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State of the Art

Semen Collection, Evaluation, and


Cryopreservation in Exotic Animal Species: Maximizing
Reproductive Potential

Barbara S. Durrant

Introduction holding space requirements and increase the number of


species that can be conserved through captive breeding.
The judicious assimilation of genetic material from frozen
In 1980, Thomas Lovejoy of the World Wildlife Fund stat- reserves into extant breeding populations will ensure con-
ed that reduction in the biological diversity of the planet is tinuance of genetically diverse species in captivity.
the most basic issue of our time and that slowing this pro- Importation of wild-caught individuals of endangered
cess of "bioa'c impoverishment" is a great challenge to the species is now greatly restricted. When cryopreservation
ingenuity of biologists. Extinction of a species represents protocols have been perfected, it will be feasible for
the loss of a resource that has evolved through thousands, researchers to collect germ plasm "in the field" to sustain
perhaps millions, of years of mutation and natural selec- captive breeding programs. Incorporation of germ plasm
tion. The genetic diversity that now exists in captive exotic from individuals remaining in the wild into captive popula-
animals will steadily dwindle through random processes tions will increase the genetic diversity of captive groups
without continuous input from wild populations. The without the losses that would occur by removing free-rang-
National Research Council (1978) has stressed the impor- ing animals from the wild.
tance of conserving this genetic material and emphasized
Using frozen gametes and zygotes for artificially
that if immediate action is not taken, much of this vital
enhanced reproduction offers greater potential for genetic
resource will be depleted in the near future.
contribution from underrepresented captive individuals to
It is incumbent upon American zoos, as stewards of a extant and future generations. Overrepresented individuals
large proportion of the earth's captive species, to maintain can contribute germ plasm to future generations when their
them in sufficient numbers to ensure that genetic variability genes may again benefit the population. Because the
is adequate for long-term genetic health. Notter and Foose majority of captive species are within a few generations of
(1985) have calculated that nearly 50,000 individuals would genetic input from the wild, the most immediate need is to
be required to maintain 99 percent of a species' genetic preserve the genetic diversity that currently exists in cap-
diversity for 1,000 generations. Because of severe space tive populations.
limitations in zoos and animal parks, this ideal genetic
The efficacious use of cryopreserved germ plasm
diversity will not likely be attainable for any of the species
requires the development of artificial insemination (AT), in
we are attempting to save from extinction. Perhaps a more
vitro fertilization (IVF), and embryo transfer (ET) tech-
realistic goal is to preserve 90 percent of a species' diversi-
niques for each endangered species.
ty for 1,000 generations with less than 5,000 individuals.
The potential value of cryopreserved germ plasm for
Even that greatly reduced number of individuals is impos-
basic and applied research has yet to be realized in wildlife
sible to maintain in captivity.
species (Ryder and Benirschke, 1984). Once sufficient germ
However, long-term germ plasm storage can minimize cells and embryos have been preserved to assure the vigor-
Barbara S. Durrant is head of the Reproductive Physiology Division, Cen-
ous continuation of a species, excess genetic material in
ter for Reproduction of Endangered Species, Zoological Society of San these forms can be made available for biological research
Diego. to benefit the species. Frozen germ plasm will provide

ILARNEWS
researchers with materials to conduct fundamental studies niques into technologies, and finally, technologies must be
in disease transmission, genetics, sperm-egg interaction, applied to site-specific circumstances. The difficulty in
and early embryonic development transferring basic research to applied research is regarded
Eaglesome et al. (1980) have compiled considerable data as a primary impediment to the development of useful
concerning infection of the gametes and early embryos of technologies.
domestic species by viral, bacterial, and protozoan agents. The dearth of consistently successful Al or ET programs
Knowledge of the vulnerability or resistance of semen and for exotic animals illustrates the difficulty of extrapolating
embryos to various disease agents is of great importance successful protocols from domestic species or humans to
when formulating import-export regulations concerning endangered species and the need for carefully designed
the transfer of genetic material across state and national studies in male and female reproductive and gamete physi-
borders. This issue is a matter of considerable concern to ologies.
exotic animal breeders as well as commercial livestock It has been stated that the most efficient method for pre-
industries. Expansion of these studies to include exotics serving gene pools is semen cryopreservation (Gee, 1984).
will depend upon the availability of germ plasm from cap- Collecting semen is less problematic than collecting ova or
tive wild species. embryos; thus, more samples can be made available for
Heterologous in vitro fertilization can be used to evalu- cryopreservation studies. In addition, semen thawing and
ate the karyotype of sperm cells (Rudak et al., 1978). The Al require less training and equipment than do embryo
technique could potentially facilitate identification of lethal thawing and ET. Therefore, Al is feasible for more zoologi-
or undesirable chromosomal aberrations and, by removing cal institution personnel. For example, American cattle
affected males' semen from the breeding population, the breeders performed 200,000 ETs in 1985 primarily using
elimination of the defect from the population. fresh embryos collected and transferred by reproductive
Embryo sexing by staining or karyotypic analysis prior physiologists or veterinarians. In contrast, almost all of the
to ET will allow the maintenance of the most favorable sex 10.5 million dairy calves produced by Al that year were
ratios in captive breeding groups. For species that are nor- conceived with frozen semen inseminated by farm person-
mally housed in single male groups (i.e., Przewalski's hors- nel and trained technicians (U.S. Congress, Office of
es [Equus przewalsHi] and lion-tailed macaques [Macaca Technology Assessment, 1987). Because most zoos do not
silenusj), problems associated with housing excess males employ reproductive physiologists or veterinarians trained
could be alleviated by embryo sexing. in embryo transfer techniques, Al offers the opportunity for
Intraspecies in vitro fertilization studies may elucidate more zoos to cooperate in programs designed to maximize
mechanisms of fertilization or early embryonic develop- the genetic diversity of captive species.
ment that can be enhanced for greater reproductive success
or inhibited for contraception.
Our future dependence on cryopreserved germ plasm to
Semen Collection
maintain genetic diversity obligates the curators of "frozen
zoos" to store only the highest quality samples. Although
the successful management and propagation of endangered The intractability of most exotic species severely limits
species depends on the long-term storage and discriminat- our ability to collect semen without chemical restraint.
ing use of cryopreserved germ plasm, the technology to Hence, electro-ejaculation (EE) has become the standard
properly preserve it is not yet in place. To achieve the goal collection technique in zoos and primate centers (Figure 1).
of functional germ plasm depositories, extensive research The physiological mechanisms of EE are well understood
must be undertaken to optimize cryopreservation tech- (Martin, 1978), and the prerequisite tranquilization or
niques for each species. Although individual research facil- immobilization appears to be the most significant health
ities and zoological institutions are beginning to establish concern associated with the procedure.
and stock private germ plasm banks, no large-scale central- Adherence to a regimented protocol of stimulation for
ly organized or coordinated program exists for sampling, each EE procedure has been suggested (Wildt et al., 1983).
evaluating, preserving, and using available sources of germ However, because response to EE can vary greatly between
plasm (Council for Agricultural Science and Technology, males of the same species and even between successive
1984). attempts with the same male, standard EE practice calls for
The U.S. Congress Office of Technology Assessment, in modification of the protocol during each procedure based
its 1987 report, "Technologies to Maintain Biological on the animal's response (Median et al., 1981). Voltage and
Diversity," describes a series of steps through which useful number of stimulations required for erection, ejaculation,
technology emerges: first, basic research provides an or both is affected by the animal's plane of anesthesia. Gen-
understanding of the nature of a biological system. As a erally, the deeper the anesthetic plane, the more stimulation
result of these basic studies, researchers define require- is required (Gould et al., 1978). Some tranquilizers and
ments and develop techniques to manage a species or its anesthetics are contraindicated for EE (Meltzer et al.,
genetic resources. Next, researchers must translate tech- 1988). Even identical doses of anesthetics are not always

Volume 32, Number 1 Winter 1990


gorilla) has been reported (Fussell et al., 1973; Gould et al.,
1985 ). In San Diego, a hand-raised cheetah (Acinonyx
jubatus) contributed 200 semen samples to a cryopreserva-
tion program over 4 years using an AV, and a second chee-
tah contributed 30 samples over 9 months (Durrant et al.,
1989). Three mother-raised cheetahs were sufficiently
nonaggressive to permit routine collection with an AV.
An alternative opportunity for noninvasive semen col-
lection is the use of ejaculates obtained by masturbation
(Gould et al., 1985; Martin et al., 1978). At San Diego a
number of primate species, including the drill baboon

V d (Papio leucophaeus) and the lion-tailed macaque, have


provided ejaculates for detailed evaluation and cryopreser-
vation. Rewarding successful masturbatory behavior
(preferably in an off-exhibit area) may be, for certain
males, a most efficient method of obtaining large numbers
of samples for cry ©preservation, artificial reproduction
studies, or both.

O
Figure 1 Semen collection by electroejaculation of tranquilized
(top) cheetah (Acinonyx jubatus) and (bottom) cimitar-horned oryx
(Oryx dammah).

similarly effective, probably due to physiological differ-


ences between males, including those differences resulting
from the stress of capture and restraint. The small body of
literature describing the influence of anesthesia on EE must
be greatly expanded to provide guidelines for exotic
species.
Semen collection with an artificial vagina (AV) offers
the advantage of frequent sampling without the stress of
chemical or physical restraint (Figure 2; Durrant et al.,
1985). The contact required to train a male to service an AV
excludes all but the most tractable animals. AV semen collec- Figure 2 Collection of semen from (top) cheetah (A. jubatus) and
tion in chimpanzees (Pan troglodytes) and gorillas (Gorilla (bottom) tapir (Tapirus pinpinchaque) using an artificial vagina.

ILAR NEWS
gy, and behavior. It is a complex continuum that begins
with spermatogenesis and proceeds to sperm maturation in
the epididymis, ejaculation, sperm transport through the
female reproductive tract, and finally, penetration of the
ovum. Each step in this scheme (greatly oversimplified) is
the subject of intense scientific investigation aimed at
defining the myriad biochemical processes leading to nor-
mal fertility and the perturbations that result in subfertility
or sterility. The biological basis of male infertility is still
not well understood even in the most extensively studied
species.
The ultimate test of male fertility is, of course, concep-
tion. The fertilizing capacity of sperm can be evaluated by
examining ova for evidence of fertilization following natu-
Figure 3 Collection of semen from a pheasant (Tragopan temmincld) ral breeding or artificial insemination. Unfortunately, for
using the massage technique. the majority of exotic mammals, the time of ovulation is
not known, and artificial insemination techniques have not
been perfected. Removal of ova from females for micro-
Semen collection in avian species is not widely prac- scopic examination is not a procedure that could be justi-
ticed in zoos despite the relative ease of capture and physi- fied for routine fertility assessment An alternative strategy
cal restraint. Imprinted birds have been trained to deposit would be to inseminate ova in vitro. However, the ova of
semen on or in appropriate receptacles (Boyd and endangered species are not readily available for such stud-
Schwartz, 1983). Manual "massage" collection has been ies. These difficulties also plague researchers working with
described for various species (Figure 3), including cranes human subjects and have led to attempts to develop labora-
(Grus conodensis; Gee, 1983), budgerigars (Melopsittacus tory tests of semen quality that are correlated with fertility.
undulatus; Samour et al., 1986), and pheasants (Lophopho- Traditional parameters of semen quality include volume
rus Ihuysii; Spiller et al., 1976). Although a relatively and sperm motility, concentration, and morphology. As sin-
uncommon technique in birds, EE has been reported in par- gle predictors of fertility, none of these measurements are
rots (Harrison and Wasmund, 1983) and ducks. The latter sufficiently sensitive. When these parameters are com-
have been shown to respond to EE with larger volume and bined, the accuracy of fertility assessment is enhanced but
higher concentration ejaculates than with the massage tech- still incomplete (as none of these criteria can predict the
nique (Watanabe, 1957). ability of sperm to perform effectively in the female repro-
There are very few published reports of semen collec- ductive tract).
tion in reptiles. Cloacal massage and Al of resultant semen Truly comprehensive semen analysis involves evalua-
were reported in snakes by Mendgen et al. in 1980. EE tion of the ability of sperm to
yielded semen samples heavily contaminated with urates.
• reach the site of fertilization,
A combination of EE and manual penile massage was suc-
• undergo capacitation and the acrosome reaction,
cessful in producing ejaculates in unanesthetized Galapa-
gos tortoises (Geochelone elephantopus) and Aldabra tor- • penetrate the zona pellucida, and
toises (G, gigantea) (Durrant, unpublished data, 1982). • fuse with the ooplasm and decondense (Bedford,
Larsen and Cardeilhac (1984) did not find EE useful in alli- 1981).
gators (Alligator mississippiensis), but were able to collect In vitro tests have been developed for each of the above
sperm by penile groove cannulation and postmortem epi- parameters, and their correlation with fertility has been the
didymal extraction. much-disputed topic of many research reports.
Postmortem extraction of sperm can be developed as an Motility, once considered an adequate predictor of fertil-
important source of germ plasm. Sperm from a pygmy ity, is not consistently correlated with in vitro or in vivo
chimpanzee (P. paniscus), lion-tailed macaque, and patas fertilizing capacity (Anderson et al., 1980; Hall, 1981).
monkey (Cercopithecus patas) frozen as long as 12 hours Recognition that motility of sperm in culture medium on a
after death have shown acceptable postthaw motility and glass microscope slide may not represent its ability to tra-
ability to penetrate hamster ova in vitro (Durrant, 1987). verse the female reproductive tract prompted development
of the postcoital test (Tredway et al., 1978). Quantification
of sperm numbers and motility in human cervical mucus
following insemination gives a more accurate measure of
Semen Evaluation the functional motility of sperm. For exotic animals, recov-
ery of cervical mucus following breeding is impractical or
Male fertility is a symphony of physiology, endocrinolo- impossible. The collection of estrous cervical mucus for in
Volume 32, Number 1 Winter 1990
vitro testing is likewise unfeasible. Gaddum-Rosse and
associates (1980) offered an alternative by recording the
progress of human sperm through estrous bovine cervical
mucus (BCM). Keel and coworkers (1987) found that, for
humans, BCM penetration was superior to the postcoital
test in its ability to evaluate the functional motility of
sperm and predict pregnancy. The BCM penetration test is
simple to perform with commercially available mucus
preparations, and its usefulness in semen evaluation of
exotic animals warrants investigation.
The latest entry in the field of semen evaluation is com-
puter assisted semen analysis (CAS A). Its greatest advan-
tages are elimination of the subjective nature of routine
semen evaluation and the addition of detailed motion anal-
ysis unquantifiable by visual examination. Reports corre-
lating CASA parameters with fertility are scarce. However,
Mathur and coworkers (1986) reported a significant differ-
ence in the swimming speed and linearity of sperm from
fertile versus infertile men as calculated by CASA. These
differences could not be detected by manual analysis. Figure 4 Penetration of cheetah (A. jubatus) sperm into hamster
Amann (1989) reported a significant multiple correlation (Mesocricetus auratus) ova in the heterologous sperm penetration
between six swimming parameters and a competitive fertil- assay. Three decondensing sperm heads are seen within the
ooplasm (400 x).
ity index in bulls. Because the sperm of each species varies
in size and morphology, CASA programs written for
human or bovine semen are not immediately applicable to
most exotics. The reprogramming necessary to analyze cess in humans (Yanagimachi et al., 1976). When fertile
each sperm type likely to be encountered in a zoo physiolo- and infertile men were compared by the SPA and by tradi-
gy laboratory is, at least at this time, prohibitive. In addi- tional spermiogram, Rogers (1985) found far fewer false
tion, the extensive analysis necessary to correlate CASA positives and false negatives with the SPA. Experiments by
parameters with fertility will be difficult to carry out with van Kooij and coworkers (1986) demonstrated the ability
the limited number of samples available for exotic species. of the SPA to differentiate infertile men with normal
Tests that evaluate sperm's ability to reach the site of spenniograms from fertile men. The heterologous nature of
fertilization must be considered to be only one facet of the assay dictates caution when interpreting results, as
comprehensive semen analysis. The ability of sperm to mechanisms of oolemma fusion and decondensation may
capacitate, acrosome react, and penetrate the zona pelluci- differ from homologous fertilization in vivo. However,
da and the ovum must also be assessed. The mammalian there is general agreement that the SPA is a valid indicator
zona pellucida is rarely if ever penetrated by sperm of other of sperm's ability to capacitate, acrosome react, fuse with
species. Thus, to assess the ability of sperm to negotiate the the oolemma, and decondense (Prasad, 1984).
zona, homologous ova are required in the assay system. The sperm of numerous exotic species—including dol-
Postmortem collection of ova with intact zonae may pro- phin (Tursiops truncatus; Fleming et al., 1981), marmoset
vide an occasional opportunity to perform homologous (Callithrix jacchus; Moore, 1981), bat (Lambert, 1981),
zona penetration assays. Perhaps a more feasible system cynomolgus monkey (M.fascicularis; Hoffman and Curtis,
would be to determine if sperm of the target species were 1984), rhesus macaque (M. mulatto; Boatman and Bavister,
capable of penetrating zonae of closely related species 1984), budgerigar (Samour et al., 1986), patas monkey,
from which they could be routinely collected and frozen or lion-tailed macaque, and pygmy chimpanzee (Durrant,
salt stored (Yanagimachi et al., 1979). 1987), tiger (Post et al., 1987), and cheetah (Durrant et al.,
Ruling out in vivo fertilization assays and penetration of 1989)—are capable of penetrating hamster ova with vary-
homologous ova in vitro for logistical reasons, heterolo- ing success. However, no attempt has been made to corre-
gous ova must be substituted for tests of fertilizing capaci- late the SPA with fertility in any of these species.
ty. In 1972, Yanagimachi reported the penetration of zona- The SPA may find its greatest usefulness in the evalua-
free hamster ova by guinea pig sperm. Since then, the tion of various cryopreservation protocols. Combined with
unique ability of hamster ova to be penetrated by sperm of other semen analysis parameters, the loss or retention of
other species has been extensively explored as a means to ability to penetrate hamster ova could be used to discrimi-
determine the fertilizing capacity of sperm. This heterolo- nate between freezing methods and aid in the development
gous sperm penetration assay (SPA; Figure 4) has been of optimal cryopreservation protocols for endangered
developed for widespread use in the prediction of IVF suc- species.

ILAR NEWS
The acrosome reaction is considered an end point of semen samples in the field for later examination under a
successful sperm capacitation. Human sperm must be acro- light microscope, this simple technique may be a valuable
some reacted prior to penetration of zona-free hamster ova addition to semen analysis in exotic species.
(Yanagimachi, 1984). Thus, the SPA may be used to for- A relatively new technique that measures the functional
mulate in vitro capacitation techniques. The ability to or physiological integrity of the sperm plasma membrane is
capacitate sperm in vitro is essential for FVF studies or for the hypoosmotic swelling test (HOS; Jeyendran et al.,
AI using immature sperm collected from the epididymis. 1984). The test, which is easily read with a light micro-
Other less complex assays of the acrosome reaction are scope, assesses the ability of the membrane to transport
available. For many species, light microscopic examination water. If the membrane is functional, the sperm tail will
is not sufficient to visualize the unstained acrosome. coil within the swollen membrane. These authors reported
Because electron microscopy is expensive and labor inten- a significant correlation between the percentage of swollen
sive, several acrosome stains have been developed that can tails and percentage of human sperm penetrating in the
be evaluated with light microscopy. The divalent cation SPA. This straightforward technique—in combination with
ionophore A23187 is often used to induce the acrosome other evaluation techniques—may be a useful adjunct to
reaction in vitro to confirm that the stain technique being semen evaluation in exotic species. It has been shown to
tested can differentiate reacted from unreacted sperm correlate well with sperm motility in cheetahs and pheas-
(Green, 1978). A triple stain designed by Talbot and Cha- ants (Durrant, unpublished data, 1989).
con (1981) is frequently used to assess spontaneous (nor- No single semen evaluation parameter is likely to be
mal) acrosome reactions and those occurring as the result proven predictive of fertility. However, combinations of the
of sperm degeneration. Although this technique requires above techniques, such as a fluorescent acrosome stain, a
only a light microscope for acrosomal status evaluation, it supravital stain, and the HOS test, may give reasonably
is time consuming. A faster method uses chlortetracycline accurate profiles of potential fertility.
fluorescent stain (Ward and Storey, 1984), which causes The direct comparison of various analyses using the
reacted sperm to fluoresce in the region of the acrosome. same samples (or at least the same males) will streamline
The disadvantage of this technique and other fluorescent evaluation of semen of each species by eliminating redun-
stains is the requirement for a microscope equipped with dant tests and demonstrating which are most highly corre-
epifluorescence optics. The potential toxicity of fluorescent lated with fertility. For example, the SPA, HOS, and triple
stains can be assessed by incorporating a supravital stain acrosome stain were all applied to aliquots of the same
into the assay. ejaculates from men of known fertility or infertility (van
Exclusion of a supravital stain is a measure of the struc- Kooij et al., 1986). Only the SPA was significantly differ-
tural integrity of the sperm plasma membrane (Figure 5; ent between fertile men and infertile men with normal
Schrader et al., 1986) and is a well-studied adjunct to tradi- spermiograms. Multivariate discriminant analysis has been
tional semen evaluation (Eliasson and Treichl, 1971; used to select semen parameters capable of predicting fer-
Lasley et al., 1942). Supravital stains are not equally appli- tility. One study with human subjects found semen volume,
cable to sperm of all species (Varner et al., 1987), and the sperm motility, the log of sperm concentration, the SPA,
suitability of each stain must be determined for each and the HOS to be significant discriminating factors,
species. Because supravital stains are easily applied to which, when taken collectively, predicted the fertility of
these men with 77.6 percent accuracy (Wang et al., 1988).
Analysis of sperm morphology has long been part of tra-
ditional semen evaluation. Individual males with high pro-
portions of severe sperm anomalies, such as detached
heads, would be expected to be subfertile due to impaired
motility (Figure 6). The relationships between less serious
sperm defects and fertility are not clear. In one study of
domestic stallions, the male with the highest number of
abnormal sperm had the highest pregnancy rate (Voss et al.,
1981). Two clouded leopard (Neofelis nebulosa) males of
proven fertility had extremely high proportions of sperm
abnormalities, as did six other males of unknown fertility
(Wildt et al., 1986). Free-ranging and captive cheetahs
have uniformly poor semen quality, including a large per-
centage of morphological abnormalities (Durrant et al.,
1985; Wildt et al., 1983). All proven sires at the Zoological
Society of San Diego exhibit these semen characteristics
Figure 5 Supravital staining of hamster (M. auratus) sperm. Live that, if observed in domestic felids, would signal potential
sperm has excluded the stain; dead sperm is stained (100 x ) . infertility (Durrant et al., unpublished data, 1988).

Volume 32, Number 1 Winter 1990


must be established. Comprehensive semen analysis before
and after freezing is essential for assessing damage and dis-
criminating between various cryopreservation protocols.
Each of these steps represents a significant investment of
time, money, and semen.
Numerous reports in the literature claim the successful
freezing of semen of exotic species (see reviews by Gra-
ham et al., 1978, and Wildt, 1986). The end point of most of
these early experiments was the demonstration of postthaw
motility, however slight. (For purposes of this paper, the
birth of offspring following AI with frozen semen will
define successful semen freezing.)
Few studies have involved evaluation of frozen semen
by any parameter other than motility. Haigh and coworkers
Figure 6 Sperm of the greater kudu (Tragelaphus strcpsiceros) illus- (1986) compared three media with and without EDTA and
trating common morphological abnormalities (detached heads and estimated acrosomal damage of cryopreserved wapiti
coiled tail; 100 x).
(Cervus elaphus) sperm with light microscopy in addition
to motility at thaw. Motility differed in only one medium
when EDTA was added, but acrosomal damage was
Correlation of sperm or semen characteristics with fer- decreased in two media when it was added, which perhaps
tility should not be mistaken for fertility prediction. The indicates the superior discriminating power of acrosomal
final step required to develop predictive semen analysis is assessment. Howard and colleagues (1986) studied sperm
to apply techniques shown to be correlated with fertility in motility, duration of motility, and acrosomal damage of
one set of samples to different sets of samples from males African elephant (Loxodonta africana) sperm frozen in
of unknown fertility (Amann, 1989). This step has not been several freezing media and in two thawing media. The per-
carried out for any species. Without large numbers of sam- centage of normal acrosomes was the most effective
ples from known fertile and infertile males and the ability parameter for discriminating between the different freezing
to use aliquots of the same ejaculate for evaluation and AI, media. Cryopreservation of ferret (Mustela putorius)
it may not be possible to use semen evaluation parameters semen in three media, by two freezing methods and two
to predict fertility in most exotic animals. thawing methods, was evaluated by postthaw motility and
acrosomal integrity (Howard et al.,1989). One protocol was
selected based on these studies, and a 70 percent pregnancy
Semen Cryopreservation rate was achieved following AI. Sixteen cryopreservation
protocols for cheetah semen compared extender composi-
tion and pH, freeze rates, and packaging methods (Durrant
Semen cryopreservation is a complex and poorly under- et al., 1989). Postthaw motilities did not differ between
stood technique that is consistently successful in a very cryopreservation methods, and the SPA was not satisfacto-
limited number of species. Because the biochemical prop- ry as an indicator of retained fertilizing capacity in this
erties of semen vary widely between species, protocols species. These preliminary studies represent the beginning
must be redesigned for each new animal under investiga- of a new field of study.
tion.
In 1972 the first pregnancies were established in an
The remarkable success enjoyed by the dairy industry exotic species—the fox (Vulpes vulpes)—with frozen
using frozen semen for AI is the result of many years of semen (Aamdal et al., 1972). A slowly emerging literature
research and field trials with thousands of experimental reports successful pregnancies in the following exotic ani-
animals. Additional factors are the relative resistance of mals: reindeer (Rangifer tarandus; Dott and Utsi, 1973),
bovine sperm to damage during the freezing and thawing wolf (Cards lupus; Seager et al., 1975), crane (Sexton and
process and intense selection of males for semen quality Gee, 1978), red deer (C. elaphus; Kelly and Moore, 1981),
and high fertility. wapiti (Haigh et al., 1984), addax (Addax nasomaculatus;
From this history of cryopreservation protocol develop- Densmore et al., 1987), kestrel (Falco sparverius; Brock et
ment in cattle, the basic experimental parameters may be al., 1984), falcon (F. peregrinus; Parks and Hardaswick,
derived. First, an appropriate extender or diluent must be 1987), and budgerigars (Samour et al., 1988).
formulated for each species. Second, the correct concentra-
For many exotic species, semen freezing techniques
tion of the proper cryoprotectant equilibrated at the right
probably will be similar to those developed for closely
temperature for the optimum period of time must be added
related domestic species. Taxonomically unique species,
to that medium. Finally, freeze rates and thaw methods
such as the cheetah, however, may require extensive

ILAR NEWS
research to elucidate appropriate protocols. For example, Durrant, B., T. Schuerman, and S. Millard. 1985. Non-invasive semen col-
lection in the cheetah (Acinonyx jubatus). Proc. Am. Assoc. Zool.
studies with antelope or gazelle semen will logically begin Parks Aquar. 1985:564-567.
with cryopreservation techniques developed for domestic Durrant, B. S., J. K. Yamada, and S. E. Millard. 1989. Development of a
cattle, the semen of wild equids may freeze as well as semen cryopreservation protocol for the cheetah. Cryobiology
26(6):542-543.
domestic stallion semen using the same methods, and Eaglesome, M. D., W. D. D. Hare, and E. Singh. 1980. Embryo transfer:
human semen freezing methods will serve as models for A discussion of its potential for infectious disease control based on a
freezing primate sperm. Comprehensive evaluation of review of studies on infection of gametes and early embryos by vari-
sperm through the application of multiple analyses both ous agents. Can. Vet. J. 21:106.
Eliasson, E., and L. Treichl. 1971. Supravital staining of human spermato-
before and after freezing is essential to the development of zoa. Fertil. Steril. 22:134-137.
freezing protocols, as well as the identification of appropri- Fleming, A. D., R. Yanagimachi, and H. Yanagimachi. 1981. Spermatozoa
ate evaluative procedures for sperm of each species. of the Atlantic bottlenose dolphin, Tursiops truncatus. J. Reprod. Fer-
til. 63:509-514.
Fussell, E. N., L. F. Franklin, and R. C. Frantz. 1973. Collection of chim-
panzee semen with an artificial vagina. Lab. Anim. Sci. 23:252-255.
Gaddum-Rosse, P., R. J. Blandau, and W. I. Lee. 1980. Sperm penetration
Summary into cervical mucus in vitro. I. Comparative studies. Fertil. Steril.
33:636-643.
Gee, G. F. 1983. Crane reproductive physiology and conservation. Zoo
Biol. 2:199-213.
The importance of semen cryopreservation to the future Gee, G. F. 1984. Value of frozen tissue collections for gene pool preserva-
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