, for example) attached to a
suction device (propipette) works well to pool ejacu-
lates of 100 ± 500 ml (Gee et al., 1993).
The tip of any collecting device should be kept small
to reduce dehydration and reduce semen losses on the
wall of the tube. A small tomcat catheter (5 inches)
attached to the Luer end of a 1-ml syringe works well
to reduce surface tension and reduce semen loss.
One can store small (10 ± 100 ml) samples in capil-
lary tubes (sealed at both ends to prevent semen loss)
or store pooled samples in covered 2-ml pointed tubes.
The duration of storage depends on many variables
like temperature, species, diluent, etc. We place a wax
®lm over the open end of the 2-ml tube to reduce
evaporation and contamination from dirt and dust.
Tomcat catheters can be used to remove the semen
from the tubes (Gee and Mirande, 1996).
Technicians can make two-milliliter pointed tubes
from Pasteur pipettes by melting off the long narrow
glass tip where the pipette begins to widen. This leaves
a smooth rounded but small tip about 1 mm long.
A capillary tube or a 2-ml tube can be placed inside
a larger (4-ml) round-bottomed tube to prevent rapid
temperature ¯uctuations. We place the ``tube inside a
tube'' vertically in a rack in a cool (about 5 C),
insulated water bath. Semen from some species may
survive better at warmer temperatures (Smyth, 1968).
Therefore, we test each new bird species to ®nd the
best in vitro storage temperature.
Most bird semen will remain alive for 12 or more
hours in vitro (Weaver and Cade, 1985; Gee and
Mirande, 1996). However, it is best to evaluate the
semen within one hour of collection. Fertile eggs can
result from diluted semen shipped in insulated
containers on commercial airlines to distant locations.
These inseminations occur hours after collection
(Weaver and Cade, 1985; Gee and Mirande, 1996).
A number of papers have provided data on semen
evaluation parameters in ostriches (von Rautenfeld,
1977; Hicks, 1990; Bertschinger et al., 1992; Irons,
1995; Irons et al., 1996; Hemberger, 1996; Hemberger
et al., 2001). For the ostrich, volume, color, pH,
consistency, mass motility, individual motility, sperm
morphology and concentration prove the most useful
factors in evaluating semen (Bertschinger et al., 1992).
76 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Normal semen color varies from white to ivory and
consistency from thin to thick creamy. However,
semen consistency and color of the sample are decep-
tive. Consistency was not correlated with concentra-
tion, mainly due to the presence of granular-like
particles in the samples. The origin of these micro-
scopic granules is unknown and are found in other
nondomestic bird semen. Creamy samples were often
without sperm. Normal semen color is white to ivory.
Brownish samples were more frequently observed out-
of-season and were associated with dead sperm and
sperm debris (Bertschinger et al., 1992). Reddish
samples indicated the presence of blood due to
trauma during semen collection. The pH varies from
6.0 to 8.0. Good motility and concentration was most
often associated with a pH of 6.4. Urine contamination
pushes the pH into the more alkaline range.
The range of osmolalities observed in ostrich ejacu-
lates also provides proof of urine contamination. The
expected osmolality of semen should be close to that of
blood plasma and a mean value of 312 mOsmyl has
been observed. Osmolality, however, has been seen to
range from 180 to 893 mOsmyl (Bertschinger et al.,
1992).
Sperm number and concentration
Semen volume and sperm concentrations can vary
dramatically between species (Table 3) but most
birds produce semen concentrations that range from
2 to 106109 per ml. Sperm concentration in the
ostrich varies considerably from 1.67 to
6366106 yml. The mean total sperm count per ejacu-
late was 2006106 without and 4206106 with an
oxytocin injection. Hemberger et al. (2001) report
sperm concentration counts which range from 3.9 to
36.0 millionyml, with a mean value of 7.3 millionyml.
The American kestrel produces little semen (10 ml) per
ejaculate with a low sperm count (336106 ) yet
insemination with a single ejaculate results in good
fertility (Gee et al., 1993; Brock, 1986). Most nondo-
mestic bird ejaculates contain more sperm than that
needed for one insemination (Gee and Sexton, 1978)
and the ejaculate can be divided for multiple insemina-
tions. However, one must inseminate enough sperm to
insure fertility. The minimum number appears to be
about 1 million for the American kestrel (Brock, 1986;
Gee et al., 1993), 16 million for cranes (Gee and
Sexton, 1978; Gee et al., 1985), 150 million for
chickens, 150 million for turkeys, 80 million for
Guinea-fowl, and 70 million for ducks (Brillard,
1995). Repeated ejaculations decrease the sperm avail-
able and can exceed the rate at which the bird replaces
cells (Birkhead et al., 1995).
If a sperm count is impractical for your AI
program, technicians can score concentrations in a
capillary tube or on a hanging drop slide under a
microscope at 4306. One scores concentration as
follows: 0 ˆ no sperm; 1 ˆ few sperm with large
empty spaces; 2 ˆ many sperm with moderate
spacing between them; 3 ˆ sperm numerous with
little space between them; and 4 ˆ packed sperm,
hard to detect single sperm. In many nondomestic
species, diluted (1 : 1) semen with scores of 3 or 4
produce good fertility when inseminated.
When collecting semen, one needs to accommodate
sperm concentration and morphology in ways to
maximize collections of intact cells. For example,
dilute semen spreads throughout the cloaca.
Collecting semen at the base of the seminal papillae
increases recovery of sperm from dilute semen. Also,
semen with few sperm should not be diluted as much
as a concentrated sample. The insemination dose in a
diluted sample can exceed the bird's ability to retain
enough of the sperm inseminated to produce fertility.
Concentrated samples of sperm with long or stiff tails
need to be diluted before handling to decrease the
incidence of broken tails or other types of ¯uid sheer
damage.
For laboratory examination you can collect a small
quantity of undiluted semen in a plain (non-hepari-
nized) microhematocrit tube. The sample is taken by
quickly stabbing an uncontaminated part of the semen
in the collection device. Next, move the sample to the
middle of the capillary tube and seal with vinyl plastic
putty. You can label the sample with bird number by
writing on a small piece of transparent tape (¯ag)
attached to the capillary tube. Count the cells after
the appropriate dilution using the standard hemocyt-
ometer (Bakst and Cecil, 1989).
For species that provide large ejaculates with high
sperm count, the Unopette System (Becton-Dickson,
Rutherford, NJ) can be used to count the cells. After
the semen is drawn and gently mixed into the Unopette
System, add the diluted semen (1 : 200 dilution) to the
Neubauer hemocytometer counting chamber and allow
it to settle for a few minutes. Count the total number of
sperm in the four corner and central squares of the
counting chamber and multiply by 10,000 to obtain the
total sperm count per microliter.
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 77

Diluents can be used to extend the number of Tinamou

(spade-like
inseminations per ejaculate (Rowell and Cooper, Killdeer acrosome Chickens Doves
1960). However, dilution can reduce fertility when
the semen is diluted extensively. In the domestic acrosome

fowl, dilution of 1 part semen to 10 parts extender

reduces fertility (Friars and Mullen, 1971; Pistenma head
et al., 1971). Sexton (1979) recommends dilution not
to exceed 1 part semen to 5 parts chicken extender.
The physiology involved in the ``dilution effect'' is Ducks
(conical
mid-piece
poorly understood. Dilution does stimulate sperm midpiece)
Sooty Tern
motility and metabolic activity and may shorten cell tail
life in vitro. Steele and Wishart (1996a,b) showed that
excessive dilution removes sperm surface proteins but
does not otherwise harm the cells. Yet, this lack of American Kestrel

protein appears to interfere with the cells reaching the

SST.

Sperm morphology

Avian sperm vary from the simple sauropsid form to

a complex helical type with exterior ribbon like Woodhewers Corvids Violet Green Swallow
Ovenbirds Shrikes Tuffed Titmouse
membrane and long tail (McFarlane, 1962 and Robin
Wedge-tailed Summer Tanger
1971) (Figures 18a and 18b). More primitive Ant-thrushes Shearwater Common Snipe
Flycatchers
species have the simple form and the more recently Western Kingbird
evolved passerines the more complex form (Table 4).
The round to slightly ¯attened sperm in the American
kestrel is the exception to the rule (Brock and Bird, acrosome
ribbon
1991; Gee et al., 1993). Sperm size and relative ratios
of the various structures within sperm vary tremen-
dously and are species diagnostic (Briskie et al.,
head
1997; McFarlane, 1962, 1971). The reed bunting
has one of the longest sperm of any avian species
(291 mm) (Dixon and Birkhead, 1997). The sooty tern
has one of the shortest sperm of any avian species
mid-piece
(60 mm) (McFarlane, 1971). When handling avian
semen, one must be aware of sperm size,
morphology, and locomotion differences between
tail
species to avoid injuring the cells.
Gross sperm morphology provides a useful indica-
tion of the quality of the semen collected, damage
caused in collection and handling, and the effective-
ness of conditions used in storage. In some species, a Fig. 18 Avian sperm types (a) sauropsid and round. (b) Helical.
variety of unusual sperm types are common in semen.
These include giant cells, multiple tails, swollen
heads, broken tails and droplets (Gee and Temple, However, normal morphology showed a positive
1978; Russman, 1981; Gee et al., 1985; Gee and correlation with egg fertility (r ˆ 0:32). This informa-
Sexton, 1990; Marquez and Ogasawara, 1975; Soley tion would seem to indicate that morphology is a
and Els, 1993; Soley et al., 1996). There was no more reliable criterion for the selection of fertile male
correlation between egg fertility and percentage ostriches than other parameters. These abnormalities
motile sperm in the ostrich (Soley et al., 1991). may be more common very early in the reproductive
 78
Table 4 Avian sperm types
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Struthioni- Struthio Ostrich Sauropsid 70 2 11 3 2 Looks much like
formes camelus chicken sperm
Casuarius Double- Sauropsid 10
casuarius wattled and
Cassowary 14
Tinami- Cryturellus Tataupa Sauropsid 1 Spade-like anterior

G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

formes tataupa timamou projection on
acrosome
Procellarii- Diomedea Laysan Sauropsid 1 Blunt, rounded
formes immutabilis albratros acrosome
Oceanodroma Storm Sauropsid 1 Blunt, rounded
petrels acrosome
Puf®nus Wedge- Sauropsid Acro- 1 Helical acrosome
pacicicus tailed some
shearwater
Pelecani Tropic Sauropsid 1
formes birds,
gannets,
darters,
frigate-
birds
Ciconii- Butorides Green Sauropsid 10
formes virescens heron
(striatus)
Anseri- Waterfowl Sauropsid 1 and Pointed acrosome,
formes 10 tapered head and
mid-piece
Somateria Common Sauropsid 1 Pointed acrosome,
mollissima eider tapered head and
conical mid-piece
Falconi- Vulture, Sauropsid 1
formes hawks,
falcons
Falco American Round 3.3 3.4 9 Round, somewhat
sparverius kestrel ¯attened head
Table 4 Avian sperm types
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Galiformes Fowl Sauropsid 1 and

Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
12
Numida Helmeted Sauropsid 77.5 1.8 12.8 5
meleagris guinea
fowl
Lophura Silver Sauropsid 82 1.8 8.3 3
nycthemera pheasant
Galus Chicken Sauropsid 108 2 0.5 4 1,3, Pointed acrosome
galus 4, and tapered to head
12 and head tapered to
tail
Alectoris Rock Sauropsid 90 1.8 11.3 3.7 1 Sperm encased by
graeca partridge cytoplamic membrane
Meleagris Turkey Sauropsid 80 2.6 11 6 3,6
gallopavo
Gruiformes Cranes Sauropsid
Grus Florida Sauropsid 13.8 7,8
canadensis Sandhill
pratensis
G. c. Greater Sauropsid 13.8 7,8
tabida Sandhill
G. c. Lesser Sauropsid 11.3 7,8
canadensis Sandhill
G.c. Mississ- Sauropsid 12.1 7,8
pulla ippi
Sandhill
G. Red- Sauropsid 7.5 7
japonensis crowned
G. vipio White- Sauropsid 7.7 7
naped
G. Whooping Sauropsid 6.9 7
americana
G. grus Common Sauropsid 7.8 7
G. Indian Sauropsid 7.2 7
antigone Sarus
antigone
G. a. Eastern Sauropsid 6.5 7
sharpii Sarus

Anthropoides Demoi- Sauropsid 8.1 7

virgo selle

79
Table 4 Avian sperm types

80
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Gruiformes (continued)

Anthropoides Blue Sauropsid 7.2 7

paradisea
G. rubicunda Brogla Sauropsid 8 7
G. Siberian Sauropsid 7.7 7
leucogeranus
Bugeranus Wattled Sauropsid 7.8 7
carunculatus

G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Chardrii- Shorebirds, Sauropsid 1 Cylindrical shape
formes gulls, of head and
alcids slightly smaller
mid-piece, except
snipes and
sandpipers
Larus Black- Sauropsid 1 Thick head, button
marinus backed like acrosome
gull
Sterna Sooty tern Sauropsid 60 0.9 7.2 2.2 1 Sperm encased by
fuscata cytoplasmic membrane
Charadrius Killdeer Sauropsid 1 Cylindrical nucleus
vocifeus and blunt acrosome
Fratercula Atlantic Sauropsid 1 Narrow head and
artica (common) small button like
puf®n acrosome, mid-piece
one-half length of
head
Galachelidon Gull- Sauropsid 1 Small button like
nilotica billed acrosome, mid-piece
tern one-fourth length
of head
Himantopus Black- Sauropsid 1 Large button-like
himantopus necked acrosome, short
stilt head
Charadrius Wilson's Sauropsid 1 Button-like
wilsonia plover acrosome
Capella Common Helical 1 Helical acrosome
gallinago snipe and nucleus
Actitis Spotted Helical 1 Helical acrosome
macularia sandpiper and nucleus
Columbi- Claravis Blue Sauropsid 1 Mid-piece extends
formes pretiosa ground- down tail
dove
Columba Rock dove Sauropsid 160 2.4 16 105 1 Mid-piece extends
livia down tail
Table 4 Avian sperm types
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Psittaci- Parrots Sauropsid 10

Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
formes and
11
Poicephalus Senegal Sauropsid 10
senegalus parrot
Amazona Puerto Sauropsid 10
vittata Rican
amazon
Eclectus Electrus Sauropsid 10
roratus parrot
Amazona Hispaniol- Sauropsid 10
ventralis an Amazon
Melopsit- Budgerigar Sauropsid 11
tacus
undulatus
Cuculi- Cuckoos Sauropsid 1
formes Roadrunne,
Anis
Strigi- Owls Sauropsid 1
formes
Caprimulgi- Goat- Sauropsid 1
formes suckers
Apodi- Swifts, Sauropsid 1
formes humming-
birds
Trogoni- Trogan Collard Sauropsid 1 Button-like
formes collaris trogon acrosome,
cylindrical mid-
piece
Coracii- Megaceryle Belted Sauropsid 1
formes alcyon king®sher
Piciformes Wood- Sauropsid
peckers
Centurus Red- Sauropsid 90 1.2 13 5.4 1
carolinus bellied

81
Table 4 Avian sperm types

82
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Passeri- Perching Helical 1 Helical progress
formes birds from a simple
revolution to
tightly wound
cockscrew
Dendroco- Wood- Helical 1 One simple
Laptidae and creeper and revolution
Furnariidae ovenbirds

G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Formicari- Ant- Helical 1 Tightly wound coil
idea, thrushes,
Pipridae, cotingas,
Cotingdae and manakins,
Tyrannidae tyrant
¯y-
catchers
Corvidae and Crows Helical 1 Helix restricted to
Laniidae jays, nucleus, mid-piece
shrikes not in spiral
position
Deconychura Long- Helical 1 One simple
longicauda tailed revolution
typica wood-
creeper
Myiarchus Crested Helical 50 2.5 18.5 3.3 Acro- 1 Flattened and
crinitus ¯y- some twisted cell
catcher
Corvus American Helical 1 Helix restricted to
brachy- crow nucleus, small
rhychos acrosome, mid-piece
not in spiral
position

Parus Tuffed Helical 90 7.1 5.8 50 Acro- 1 Narrow ribbon

bicolor titmouse some length of acrosome
Viero Red-eyed Helical 80 8 5.5 10 Acro- 1 Narrow ribbon
olivaceus vireo some discontinues over
and nucleus. Continues
tail short distance down
tail
Table 4 Avian sperm types
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Passeri-

Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
formes (continued)

Piranga Summer Helical 170 12 3 146 Acro- 1

rubra tanger some
Tangara Bay-headed Helical 148 Nearly 1
gyrola tanger
complete

Trudus Robbin Helical 70 6.7 2.5 46 Nearly 1

migratorus
complete
Procnias Three- Helical 1 Extreme helical
tricarun- wattled con®guration
culata bellbird
Swallow Helical Mid-piece continues
with head
Tachycineta Violet- Helical 285 13.5 4.5 Acro- 1 Ribbon substantial
thalassina green some on acrosome
swallow
Petrocheli- Cliff Helical 81 10.3 1
don swallow
pyrrhonota
Hirundo Barn Helical 89 13 1
rustica swallow
Riparia Bank Helical 119 11.7 1
riparia swallow
Iridoprocne Tree Helical 233 12.6 1
bicolor swallow
Warbler Helical
Dendroica Prairie Helical 238 12.8 1
discolor warbler
D. Yellow- Helical 253 13.2 1
dominica throated
D. Black- Helical 260 13 1
caerulescens throated
blue
D. cerulea Cerulean Helical 263 13.4 1
D. castanea Bay- Helical 277 14.8 1
breasted
D. coronata Yellow Helical 284 13 Complete 1 Broad ribbon from
runmped acrosome to tip of
tail

83
Table 4 Avian sperm types

84
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Passeri-
formes (continued)

D. fusca Blackburian Helical 285 12.5 1

Seirus Oven Bird Helical 1 Appears ¯at but

aurocapillus the cell from
acrosome to mid-
piece completes one

G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

spiral
Tyrannus Eastern Helical 1 Helical, no ribbon,
tyrannus kingbird short acrosome
Tyrannus Western Helical 1
verticalis kingbird
Passer House Helical Complete 1 and
domesticus sparrow 10
Passer Tree Helical 1
montanus sparrow
Uroloncha Munia Helical 1
striata
Fringilla Chaf®nch Helical 1 and
coelebs 13
Taeniopygia Zebra Helical 1
castanotis ®nch
Spinus American Helical 10
tristis gold®nch
Spinus Pine Helical 1 Narrow ribbon
pinus siskin discontinues over
nucleus
Ammospiza Seaside Helical 10
maritima sparrow
Table 4 Avian sperm types
Order Bird or Bird Shape Sperm Acrosome Nucleus Mid-piece Ribbon Ref. Comments
species m m m m
Agelaius Red-winged Helical 14.2 1

Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
phoeniceus blackbird
Sturnus Starling Helical 10
vulgaris
References
1. Mcfarlane, 1962, 1971
2. Soley, 1992
3. Rose, 1996
4. Johnson, 1986b
5. Thurston et al., 1982
6. Marquez and Ogasawara, 1975
7. Russman, 1981
8. Sharlin, 1976
9. Brock, 1986
10. G. Gee, personal observations
11. T. Hargrove, personal communication
12. Lake et al., 1968
13. Furieri, 1962 ± 63
14. C. Picket, personal communication.

85
86 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
season in some birds (Bird et al., 1976; Blanco et al.,
2001) but not in others (Gee and Temple, 1978; Gee
and Sexton, 1990; Russman, 1981). Abnormal and
immature sperm, including spermatogonia and sper-
matids, are more commonly observed in peregrine
falcon ejaculates during the early stages of the
breeding season (Blanco et al., in press).
In nondomestic species that have experienced little
selection pressure, sperm morphology can have posi-
tive and negative in¯uences on fertility. Sharlin (1976)
and Sharlin et al. (1979) reported that within the
normal sperm population, cranes with sperm with
slightly longer heads had signi®cantly better fertility.
When counting cell types, one must insure that the
cell number is not changing. Many situations can cause
abnormal cells to burst leaving behind the more normal
cells (Gee et al., 1993). This can leave the impression
that the sample is better than another sample where the
abnormal cells remain.
Semen smears for the study of ostrich sperm
morphology can be prepared in two ways. The
ejaculate can be diluted with phosphate buffer,
making the smear, air-drying it and then ®xing
with glutaraldehyde (2% in Millonog's phosphate
buffer). Or the ejaculate can be stained with
eosinynigrosin before making the smear and then
air-drying it. The glutaraldehyde-®xed smears are
mounted with water and examined with a phase
contrast microscope. The glutaraldehyde method
provides the best microscopic sperm detail.
Eosinynigrosin smears give best results when
mounted under a cover slip with a commercial
mounting medium such as EntellanÕ (Merck)
(Bertschinger, unpublished data) before evaluating
either under bright ®eld or with phase contrast.
Ostrich sperm are of the simple, sauropsid type and
their structure has been described by a number of
authors (Retzius, 1911; von Rautenfeld, 1977; Soley,
1992). Ostrich sperm differ in a number of respects
from that of other non-passerine birds but show a close
similarity to sperm of the rhea and crested tinamou
(Asa et al., 1986; Phillips and Asa, 1989). The cells are
70 mm in length and vermiform (worm-like) in appear-
ance. The slender head is gently curved although
crescent-shaped and convoluted forms are also
observed (Soley, 1992). More detailed descriptions of
ostrich sperm morphology are available and again
illustrate the species speci®c differences that exist
among birds (Asa et al., 1986; Baccetti et al., 1991;
Phillips and Asa, 1989; Retzius, 1911; Soley, 1989,
1992, 1993, 1994; Soley and Roberts, 1994; von
Rautenfeld, 1977).
Live-dead cells
The eosin-nigrosin stained sperm slides have been
used to distinguish between live and dead sperm for
decades. The intact live cell membrane excludes the
eosin and the dead one does not. An intact membrane,
live or dead, excludes the nigrosin. The nigrosin
provides a blueyblack background on the slide. Live
cells appear white on the slide and dead appear red
(Bakst and Cecil, 1997b). Eosin-nigrosin smears are
best when mounted under a cover slip with a commer-
cial mounting media (Bakst and Cecil, 1997b). The
stain works well for most avian species and the
aviculturist can prepare the slides under most ®eld
conditions. In some species, dead cells also exclude the
eosin stain or in other ways fail to stain reliably.
Hargrove (1986) found this phenomenon most
pronounced in the Budgerigar (Melopsittacus undu-
latus). The American kestrel is another species that
fails to stain properly with eosin-nigrosin stains
(Brock, 1986; Brock and Bird, 1991; Gee et al.,
1993). Therefore, one should validate the eosin-
nigrosin staining process for each species.
The stained slides keep for a long time in the
laboratory and can be used for morphological exam-
inations (Gee and Temple, 1978). Technicians can
achieve repeatable results if they take care to prepare
the slides within standard protocol. Slides made in a
humid environment (common for nondomestic birds)
may dry slowly and cells die before the eosin has
dried. A small alcohol lamp or other drying device will
produce more uniform results.
A variety of ¯uorescent dyes are available like
ethidium bromide that penetrate dead cells but not
live cells (Bakst and Cecil, 1997c). Using a ¯uorom-
eter, ®rst the dead cells are measured. Then a chemical
added disrupts the membranes on all the cells. The
¯uorescence when measured again provides the total
cell ¯uorescence and an estimate of the ratio of live to
dead cells.
The microscope can be used to count live and dead
cells using ¯uorescent solutions that stain dead cells
and live cells differently. Differences are quite vivid
and anyone not hindered by colorblindness can count
the cells. These ¯uorescent tests allow one to simulta-
neously count live and dead cells (Donoghue,
1997a,b). Usually 100 cells are counted and the
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
percentages live calculated. Examinations like this
need to be tested with nondomestic species and
adapted to species speci®c differences.
The ¯uorescent tests are quick but short-lived. Time
can be a factor in getting repeatable results. But, if
done properly, these tests compare favorably with the
eosin-nigrosin estimates. Although not yet tested on
the sperm that give poor results with eosin-nigrosin,
they may be a better way to measure dead cells.
One can also stain cellular components like mito-
chondria. Fluorescent probes like rhodamine 123
(Sigma, St. Louis, MO) speci®cally binds to active
mitochondria that stains green under green light spec-
trum (546 nm). Most of these cell components are
located in the midpiece of the sperm, a structure
highly susceptible to the cryodamage (Bakst and
Sexton, 1979; Bakst and Cecil, 1997c). Damage to
the mitochondria greatly affects sperm motion in¯uen-
cing fertility. In addition, degree of mitochondrial
activity can be monitored using Mitotrack (Firelight
from Molecular Probes). This probe ¯uoresces red
when activity is high and turns to green when activity
is low (Blanco, unpublished).
Motility
Most non-passerine cells swim within the supporting
¯uids, propelled by the tail. Passerine sperm shoot
from side to side like darts propelled by the auger-like
movement of cell and the ribbon-like membrane
surrounding the cell.
The motile sperm can be determined using a hanging
drop slide, a slide under a cover slip or even in a
capillary tube. More high tech video camera systems
can provide computer scored results. A variety of other
motility tests are available and may ®nd use in some
nondomestic bird research. Dilution, extender,
temperature, light, vibration, chemical environment
(especially pH and osmolality) and observer in¯uence
motility scores (Bakst and Cecil, 1997a). When
making comparisons, carefully controlled, standar-
dized protocols should be used to reduce variability.
Most laboratories evaluating nondomestic avian
species do not have the equipment available to make
motility studies. However, a subjective motility score
when used by trained technicians produces repeatable
results. To do this, progressive motility is scored three
times to reduce variability. One scores the percentage
of sperm moving in a forward direction as follows:
0 ˆno motile sperm; 1 ˆless than 25% motile; 2 ˆ 25
87
to 49% motile; 3 ˆ 50 to 74% motile; and 4 ˆmore
than 75% motile.
If a sample has been held at 5 C, warm the sperm to
room temperature (about 22 C) before scoring the
sample. Sperm are less active when cold. We get
good fertility in cranes when the average concentration
and motility score is 3 or better, and in some crane
species 2 or better, if the entire ejaculate is used for
insemination (Gee and Mirande, 1996).
Ostrich semen does not demonstrate signi®cant mass
motility and individual motility is also poor in compar-
ison to chickens and turkeys (Bakst and Cecil, 1997a)
and mammalian sperm (Bertschinger et al., 1992).
Hemberger et al. (2001) reported signi®cant mass
motility in only 12% of ejaculates examined, with
little mass movement in 46% of the samples and no
movement in 42% of the samples. However, they did
record good individual motility which ranged from 42
to 96%, with a mean value of 78%. No proper wave
motion was observed with a hanging drop in the
ostrich. Instead we assessed individual motility under
a cover slip on a heated stage at 37 C. The % sperm
showing progressive or linear motility was assessed
subjectively. Normal ostrich sperm show typical spiral
movements as they progress in a linear direction. The
¯agellar movements, at least after collection, are very
rigid and do not show whip-lash patterns. This may
change once they have entered the female tract or have
been in the storage sites for some time.
Sperm-egg interaction assay
The sperm-egg interaction assay (Robertson et al.,
1997, 1998) may have use in nondomestic bird
research. This assay uses the chicken-egg inner peri-
vitelline layer to detect sperm with fertilizing ability.
We have used this assay successfully with crane sperm
using the perivitelline layer from chicken eggs.
FRESH SEMEN PROTECTION
Semen needs protection when collected. To maintain
viability, semen samples are quickly diluted with an
appropriate extender, the collection vessel covered or
sealed, and the semen stored in a cool, dark (4 C)
place. In general, this is a good way to start but the
aviculturist needs to tailor these conditions to the
species under study.
Several extenders used with domestic poultry
(BPSE, Minnesota Turkey Growers Association,
88 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley
Universal and Lake's) support most avian semen
samples (Sexton, 1977; Giesen and Sexton, 1983;
Blanco et al., 2001; Minnesota Turkey Growers
Association, St. Paul, MN 53114). Adjusting the pH
and osmolality of the domestic species buffers to the
ejaculated pH and osmolality of the nondomestic
species may improve in vitro sperm quality. Sperm
in Guinea fowl semen extended with a phosphate
buffer retain motility for up to ten days (Fujihara
et al., 1989) but for most birds sperm survive for a
day or two. In the ostrich, Hemberger (1996) claimed
good results using a new medium, which she referred
to as ``Triladyl-ostrich egg yolk-saline 0.7 sperm
diluent''. Although sperm survive for many hours,
fertility with fresh semen usually exceeds that from
stored semen.
Fecal and urate contamination causes problems in
some birds. Removing visible fecal and urate contam-
ination before collecting the semen often produces
good fertility (Gee and Mirande, 1996). Antibiotic
treatments in the extender may help maintain sperm
in contaminated samples (Gale and Brown, 1960;
Sexton et al., 1980). We usually avoid vaginal inse-
mination with contaminated samples, placing the
semen in the cloaca near the opening to the oviduct
(see eagle insemination for exception).
SEMEN PRESERVATION
Use
Properly frozen semen and other tissues provide
scientists and aviculturists with material for systematic
studies, for exchanges between zoological parks,
environmental monitoring and for retrospective
studies, forensic studies, and gene pool preservation.
Semen cryopreservation is the most practical proce-
dure for the preservation of avian gene pools. The
space required to store a frozen semen pool is gener-
ally small and the cost low compared to maintaining
live animals. Molecular components, especially
proteins and nucleic acids from frozen tissues,
support population genetic studies (Dessauer et al.,
1984) and retrospective pollution and disease studies
(Smith and Hafner, 1984; Carpenter et al., 1987).
Semen stains on clothing or other surfaces provide
species-speci®c markers that can be stable for long
periods. Scientists using frozen semen of known origin
identify these stains through comparative biochemical
tests (Dessauer and Goddard, 1984) and sperm through
microscopic examination (Gee, personal observation,
McFarlane 1962, 1971).
Studies of frozen semen before and after environ-
mental perturbations should play an important role in
environmental monitoring programs (Smith and
Hafner, 1984). Cryopreservation makes it feasible to
store tissues, blood, and gametes and to thaw them
when needed with little loss in viability.
Aviculturists can use frozen semen to minimize
inbreeding, maximize the number of genetically effec-
tive individuals, equalize the contribution from
founder individuals, and keep population sizes large
enough to reduce the losses of genetic diversity over
time (Ryder and Benirschke, 1984). Frozen semen
provides a way to exchange genetic lines between
captive populations without moving animals, a valu-
able resource when managing small populations. When
fresh semen is limited, frozen semen can be used in
combination with fresh semen to improve the chances
for fertility. Archibald and Gee used frozen and fresh
semen to inseminate an unmated whooping crane to
produce a chick, saving the whooping crane line
``Tex'' from extinction (United Press International,
1982).
Frozen semen can be used to eliminate deleterious
alleles by careful selection (Hanset, 1988; Schnatterly
and Hogge, 1989), to rejuvenate gene pools, to make
more effective use of proven sires, and to reduce
problems associated with disease transmission.
Frozen semen can be used when males and females
are reproductively active at different times of the year
or separated by great distances. Cryopreserved semen
can be used to prolong the use of sires after their death.
Gamete preservation makes it possible to maintain the
integrity of a species during periods of great risk or
prolonged restoration efforts (Herman, 1968; Ryder
et al., 2000).
However, we need much additional research on
freezing and storing gametes and embryos if germ
plasma cryopreservation is to become a viable
management tool for nondomestic birds (Gee, 1984;
Hammerstadt, 1995). Avian semen cryopreservation
programs have failed to meet the goals envisioned 20
to 30 years ago by the poultry industry (Hammerstadt,
1995). A factor in the failure was preservation tech-
nique. Available chicken semen preservation techni-
ques are complicated and make chicken AI more
expensive. Also, chicken breeders have made rapid
genetic progress and holding genetic lines of special
value has not been a priority. Although the turkey
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
industry could bene®t from frozen semen technology,
cryopreservation of turkey semen proved more dif®-
cult. With the end of many standard bred lines and
breeds of chickens, a frozen gene pool makes sense if
we want to retain diversity in the gene pool. A few
nondomestic-bird, frozen-semen banks do exist. The
lack of techniques and inadequate funding restrict
these to a few large institutions (Ryder et al., 2000).
Cryopreservation of avian sperm
Scientists have recognized the potential of long-term
preservation of avian sperm for decades. Shaffner
et al. (1941) was the ®rst to successfully freeze-
thaw fowl sperm. The ®rst semen to be frozen-
thawed and used to produce live offspring followed
the serendipitous discovery that glycerol was an
effective cryoprotectant (Polge, 1951). Glycerol has
been widely used as a cryoprotectant for the sperm of
both poultry and nondomestic species. However, the
associated protocols remain troublesome due to the
need for multiple procedural steps during freezing
and thawing. One must remove glycerol before
insemination because of its contraceptive effects in
birds (Hammerstedt and Graham, 1992; Hammerstedt,
1995). Fertility after thawing also is less than
optimal. Less than 2% of poultry sperm are capable
of fertilizing after thawing when compared with fresh
semen (Wishart, 1985).
Other researchers encountered similar problems
with cryopreserved sperm from wild species.
Arti®cial insemination in the greater sandhill crane
with sperm cryopreserved in 6% dimethylsulfoxide
(DMSO) yielded 50% fertile eggs (Sexton and Gee,
1978; Gee et al., 1985; Galem, 1987) compared to
90% fertility using the same number of fresh sperm
(Figure 19). In the American kestrel, 56% of the eggs
were fertile after insemination with fresh semen and
30.4% with frozen-thawed semen containing 12.3%
dimethylacetamide (DMA) (Brock and Bird, 1991).
This low rate of fertility obtained with cryopreserved
avian sperm may result from sensitivity to the freeze-
thaw process and the less than optimal methods of
preservation (Hammerstedt and Graham, 1992) or
possibly other semen components (Gee, personal
observation).
Additionally, sperm function, transport and storage
in the hen's reproductive tract appear compromised
after cryopreservation (Donoghue and Wishart, 2000).
Normally, birds maintain sperm in sperm storage
89
tubules within the hen's reproductive tract for days
to weeks. Refrigerated storage or cryopreservation of
sperm signi®cantly reduces the duration of fertility
(Donoghue et al., 1995).
Despite limited fertility using thawed sperm,
researchers have adapted procedures to many nondo-
mestic species (Table 5). Rates of success, however,
have been highly variable across studies, in part, due
to the lack of systematic, cryobiological approaches.
Factors that in¯uence results include cryoprotectant,
time of equilibration, semen quality, semen concen-
tration, and insemination volumes, timing and
frequency. Especially important is species speci®city,
which can vary remarkably even among closely
related taxa on the evolutionary tree. For example,
in a study evaluating sensitivity of sperm to freezing
rates among chicken, turkey and four raptor species,
signi®cant species differences were evident. Rapid
cooling was detrimental to sperm from Bonelli's
eagle, golden eagle, peregrine falcon and turkey yet
was advantageous to cryopreserving Imperial eagle
(Aquila adalberti) and chicken sperm (Blanco et al.,
2000). The crane follows a pattern similar to the
turkey for which rapid cooling is detrimental to
sperm survival compared to slower rates (70:5 to
72 Cymin). Domestic bird species protocols are
used routinely (or modi®ed only slightly) for nondo-
mestic species. Yet this study clearly demonstrated
that bird species vary so signi®cantly in fundamental
sperm sensitivities that each requires signi®cant basic
research before sperm cryopreservation is effective.
Although there is a need to address issues at the
cryolevel, practical issues remain a concern. For
example, urate contamination is common in ejaculates
collected from Aleutian Canada geese (57%) (Gee and
Sexton, 1990), Magellanic penguin (62%) (O'Brien
et al., 1999); golden eagle (37%); Imperial eagle
(43%); Bonelli's eagle (Hieraaetus fasciatus) (29%)
and the peregrine falcon (48%) (Blanco et al., 2000).
Urate contamination reduced sperm survival in frozen-
thawed Aleutian Canada goose (Branta canadensis
leucopareia) semen (Gee and Sexton, 1990).
However, semen from Aleutian Canada geese was
frozen and used to improve genetic diversity in a
founder population sent to Japan (Figure 20).
Although one can physically separate semen from
fecal and urate contamination, some semen may be lost
or impossible to separate (urine). Washing urine-
contaminated semen has avoided some fundamental
challenges of contaminated ejaculates when followed
90 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Fig. 19 First crane from frozen semen (Grus canadensis pratensis). Bruce Williams holds the ®rst crane
produced through AI with frozen-thawed semen. Since the 1970s many cranes and other species of nondomestic
birds have been produced from insemination with cryopreserved semen.

by intramagnal insemination described earlier (Blanco Cryoprotectants

et al., 2000). Inseminating in the cloaca and not the
vagina helps to avoid infections from contaminated
semen (Perek et al., 1969). Basic cryobiological
research combined with the continued search for
novel sperm collection, storage and insemination
technologies will allow assisted breeding to play a
more signi®cant role in the management and conserva-
tion of rare bird species.
There are many nonpenetrating and penetrating cryo-
protectants available for avian semen preservation.
Things like glucose, sucrose, and polyvinylpyrrolidone
are nonpenetrating cryoprotectants. A nonpenetrating
cryoprotectant (levulose) was the ®rst cryoprotectant
used in domestic fowl (Shaffner et al., 1941; Shaffner,
1942).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation 91

Table 5 Successful cryopreservation of nondomestic bird semen

Buckland, 1977; Tereshchenko, 1985; Tajima et al.,
Species Sperm Chicks Source 1990; Brock and Bird, 1991; Blanco et al., 2000).
survive produced
Sperm freeze-thaw survival depends on factors other
Aleutian Yes Yes Gee and than cryoprotectant. Each species and sometimes,
Canada goose Sexton, 1990 members of the same species may react differently
Andean condor Yes No Gee, 1995
depending on their physiological ability to overcome
American Yes Yes Brock and
kestrel Bird, 1991
or prevent damage from the freeze-thaw process. The
Gee et al., major factors that affect sperm survival include
1993 osmotic stress, the cryoprotectant and its concentra-
Budgerigar Yes Yes Samour et al., tion, time of equilibration before freezing, temperature
1988
at equilibration, the diluent, container, rate of freezing,
Crane Yes Yes Gee et al.,
1985 presence or absence of energy sources etc. (Blanco
Bonelli's Yes No Blanco et al., et al., in press). Centrifugation, washing, dilution, and
2000 ®ltering associated with some cryopreservation
Imperial eagle Yes No Blanco et al., processes may limit the fertilizing ability of frozen-
2000
thawed sperm (Sexton, 1973; Steele and Wishart,
Golden eagle Yes Yes Blanco et al.,
2000; Blanco 1996a,b).
et al., 2001; Many cryprotectant extenders have been used to
Knowles-Brown improve sperm survival during freezing and thawing.
and Wishart,
2001
These include chemically de®ned solutions like BPSE,
Peregrine Yes Yes Parkes et al., and natural products like milk and egg yolk. We prefer
falcon 1986; Samour, to use some variation of BPSE with either DMSO or
1988 DMA as the cryoprotectant. Yet, several other diluents
Megellanic Yes No O'Brien et al., are effective including Lake's, MTGA, and Biggers,
penguin 1999
Whitten and Whittingham's medium (Lake, 1968;
Chinese Yes No Durrant and
pheasant Burch, 1991; Samour et al., 1988). These extenders can make a
Durrant et al., difference. Please see Lake and Ravie (1984) for a
1995 review of diluents used effectively in poultry.
Edward's Yes Yes Rose, 1996 Most investigators freeze semen in small French
pheasant
Silver Yes Yes Rose, 1996
straws (0.5 ml French straw [Fiche de gontrols]
pheasant Edwards Agri-sales Inc. Baraboo WI) (Gee, 1996)
Piping guan Yes No DeMatteo et al., but recently quick freezing semen in a pellet has
1998 proven successful in chickens (Tselutin et al., 1995).
Houbara Yes Yes Hartley et al., Gee (1987) provides a detailed account of the actual
bustard 1999
freezing process using straws and DMSO with cranes
(Figure 19). Pursel (1979) developed the ®rst
successful pellet freezing technique in swine at the
Penetrating cryoprotectants include things like Beltsville Agricultural Research Center in the 1970s.
glycerol, ethylene glycol, DMSO, and DMA (Sexton, In the 1990s, Tselutin developed similar pellet tech-
1973, 1975, 1976; Brock, 1986). A great variety of nique for domestic chickens in the Russian Research
other cryoprotectants are available like polyethylene Institute of Farm Animal Breeding and Genetics. The
oxide, formamide, methyl formamide, dimethyl forma- process uses DMA as the cryoprotectant (Tselutin
mide, propanol, and methyl pyrrolidone (Sexton, 1973, et al., 1995).
1975; Oderkirk and Buckland, 1977; Graham et al., With the help of Tselutin, we successfully cryopre-
1982a,b; Lake and Ravie, 1984). served chicken semen using the pellet technique.
Comparisons of cryoprotectants prove dif®cult Success was limited when we tried to cryopreserve
because of differences in freeze-thaw methods, AI turkey, and crane semen using the pellet technique.
systems, dose rates, and in evaluations of the results. These studies should continue until we ®nd the reasons
Several authors have attempted to compare cryopro- pelletizing works in the chicken and fails in turkeys
tectants in birds (Sexton, 1973; Oderkirk and and cranes. It appears from recent results that avian
92 G.F. Gee, H. Bertschinger, A.M. Donoghue, J. Blanco and J. Soley

Fig. 20 Brood of Aleutian Canada geese from arti®cial insemination with frozen semen. Frozen semen produced
broods of the endangered Aleutian Canada goose with greater diverse paternal lineage than possible through
natural matings. Japan used these birds to establish a captive ¯ock and their young for a reintroduction program.

sperm differ in the ability to survive rapid freezing
even in closely related species (Blanco et al., 2000).
Glycerol, DMSO and DMA protect avian semen
during cryopreservation (Sexton, 1979; Gee et al.,
1985; Fujihara and Buckland, 1987; Brock and Bird,
1991; Tselutin et al., 1995). Glycerol has a contra-
ceptive affect when inseminated with sperm and one
must remove it after cryopreservation and before
insemination (Hammerstedt and Graham, 1992;
Hammerstedt, 1995). Recently developed equipment
for the dialysis of semen samples provides an effective
way to remove glycerol from large semen samples
(Buss, 1993). Glycerol has been used to produce fertile
eggs in nondomestic birds: 33% fertile in the peregrine
falcon (Parkes et al., 1986) and 11% fertile in the
American kestrel (Brock and Bird, 1991).
Although glycerol has been used in nondomestic
birds, DMSO and DMA hold more promise with many
nondomestic birds because of the small semen samples
collected in many species. Also, aviculturists can use
semen directly, without removing the cryoprotectant
(Table 5).
 Reproduction in nondomestic birds: physiology, semen collection, arti®cial insemination and cryopreservation
Regardless of the cryoprotectants and extenders
used, semen survival seldom exceeds 50%.
Laboratory and insemination trials are under way
with both DMA and DMSO in our labs. We are
moving these studies from testing changes in tech-
nical conditions in freezing trials to phenomenolo-
gical and mechanistic models using the comparative
approach across avian taxa (Blanco et al., 2000).
Good fertile egg production from frozen-thawed
semen will be the ®nal test of any of these
approaches.
SUMMARY
There has been progress in understanding the fasci-
nating reproductive strategies of wild birds. Yet, there
are more than 9000 bird species, and only a handful
has been studied. Early studies have recognized a
remarkable variation in reproductive mechanisms
among species. These things considered, the magni-
tude of the challenge becomes apparent. Birds evolved
to survive in many microenvironments around the
world. These adaptations brought changes in reproduc-
tive strategies and physiological functions that ensure
self-perpetuation. Without a comprehensive under-
standing of these specialties in natural reproduction
we may miss valuable knowledge, important scholarly
information and information useful in management
and conservation.
Thus, understanding the reproductive physiology of
birds can help species management. These bene®ts
range from providing knowledge for animal curators
to improve husbandry, to `assisted breeding' espe-
cially through AI. `Assisted breeding' helps overcome
the problem of captive birds failing to copulate due to
physical and behavioral incompatibilities. Combined
with effective sperm storage (cryopreservation), AI
would permit easy movement of genes between
distant breeding facilities, avoiding the need to trans-
port stress-sensitive birds while ensuring that every
genetically valuable individual has the chance to
reproduce. In theory, it also may be possible in the
future to inseminate wild birds (from small, frag-
mented populations) using sperm from unrelated
males, thereby promoting genetic diversity.
However, for now, we need a better understanding
of how sperm respond to different cryoprotectants. It
is also time to consider how to preserve the female
gamete and studies should begin using domestic
poultry as a ``model'' species. Finally, the routine
93
storage of gametes from endangered birds through the
development of genome resource banks would
provide a hedge against extinction in the face of
unforeseen catastrophes (e.g., disease epidemics,
loss of prey, drought or ¯ood). An avian frozen-
sperm bank is the most practical approach for now.
Similar systematic banks of sperm have been estab-
lished for certain mammals, both domesticated and
wild.
ACKNOWLEDGEMENTS
Special thanks for ®nancial support to the USDA
Foreign Agricultural Services Project #SP33, the
USGS Patuxent Wildlife Research Center, the Junta
de Comunidades de Castilla-La Mancha, and the
University of Pretoria.
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