SURVIVAL OF CANINE EPIDIDYMAL SPERM UNDER COOLED AND
FROZEN-THAWED CONDITIONS
A Thesis
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
Requirements for the degree of
Master of Science
In
The Department of Animal Sciences
By
Karla Stilley
B.S., Centenary College of Louisiana, 1994
December 2001
ACKNOWLEDGEMENTS
The author would like to express her appreciation to her major professor,
Dr. Robert A. Godke for his support and guidance during her graduate study. The
author would also like to thank Dr. Earle Pope and Dr. Dale Paccamonti, members
of the examining committee, for their assistance with this project.
The author would also like to thank her fellow graduate students: Joel
Carter, Richard Cochran, Mike Collins, Glen Gentry, Aidita James, Brett Reggio,
Allison Landry, Oscar Perez, Stacy Hoffman and Heather Green for their unending
help and support. And a special thanks is extended to Darin Hylan for his excellent
example. The author also extends a special thanks to her friends: Shannon,
Tanya, Colleen, Bobby and Kellie for their caring and encouragement.
The
author would also like to recognize Dr. Kevin Thibodeaux, Dr. David Gardner, Dr.
Michelle Lane and Mrs. Angela Hunsinger without whose help, this would not have
been possible. The author expresses her deepest gratitude to her family for
always believing in her. With a very special thanks to her sister, Tiara Harper, her
greatest protector and best friend. And finally, the author would like to thank Jody,
for being her best motivation.
ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS ................................................................................ ii
LIST OF TABLES .......................................................................................... v
LIST OF FIGURES....................................................................................... vi
ABSTRACT ..................................................................................................vii
CHAPTER
I. INTRODUCTION ................................................................................. 1
II. LITERATURE REVIEW ....................................................................... 4
Assisted Reproductive Technologies in the Dog and Wild Canids .. 4
Domestic Canine Reproductive Physiology................................. 4
Exotic Canine Reproductive Physiology ...................................... 5
Oocyte Maturation, IVF and ET ................................................... 6
Semen Collection and AI ........................................................... 10
Cryopreservation ........................................................................... 12
The Effects of Cryopreservation on Sperm Cells....................... 12
Cryopreservation of Dog Sperm ................................................ 14
Epididymal Sperm ......................................................................... 15
Role of the Epididymis............................................................... 16
Use of Epididymal Sperm in ART .............................................. 17
Epididymal Sperm of Exotic Species ......................................... 22
Problems with the Reproduction of Endangered Species.............. 24
Small Population Sizes.............................................................. 24
Loss of Heterozygosity .............................................................. 26
The Effects of Inbreeding on Reproduction ............................... 28
Need for New Founder Animals ................................................ 31
Problems Faced by Zoos........................................................... 32
How ART Can Address These Problems .................................. 34
III. THE EFFECTS OF STORAGE AT 22°C VS. 4°C ON THE
SURVIVAL OF EPIDIDYMAL MOUSE SPERM ................................ 36
Introduction.................................................................................... 36
Materials and Methods .................................................................. 39
Experimental Animals................................................................ 39
Experimental Design ................................................................. 39
Sperm Extraction and Evaluation .............................................. 40
Statistical Analysis..................................................................... 44
Results .......................................................................................... 44
Discussion ..................................................................................... 51
iii
IV. THE EFFECTS OF STORAGE AT 22°C VS. 4°C ON THE
SURVIVAL OF EPIDIDYMAL CANINE SPERM ................................ 59
Introduction.................................................................................... 59
Materials and Methods .................................................................. 63
Experimental Design ................................................................. 63
Tissue Processing ..................................................................... 65
Sperm Analysis ......................................................................... 65
Testicle Storage ........................................................................ 67
Statistical Analysis..................................................................... 68
Results .......................................................................................... 69
Discussion ..................................................................................... 77
V. COMPARISON OF FRESH, REFRIGERATED AND
FROZEN THAWED CANINE EPIDIDYMAL SPERM ........................ 86
Introduction.................................................................................... 86
Materials and Methods .................................................................. 89
Experimental Design ................................................................. 89
Testicle Processing ................................................................... 90
Testicle Storage ........................................................................ 92
Sperm Evaluation ...................................................................... 92
Cooling, Freezing and Thawing ................................................. 95
Statistical Analysis..................................................................... 97
Results .......................................................................................... 97
Discussion ................................................................................... 101
VI. SUMMARY AND CONCLUSION..................................................... 109
LITERATURE CITED ................................................................................ 112
APPENDIX: SPERM STORAGE DATA..................................................... 133
VITA .......................................................................................................... 137
iv
LIST OF TABLES
Table 2.1. Cooling and freezing of epididymal sperm
of exotic species...................................................................... 25
Table 3.1. Experimental design for epididymal mouse sperm
stored at 22°C (Treatment A) and 4°C (Treatment B) .............. 41
Table 4.1. Experimental design for epididymal dog sperm
stored at 22°C (Treatment A) and 4°C (Treatment B) .............. 64
Table 4.2. Initial testicular measurements and epididymal sperm
parameters of canine testicles for Treatment A (22°C)
and Treatment B (4°C) ............................................................. 73
Table 5.1. Experimental design for storage of canine epididymal
sperm in cooled and frozen conditions..................................... 91
Table 5.2. Effects of storage of canine epididymal sperm in cooled
and frozen conditions on sperm motility ................................... 99
Table 5.3. Effects of storage of canine epididymal sperm in cooled
and frozen conditions on percentage of membrane-intact
sperm ..................................................................................... 100
v
LIST OF FIGURES
Figure 3.1. Effects of storage of mouse testicles at 22°C
(Treatment A) and 4°C (Treatment B) on
testicle weight........................................................................... 48
Figure 3.2. Effects of storage of epididymal mouse sperm at 22°C
(Treatment A) and 4°C (Treatment B) on
sperm motility ........................................................................... 49
Figure 3.3. Effects of storage of epididymal mouse sperm at 22°C
(Treatment A) and 4°C (Treatment B) on percent
live sperm................................................................................. 50
Figure 4.1. Effects of storage at 22°C (Treatment A) and
4°C (Treatment B) on the motility of epididymal
dog sperm ................................................................................ 74
Figure 4.2. Effects of storage at 22°C (Treatment A) and 4°C
(Treatment B) on the percentage of membrane-intact sperm of
epididymal dog sperm .............................................................. 75
Figure 4.3. Mean sperm motility and mean membrane-intact sperm (%)
mouse of canine epididymal sperm stored within the testicle
at 4°C ....................................................................................... 76
vi
ABSTRACT
The objective of the first experiment was to determine the effects of storage at
22°C vs. 4°C on the motility and percentage of membrane-intact sperm (%MIS)
of epididymal mouse sperm. Testicles were allocated into 22°C or 4°C
treatment groups and stored for 24 or 48 hours. Additional testicles were
allocated into the 4°C treatment for storage for 72 or 96 hours. Sperm was
collected and analyzed at each time point. Storage at 22°C lowered motility
and %MIS (P<0.05) when compared with control sperm and 4°C sperm at both
the 24 and 48 hours. Motility and %MIS of 4°C sperm did not decrease when
compared with the control until after 72 hours of storage. The second
experiment evaluated the effects of 22°C vs. 4°C storage for 24 and 48 hours
on epididymal dog sperm. Motility and %MIS of the 22°C sperm was lower than
that of the 4°C sperm and the control (P<0.05) at 24 and 48 hours. Motility of
the 4°C sperm was lower than the control at 24 and 48 hours (P<0.05), however
%MIS was not lower than the control until 48 hours. The third experiment
tested the effect of cryopreservation on epididymal dog sperm. Sperm was
frozen immediately (A), after 48 hours at 4°C in liquid (B) or after 48 hours at
4°C of the whole testicle (C). Both pre-freeze (PF) and post-thaw (PT) motility
and %MIS of B and C were lower than A (P<0.05). PT values were lower than
PF values in all treatments (P<0.05). PT motility of B was lower than C (0 vs.
35.0±3.6%). Storage at 4°C allows collection of motile epididymal mouse and
dog sperm for several days after death. Dog testicles can be refrigerated for 2
vii
days and epididymal sperm frozen with PT motility and %MIS of 35 and 62%.
viii
CHAPTER I
INTRODUCTION
As the human population increases and more land is claimed for human
use, more animals are being pushed toward extinction. The only hope for the
future survival of many of these species is captive breeding. Many animals in
the past have become extinct in their natural habitats and would have perished
completely without captive breeding programs. The Arabian oryx in Oman
(Price, 1986) and the Black-footed ferret in the United States (Clark, 1987) are
two examples of animals that were extinct in the wild and have now been
reintroduced into their native habitats from captive bred populations. The Grey
wolf (Canis lupus), although not completely extinct in the wild, has been absent
from many areas of its traditional range after mass extermination at the hands
of human settlers. The wolf has enjoyed a recent reintroduction into
Yellowstone National Forest where it was once common.
One of the most important aspects of a captive breeding program is to
maintain genetic diversity within the given population to avoid the problems of
inbreeding and loss of heterozygosity, which include immune system problems
and loss of fertility. This necessity has been recognized and emphasized by
conservationists and geneticists and is well documented (Kear, 1977; Ballou,
1984; Wildt and Bush, 1984; Moore, 1992). Many feel that the preservation of
germplasm through cryobanking is the next crucial step in maintaining genetic
diversity in captive populations of endangered species (Seager and Platz, 1976;
1
Durrant and Oosterhuis, 1981; Summers, 1986; Blakewood et al. 1987; Ballou,
1992; Holt, 1992; Schiewe et al., 1995).
Any use of frozen gametes either sperm, oocytes or embryos, requires
the use of assisted reproductive technologies (ART). These technologies
include artificial insemination (AI), in vitro fertilization (IVF), intracytoplasmic
sperm injection (ICSI), in vitro embryo culture, embryo reconstruction,
cryopreservation and embryo transfer (ET) procedures.
Many ART technologies are already in use in the fight to save some
endangered species through captive breeding. Efforts began with animals that
do not breed successfully in captivity and have now extended to other animals
that can breed successfully on their own, but can also benefit from the
advantages of ART. For an overview of ART in exotic species, there are many
informative articles that review gamete and embryo recovery and transfer in
exotic mammalian species as well as the importance of incorporating ART into
plans for the preservation of these animals (Durrant and Bernirski, 1981;
Durrant et al., 1986; Wildt et al., 1986; Kraemer, 1989; Bavister and Boatman,
1989; Wharton, 1984; Blakewood and Godke, 1986; Hutchins and Wiese, 1991;
Lasley et al. 1994).
Research on laboratory and domestic species has been and continues to
be the key to advances in ART for exotic species. Wildt et al. (1986) reviewed
the possibilities of applying ART developed for domestic species to their exotic
"counterparts" (e.g., cows as a model for gaur (Loskutoff et al., 1995), domestic
cats as a model for wild felids (Pope et al., 1998; Pushett et al., 2000). An
2
example of successful use of model ART in exotic species was an inter-species
transfer of embryos from an Indian desert cat to a domestic surrogate which led
to a live birth (Pope et al., 1989). The review also stressed the necessity for
researchers to use them only as a basis for their research and then establish
species specific norms for their research subjects (Wildt, 1986).
An excellent domestic animal to use as a model for endangered wild
canids is the dog. All dogs are descended from the wolf and their reproductive
cycles are nearly identical. Dogs are readily available, easy to work with and
are the focus of new avenues of research with the push to clone pet dogs.
Artificial insemination in the dog had its first success in the late 1700s with the
work of Spallanzani and is common practice today. Because of the use of AI,
there has also been extensive research into cryopreservation of dog sperm.
In an effort to further research in exotic canids, using the domestic dog
as a model, the survival of canine epididymal spermatozoa under room
temperature, refrigeration and frozen conditions is considered of primary
importance. Basic studies on the survival of sperm under various conditions
might lead to short-term storage solutions for transporting tissue and or
specimens to sperm banks. The idea being that if a male animal died in a zoo
the veterinarian could excise the testicles and ship them under refrigerated
conditions to a germ plasm bank, where a technician would process the
epididymides and freeze the sperm for future use. This would allow an
extension of that animal's genetic contribution to the population.
3
CHAPTER II
LITERATURE REVIEW
Assisted Reproductive Technologies in the Dog and Wild Canids
Domestic Canine Reproductive Physiology
The domestic dog (Canis familiaris) is a monoestreous, nonseasonal
breeder and undergoes spontaneous ovulation. The ovulatory patterns and
meiotic stage of ovulated oocytes in the bitch are unusual from most other
animals. In most mammals, luteinization of follicles occurs after the oocyte is
ovulated from the follicle, but in the bitch luteinization of the more advanced
follicles occurs before ovulation. This phenomenon exposes developing
oocytes to increasing levels of circulating progesterone prior to ovulation.
The meiotic phase of the ovulated bitch oocyte is also unique. The bitch
oocyte is ovulated with the germinal vesicle still intact (at the beginning of the
first meiotic division), while most other mammalian ova have already gone
through the germinal vesicle breakdown during maturation in the ovary and are
ovulated at the second meiotic division (M II stage). The bitch oocyte goes
through germinal vesicle breakdown shortly after ovulation and final maturation
occurs within the oviduct. The levels of progesterone rise even higher after the
oocytes are ovulated and the oocytes maturing in the oviducts are under a
different hormonal condition than that of most other mammals.
The maturation process also takes longer in the dog (thought to be 48 to
72 hours) than in other mammalian species studied. The ovum/embryo spends
4
~10 days in the oviduct before it reaches the uterus at the blastocyst stage
(Holst and Phemister, 1971; Concannon et al., 1989; Farstad, 2000a,b).
Exotic Canine Reproductive Pysiology
The fox is a canid species that is commercially valuable. The Blue fox
(Alopex lagopus) and the Silver fox (Vulpes vulpes) are both farmed for their
pelts. Desires for hybrids of these foxes for fur coats has led to reproductive
research on this species (Farstad, 1998). Like the dog, they are monoestreous
with spontaneous ovulation of primary oocytes, which mature within the
oviducts. There are slight variations in the length of the estrous stages with the
fox tending to have a longer preimplantation period, but the basic reproductive
events are very similar (Valtonen and Jalkanen, 1993).
The Grey wolf (Canis lupus and its subspecies) and the Red wolf (Canis
rufus) closely resemble the dog in their anatomy and reproductive physiology.
Female wolves are seasonally monoestreous, they mate during the winter
months and produce their young in the spring, following a 63-day gestation
length. The wolf, unlike the fox, is not commecially valuable, therefore they
have not been studied in as much detail and research into the other pertinent
reproductive events, so far, has not been done. It is well known, however, that
the domestic dog descended directly from the wolf and that they can interbreed
to produce dog/wolf hybrids (McDonald, 1985; Gloyd, 1992). The ability to
hybridize is used as an illustration of their reproductive similarities.
5
Oocyte Maturation, IVF and ET
The unique characteristics of ovulation before germinal vesicle
breakdown and the long maturation phase in the oviduct under the influence of
progesterone have made attempts to reproduce the maturation and fertilization
of canine oocytes in vitro very difficult and it has met with very limited success.
In fact, ART in the dog has lagged behind nearly all other domestic mammals,
especially laboratory species. This is ironic, considering that the first
mammalian oocyte to be described (by Karl Ernst van Baer in 1827) was
obtained from a pet dog (Farstad, 2000b).
Dogs produce offspring very proficiently and there have been no
commercial pressures in the past to fuel development of ART in the dog. New
interests in the dog will hopefully lead to new advances in ART. These interests
include the increasing importance of dogs in human medical research and the
interest in the cloning of pet dogs. Advances in ART of the domestic dog could
well lead to advances in the reproduction of endangered wild canid species.
In vitro oocyte maturation in the dog has not been very efficient. In her
review of canine reproduction, Farstad (2000a,b) quoted maturation rates of 0%
to 58% in oocytes collected from bitches at random stages of the estrous cycle.
Mahi and Yanagimachi (1976, 1978) were the first to report in vitro maturation
and sperm penetration of dog oocytes but their success was limited.
Yamada et al. (1992) attempted to superovulate dogs before oocyte
collection to determine whether maturation and fertilization rates could be
improved. The rate of fertilization of the ova was 32% by 72 hours post-
6
insemination, but of the 148 inseminated ova, only 2 developed to the 8-cell
stage. Yamada et al. (1993) continued their research with superovulated
animals comparing their oocytes to the oocytes of anestrous bitches. They
found that significantly more oocytes matured from the superovulated group
(31.9%) than in the anestrous group (12.1%). They also found that more male
and female pronuclei developed in the superovulated oocytes after exposure to
sperm (37% vs. 2%).
Hewitt and England (1997) also compared the maturation of canine
oocytes from different phases of the reproductive cycle however, they worked
with the natural cycle rather than using exogenous hormones to superovulate
the animals. Oocytes were obtained from bitches in proestrous, estrous, late
luteal and anestrous stages. No significant differences were noted in the
subsequent maturation of the oocytes collected. In an attempt to increase
maturation rates, hormones were added to the maturation media of oocytes
collected from animals in late luteal or anestrous phases. Estradiol,
progesterone and a combination of the two were tested, however, no
improvement in maturation was obtained.
Nickson et al. (1993) supplemented maturation medium with estradiol. In
oocytes with at least two layers of cumulus cells, 39% extruded their first polar
body, but no progression to M II occurred. The researchers suggested that a
sequential approach to the maturation medium might enhance oocyte
maturation.
7
In 1998, two papers were published from the Center for Reproduction of
Endangered Species in San Diego, CA. These two papers concentrated on the
use of the domestic dog as a model for endangered species. In the first paper,
Durrant et al. (1998) described a technique for isolating follicles from the
ovarian tissues collected when bitches were spayed. More preantral and early
antral follicles were obtained from larger ovaries. They also found that storage
at 4°C for 18 hours before processing of the ovaries yielded the lowest
percentage of degenerated follicles as compared with digestion of the ovaries
immediately after spaying and digestion after spaying followed by 18 hours of
storage of the isolated follicles at 4°C. In a second report, Bolamba et al.
(1998) found that culturing of preantral and early antral follicles collected by
digesting ovaries did lead to maturation, although at a low rate.
Although offspring have not been reported from IVF in the domestic dog,
recently morulae and blastocysts have been produced from modified IVF
procedures on dog ooyctes at this laboratory (Graff and Godke, 1997). Otoi et
al. (2000) reported the development of a canine blastocyst from the use of IVF.
England et al. (2001) achieved a pregnancy with 4 conceptuses after the
transfer of canine embryos produced by IVF, however, the pregnancy was lost
at 22 days. These advances not only show that canine embryos can be
produced using IVF, but also that pregnancy can be achieved and hopefully will
soon result in the birth of live pups.
8
Another encouraging approach would be the use of intracytoplasmic
sperm injection (ICSI) to fertilize dog ova. Fulton et al. (1998) reported sperm
decondensation after injection into dog ova.
IVF in exotic canids has been attempted, Farstad et al. (1993) collected
in vivo matured ova from the Blue fox and performed IVF. Only 2 of the 36 ova
were fertilized and one of them did develop to the morula stage by 144 hours
post-insemination.
Ovaries have been recovered from fox vixens and the oocytes harvested
for in vitro maturation (Wen et al., 1994). In this study, human chorionic
gonadotropin (hCG) added to the maturation medium significantly increased
maturation rates after 36 hours in culture (54%) compared with no hCG (33%)
(Wen et al., 1994). Feng et al. (1994) continued the work reported by Wen on
fox oocyte maturation and also attempted IVF on these ova. They found that
the sperm needed 5 to 8 hours of incubation time to become capacitated and
enter the ova. They also noted that the oocytes need not be mature for sperm
penetration, however, none of the immature oocytes were fertilized.
The transfer of in vitro embryos has not produced a live pup in the
domestic dog or the fox, but ET of embryos produced in vivo has met with some
success. Kraemer et al. (1979) reported the use of mid-ventral laparotomy
approach to harvest later stage embryos from the uterine horns of domestic dog
females. Using this procedure, 44 embryos were harvested from 11 collection
attempts. The same laboratory reported the birth of the first canine offspring (a
Beagle puppy) from a surgical embryo transfer of in vivo produced embryos.
9
The puppy was born on October 1, 1979 (Kinney et al., 1979). In subsequent
studies, 37 in vivo canine embryos were surgically transferred to seven
recipient females resulting in three pregnancies and four live offspring (Kraemer
et al., 1979, 1982).
Correspondingly, Archbald et al. (1980) established a successful method
of superovulating anestrous bitches using equine chorionic gonadotropin (eCG)
and described a modified method of collecting canine embryos via the cervix.
Briefly, an 8 or 10 French Foley catheter is inserted through the cervix into the
uterine horn. Medium was then flushed through the distal portion of each horn
4 cm from the uterotubal junction. This method is efficient and reduced the
chances of uterine trauma that is common with surgical embryo collection from
the donor bitch.
Jalkanen and Lindeberg (1998) surgically transferred 29 in vivoproduced Silver fox embryos into eight recipient bitches. The embryos had
been produced through artificial insemination and subsequent collection of the
embryos from the reproductive tract. The embryos were at the 16-cell to
blastocyst stages at the time of transfer. One of the recipients gave birth to two
male pups.
Semen Collection and AI
Semen can be collected from the dog using electroejaculation or digital
manipulation. Digital manipulation produces a good semen sample and is
easier on the animal therefore it is now the more common method of collection.
10
The first successful AI and was performed by Spallanzani in 1780 on the
domestic dog (Heape, 1897). In the past 2 centuries, AI in the dog has become
a common practice. Efforts soon turned to finding a way to cryopreserve
semen and use thawed semen for inseminations. This was first successful in
1969 (Seager, 1969), however, results are often extremely variable.
Aamdal et al. (1978) have reported the collection of semen in foxes using
electroejaculation and digital manipulation methods. The most commonly used
procedure for intrauterine insemination in the fox was developed by Fougner et
al. (1973). Briefly, a catheter was inserted through the cervix and into the
uterus for semen deposition. Vixens were inseminated twice per estrous
period, with 100 to 150 million sperm per dose. In this study, pregnancy rates
were as high as 79.6% with fresh semen and 73.8% with frozen-thawed semen.
By 1983, > 800,000 fox vixens had undergone AI (Jalkanen, 1993).
It is possible to collect semen from wolves with electroejaculation or an
artificial vagina (Seager, 1974; Seager et al., 1980), although electroejaculation
under anesthesia is much safer for the animal handlers and technicians and is
understandably more common. Wolf sperm cells generally have good motility
rates, ranging between 70 and 80% (Seager et al., 1980). AI from fresh semen
samples has produced several pregnancies in the wolf (Seager, 1974). A
multitude of different extenders and freezing techniques are now available for
canine semen (Platz and Seager et al., 1977; Linde-Frosburg, 1991; England,
1993). Seager et al. (1975) have reported a successful wolf pregnancy with AI
using frozen-thawed semen. The development of a detailed survival plan could
11
utilize AI to help propagate these canid species without moving animals from
zoo to zoo and could utilize genetic material from wild individuals without
bringing them into the zoo environment. These procedures could also be used
on other wild canids, such as the South American Bush dog and African Wild
dog.
Cryopreservation
The Effects of Cryopreservation on Sperm Cells
The cornerstone to preservation of genetic material is the ability to freeze
the genetic material in such a way that it can be thawed at a later time and still
be viable. Cryopreservation is widely believed to stop the enzymatic and
metabolic activity in a cell (Mazur, 1970). Freezing keeps the cells from
degrading so that, theoretically, when thawed, the cells will be in nearly the
same state they were in prior to freezing. There are many dangers inherent to
cryopreservation that cause changes and or damage to cells, including
membrane damage from osmotic changes and ice crystal formation (Mazur,
1970; Mazur, 1977; Fahy, 1986).
To help prevent these damages, cryoprotectants were used (Meryman,
1971). The three most commonly used cryoprotectants for sperm are glycerol,
dimethylsulfoxide (DMSO) and ethylene glycol, all three are penetrating
cryoprotectants (able to enter the cells) (Graham, 1996). There is great species
and even individual variability in sensitivity to these solutions (Holt, 2000).
Holt (2000) illustrated another instance when domestic animal research
could be used as a model for exotic species. Research was begun by using
12
what worked well for the domestic cow as a basis to begin research on exotic
hoofstock. For example, bull semen is usually frozen in a 4 to 8% glycerol
solution, this range has also been used in Red deer (Fennessy et al., 1990),
Scimitar-horned oryx (Garland, 1989) and gaur (epididymal) (Hopkins et al.,
1988) with good results (Holt, 2000). Even though domestic animals can be
used as a model for exotic animals, experimentation comparing the effects of
cyroprotectants and even the levels of cryoprotectants are still essential for
optimal post-thaw survival of sperm cells of exotic species.
The cryoprotectants themselves can also cause osmotic injury if added
too quickly before freezing or removed too quickly upon thawing (Goa et al.,
1995), causing shrinking and or swelling that damages membranes. The sperm
parameter that is most sensitive to osmotic injury is motility. Stepwise addition
and removal of the cryoprotectant is one technique that can help minimize
osmotic stress and injury in human sperm (Watson, 1979; Gao et al., 1995).
The rate of freezing and the rate of thawing can also have damaging effects
(Hammerstedt et al., 1990). One method, 10 minutes in liquid nitrogen vapor
followed by plunging into liquid nitrogen, has become very common, however,
each species must be tested to find the best freeze and thaw method.
One of the reasons for reduced fertility of frozen sperm listed by Watson
(2000) is surface changes that affect recognition of receptors. It has been
theorized that the cause is clustering of membrane proteins that does not fully
reverse upon thawing.
13
Cryopreservation of Dog Sperm
The effects of cooling and freezing on dog sperm cells includes
acrosome damage (the percentage of normal acrosomes can be 85% initially,
decrease to 65% after cooling and decrease to 20% after being frozen and
thawed) (Oettle, 1986). Cooling and/or freezing can also cause sperm to
become capacitated. Although sperm capacitation is a required step for sperm
to fertilize an oocyte, it is thought that these sperm become capacitated too
quickly in an AI situation and are already capacitated before they reach the
oocyte (Rota et al., 1999).
As with any type of cell, the optimal rate of cooling and the best
cryoprotectant must be discovered before optimal post-thaw fertility can be
achieved. Hay et al. (1997) reported that slow-cooling dog semen (3 hours) led
to a higher percentage of sperm bound to homologous zona pellucidae when
compared with fast-cooled semen (30 minutes). The addition of glycerol further
reduced the zona binding ability of the sperm cells, as did freezing. The level of
glycerol does not seem to have an effect on sperm longevity or acrosome
integrity of dog sperm (Pena et al., 1998) nor did glycerol effect the acrosome
status of dog sperm (Hay et al., 1997). In an early cryoprotectant study,
glycerol was found to be superior to glycine and bicarbonate-phosphate for
preserving post-thaw motility of dog sperm (Foote, 1964).
Because of the superior sperm binding of slow cooled dog sperm
reported by Hay et al. (1997), many laboratories allow dog sperm to cool for 3
to 4 hours in a diluent before it is frozen. In the dog and fox, straws are
14
generally preferred over pellets for easier identification and less chance of
contamination (Farstad, 1996). Straws are often placed on a rack that is just
above the level of liquid nitrogen to freeze (Farstad, 1984).
Although AI is a widespread practice of dog breeders, the use of frozen
sperm for AI has lagged behind the use of fresh semen for AI because of
variable pregnancy rates. This problem has been compounded by difficulty in
finding the best time of insemination and the anatomical structure of the bitch
that often hinders intrauterine insemination (England, 1993). Intrauterine
insemination is thought to be essential for optimizing fertility of frozen-thawed
dog sperm (Linde-Forsberg et al., 1999). Some groups have achieved
pregnancy rates comparable with that of fresh sperm with a pregnancy rate of
67% (Farstad and Berg, 1989).
Epididymal Sperm
Recently, exotic animal researchers have begun to show more interest in
epididymal sperm for ART. This resource can be collected either as part of a
routine neuter of an animal or from a dead animal, so that in either case his
potential for genetic contribution might be salvaged. Epididymal sperm could
even be collected from animals injured or killed in the wild. The sperm of wild
animals could then be used to produce offspring from captive animals to boost
the genetic variability of a captive species without bringing another animal into
an already overcrowded zoo system.
15
Role of the Epididymis
The epididymis is essentially a long coiled tubule that consists of several
distinct regions. The three basic regions are the caput, where fluids and solutes
are absorbed and maturation begins, the corpus where maturation is completed
and the cauda, where mature sperm are stored (Amann, 1988).
The main role of the epididymis is to cause physiologic and morphologic
changes that mature the sperm cells so that, after ejaculation, they are motile
and have the ability to fertilize an oocyte (Bishop and Walton, 1960). Amann
(1988) stated that sperm maturation, in most mammals, occurs mostly during
the 2- to 5-day transit through the caput and corpus epididymidis and is caused
by the changing fluid components in the sequential sections. These fluids are
essential for the maturation of the sperm cells and simple passage of time in
absence of these fluids will not yield mature sperm. Sperm are not completely
mature until they have acquired motility, the ability to fertilize and the ability to
induce normal embryonic development (Amann, 1988)
The sperm cells ability to fertilize increases as they travel down the
length of the epididymis. Sperm from the caput region of the epididymis have a
very low capacity to fertilize ova during IVF (>10% fertilization), the ability to
fertilize rises as the sperm travel through the corpus and reach their maximum
fertilizing ability in the cauda epididymidis (Amann, 1988). This increase in
fertility with further passage through the epididymis has been illustrated in the
guinea pig (Young, 1929; 1931), the rat (Blandau and Rumery, 1961), the rabbit
16
(Bedford, 1966), the sheep (Williams et al., 1991; Fournier-Delpech et al., 1977)
and the pig (Peterson et al., 1986).
Use of Epididymal Sperm in ART
Pregnancies and live births have resulted from ART using epididymal
sperm in several species. Laboratory animals produced from epididymal sperm
include: the mouse (Miyamoto and Chang, 1972; Hoppe and Pitts, 1973) and
rat from IVF (Toyoda and Chang, 1974), AI (Nakatsukasa et al., 2001) and ICSI
(Hirabayash et al., 2002). Farm animals produced from epididymal sperm
include: the horse (Barker and Gandier, 1957), sheep (Fournier-Delpech et al.,
1979), cow (Graff et al., 1996) and the goat (Blash et al., 2000). Humans are
routinely born after IVF and ICSI with epididymal sperm (Temple-Smith et al.,
1985 and Bonduelle et al., 1996 and 1998). Exotic animals have also been
produced from ART with epididymal sperm including: the Common marmoset
(Morrell et al., 1997; 1998) and the eland (Bartels et al., 2001). And finally, the
dog has been produced after AI with epididymal sperm (Marks et al., 1994).
The development of in vitro fertilization technology began with the use of
epididymal sperm. In a review of IVF technology, Bavister (1990) credits the
ability to capacitate epididymal hamster sperm (Yanagimachi, 1970) as the
turning point in fertilization research. This breakthrough with capacitating
hamster sperm led to successful IVF in the mouse (Wittingham, 1968) and the
rat (Miyamoto and Chang, 1973; Toyoda and Chang, 1974) using epididymal
sperm.
17
Miyamoto and Chang (1972) used epididymal mouse sperm to fertilize
mouse ova with 56% developing to blastocysts and 16% of the transferred
embryos developing into live young. Hoppe and Pitts (1973) used a chemically
defined medium to capacitate epididymal mouse sperm and fertilize ovulated
mouse oocytes in vitro, leading to the birth of live pups after ET. Toyoda and
Chang (1974) inseminated ovulated rat ova in vitro with epididymal rat sperm
and transferred the resulting 2-cell embryos to 14 recipients producing 9
pregnancies and 41 pups.
In subsequent years IVF with epididymal sperm was accomplished in the
rabbit (Ogawa, 1972). This was originally accomplished with serum to
capacitate the sperm and later in chemically defined medium (Hosoi et al.,
1981). Penetration of domestic cat ova by epididymal spermatozoa was
reported by Bowen (1977) and again by Niwa et al. (1985). Goodrowe and Hay
(1993) tested cooled domestic cat epididymal sperm against fresh epididymal
cat sperm. They found that more cooled sperm bound to hamster zonae than
fresh sperm, suggesting that acrosome damage during cooling caused the
higher rate of binding.
Lengwinat and Blottner (1994) compared fresh cat epididymal sperm to
frozen-thawed epididymal sperm. The cleavage rates for the fresh sperm were
higher than that of the frozen (40.7% and 25.3%, respectively), but the fact that
the cryopreserved sperm was capable of fertilization was encouraging. Bogliolo
et al. (2001) performed ICSI with frozen-thawed epididymal cat sperm with 82%
fertilization and 35% morula development.
18
Barker and Gandier et al. (1957) collected epididymal sperm from two
Belgian stallions at the time of castration, the sperm was cooled to 5ºC and then
frozen. Subsequent inseminations of mares with this frozen-thawed epididymal
sperm yielded a live foal from one of the seven inseminated mares. FournierDelpech et al. (1979) evaluated the fertilizing capacity of ram epididymal sperm
by performing AI with sperm from several regions of the epididymis. They
found that fertility (pregnancy rate after AI) of the sperm increased with
epididymal transit from the caput to the cauda epididymidis. The pregnancy
rate of sperm from the distal cauda epididymidis was 80%, while ejaculated
sperm yielded a 72% pregnancy rate. Sperm from the distal cauda
epididymidis also had a higher embryo survival rate with 16 of 16 implanted
embryos developing into live lambs, while ejaculated sperm resulted in 17 live
lambs from 21 implanted embryos.
A bovine pregnancy was established by Graff et al. (1996) using
epididymal bull sperm for IVF, this illustrated how the genetics of a valuable
animal could be salvaged.
Pig embryos have also been produced using epididymal sperm (Iritani et
al., 1978). In fact, frozen-thawed epididymal sperm had higher oocyte
penetration rates than frozen-thawed ejaculated sperm (Nagai et al., 1988).
Rath and Niemann (1996) found that frozen-thawed epididymal boar sperm had
higher post-thaw motility and fertilization rates than frozen-thawed ejaculated
boar sperm. One reason for higher IVF rates may be that the epididymal sperm
never came in contact with decapacitation factor(s) in seminal fluids. Putting
19
the epididymal sperm through the freeze-thaw process seems to affect the
sperm membrane so that capacitation of the cells is induced without any in vitro
treatment.
Kikuchi et al. (1998) took epididymal sperm research in the pig a step
further by storing boar epididymides at 4°C for 1 to 2 days before the sperm
were frozen. These frozen-thawed sperm were found to be able to penetrate
pig ova and form male pronuclei. Pangestu (1997) investigated if viable sperm
could be recovered from goat epididymides up to 3 days after the death of the
animal. Motility of the epididymal sperm decreased rapidly (by ~ 50% per day),
but membrane-intact, motile sperm cells were recovered even at 3 days post
mortem.
Temple-Smith et al. (1985) were first to report a human pregnancy from
IVF with sperm obtained from microsurgical epididymal aspiration.
Conventional IVF with epididymal sperm was not very effective because of the
lower concentration and motility of human epididymal sperm. With the advent
of ICSI in humans (Palmero et al., 1992), the pregnancy rates using epididymal
sperm are now comparable with those obtained using ICSI with ejaculated
sperm (Bonduelle et al., 1996). This procedure is especially important for
humans with a blockage of the vas deferens or congenital absence of the vas
deferens (Tournaye et al., 1994; Madgar et al., 1998).
Bonduelle et al. (1996) performed a follow-up study on children born
after ICSI with ejaculated, epididymal and testicular spermatozoa from fresh or
frozen-thawed embryos. Of the 877 children born from ICSI procedure, only
20
2.6% had malformations, which was within normal limits. A furthur breakdown
of the analysis to include only those children born from ICSI using epididymal
and testicular sperm showed that of 57 children born from epididymal sperm, 2
had major malformations and of the 50 children born from testicular sperm, 1
had major malformations. Both of these findings were within normal limits
(Bonduelle et al., 1998).
Blash et al. (2000) used frozen-thawed goat epididymal and ejaculated
sperm for both IVF and AI. The frozen-thawed epididymal sperm had a
cleavage rate of 40% and a blastocyst rate of 6% compared with 37% and 4%
for frozen-thawed ejaculated sperm. One pregnancy was achieved from 20 AI
attempts using frozen-thawed epididymal sperm.
Hirabayash et al. (2002) used frozen-thawed epididymal sperm from
transgenic rats to perform ICSI. In all, 181 embryos were transferred and 18
transgenic offspring resulted for the Wistar strain and 3 transgenic offspring
from 161 transferred embryos resulted for the Sprague-Dawley strain.
Morrell et al. (1997) used fresh epididymal sperm to inseminated 18
Common marmoset (Callithrix jacchus) females. Pregnancies and offspring
resulted from 6 of the 18 females inseminated. Morrell et al. (1998) then
compared insemination with fresh epididymal sperm to insemination with
frozen-thawed epididymal sperm. Six females were inseminated with frozenthawed epididymal sperm, one female conceived and gave birth to triplets.
Nakatsukasa et al. (2001) used frozen-thawed rat epidiymal sperm to
21
inseminate female rats. A total of 13 female rats were inseminated with frozenthawed epididymal sperm, 9 became pregnant resulting in 41 normal offspring.
Bartels et al. (2001) collected testicles from culled eland (Taurotragus
oryx) bulls. Epididymal sperm was collected and frozen. The sperm was later
thawed and used to inseminate 4 eland cows, one of which became pregnant
and produced a healthy calf.
Marks et al. (1994) presented a case in which a 9-year-old Boxer with
progressive, generalized seizures was euthanized and epidiymal sperm was
collected and frozen by request of his owners. The sperm cells were found to
have very poor post-thaw motility (~5%), therefore, all of the pellets were
thawed and used to inseminate a 2-year-old boxer bitch using surgical
intrauterine insemination. One female pup resulted from the insemination.
These results illustrate that epididymal sperm can result in live births for
many species using different forms of ART.
Epididymal Sperm of Exotic Species
As previously mentioned, the first birth of a Common marmoset using AI
was recently reported using epididymal sperm, both fresh and frozen-thawed
(Morrell et al., 1997, 1998). Bartels et al. (2001) were also successful using
frozen-thawed epididymal eland sperm to inseminate an eland cow and
produce a live calf.
Bartels et al. (2000) were able to fertilize ~12% of lion ova inseminated
when frozen-thawed epididymal lion sperm was used for IVF. African buffalo
embryos have been produced using oocytes collected from post-mortem
22
ovaries and sperm from the epididymides of post-mortem males (Shaw et al.,
1995). These examples show the promise of using epididymal sperm for ART
in exotic species. The banking of epididymal sperm in a frozen state could be
very important as ART research in exotic and endangered species continues.
The concept of "genome resource banks" or "frozen zoos" began with a
call for frozen embryo banks (Godke et al., 1983) and many facilities have
begun to amass collections of embryos and sperm and even somatic cells. In
fact, the frozen zoo concept at the San Diego Zoo, began with cells collected at
necropsy and now has over 600 cell strains from 11 orders of mammals
preserved (Benirschke, 1984). Wildlife biologist and researchers have begun a
flurry of activity in the area of epididymal sperm collection in exotic species.
Most of the studies at this point have looked into longevity during refrigeration
or during in vitro culture, or have done some basic research necessary to find
the optimal freezing method for these sperm. Several of these studies are
summarized in Table 2.1.
Johnston et al. (1991) explored the ability to "rescue" oocytes from exotic
felid species by collecting oocytes from ovaries of animals at the time of spay or
at the time of death. A few years later, Jewgenow et al. (1997) rescued
gametes from males of exotic felid species also utilizing tissue from surgical
sterilization or tissues from deceased animals. They collected epididymal
sperm from lions, tigers, leopards, puma and a jaguar by mincing the
epididymides and allowing the sperm to swim free of the tissue. The sperm
was frozen, thawed and used in IVF resulting in an average of 18.5%
23
fertilization rate compared with 26% for domestic cats. Although the fertilization
rate was rather low, it did illustrate that frozen-thawed epididymal sperm from
large cats was capable of fertilization. The collection and cryopreservation of
epididymal sperm either after death or at sterilization could help preserve
genetic diversity in any species. Johnston and Lacy (1995) stated that genome
resource banks "have the potential to decelerate the loss of gene and allelic
diversity in captive populations through reintroducing 'original' genetic material
through time to counter genetic drift". Wildt (1992) also advocated genetic
resource banks that should include blood, tissue, DNA and gamete samples. A
call was made for global organization of records and exchange of information
and/or specimens.
Problems with the Reproduction of Endangered Species
Small Population Sizes
The drastic decline of nearly all types of wildlife can usually be traced
back to the influence and intrusion of humans into animal habitats. The
intrusion can be direct, through outright slaughter (hunting and trapping for
food, sport or fur) and the capture of animals for display in zoos and circuses, or
to serve as pets. The intrusion can also be more inadvertent, such as habitat
destruction and introduction of new diseases and animals into an ecosystem
that decimate native species. Some experts have predicted that if habitat
destruction continues at its present rate, before this new century is over, all
24
Table 2.1. Cooling and freezing of epididymal sperm of exotic species
Storage/ in vitro
Freezing
culture studya
studyb
Species
African buffalo,
eland, Red
hartebeest,
Burchelli's zebra
White rhinoceros
Blesbok
Blesbok, impala,
springbok
Impala
Greater kudu
Waterbuck,
Greater kudu,
warthog
Cantabrian Brown
bear
African lion
4 to
7°C
18 to
28°C
28 to
37°C
CP
TM IVF
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Reference
Bezuidenhout et al.
(1995)
Williams et al.
(1995)
Winger et al. (1997)
Loskutoff et al.
(1996)
Rush et al. (1997)
Schmid et al.(1997)
Watson et al.
(1997)
X
X
Anel et al. (1999)
Bartels et al. (2000)
Friedmann et al.
(2000)
African buffalo
X
X
Black wildebeest,
Kilian et al. (2000)
X
blesbok, Roan
antelope,
gemsbok, nyala,
eland, African
buffalo
Roan antelope,
Kilian et al. (2000)
X
gemsbok,
African buffalo
White rhino,
Lubbe et al. (2000)
Burchelli's zebra
X
X
a
4 to 7°C = Refrigeration temperature ranges; 18 to 28°C = Room temperature
ranges; 28 to 37°C = Incubator temperature ranges.
b
CP = Cryoprotectant study; TM = Thawing method study.
25
exotic species that remain will be managed in captive breeding programs
(O'Brien et al., 1985). Whether the intrusion is purposeful or accidental, the
outcome of human influence is the same, reduction of a species to extinction, or
into populations that are so small and isolated from each other, that they are, in
effect, adrift on separate islands.
Just like animals on literal islands, these small populations have fewer
choices when they are choosing mates. This often leads to high instances of
inbreeding. Inbreeding reduces the heterozygosity of a population and as the
gene pool becomes more and more homozygous, the chance of a usually rare
or recessive "disadvantageous allele" becoming expressed in the species
becomes a greater threat (Fuerst and Maruyama, 1986).
Loss of Heterozygosity
One classic example of a bottleneck leading to loss of heterozygosity in
a species is the cheetah (Acinonyx jubatus). The cheetah is the only extant
member of the genus Acinonyx and has suffered from a relatively "recent"
bottleneck of unknown cause (Kingdon, 1977). O'Brien et al. (1985) discussed
the genetic problems of the cheetah and reviewed the average heterozygosities
of several cat species ranging from the domestic cat (0.082), to the tiger (0.035)
the leopard and the caracal (0.029). The cheetah had the lowest average
heterozygosity at 0.013. The almost total lack of genetic polymorphism in the
cheetah makes the entire species essentially monomorphic.
Pfeifer et al. (1983) reported a case study of an outbreak of feline
infectious peritonitis (FIP), which decimated a captive cheetah population at an
26
Oregon breeding facility. An 8-year-old female became very ill 5 weeks after
being transferred to the facility and died within a week of the onset of the
illness. Necropsy revealed that she had the effusive form of FIP caused by a
coronavirus. None of the Oregon cheetahs had titers of circulating antibodies
against the coronavirus before her arrival, but all had them by January, 1983.
Over that year more than 90% of the cheetahs showed clinical symptoms of
FIP, with 18 deaths from the virus or related complications (Pfeifer et al., 1983).
Testing showed that this was not an especially virulent strain as first assumed,
but that the reaction was specific to cheetahs because they reacted in a
“homogenously sensitive manner” (Pfiefer et al., 1983).
O’Brien et al. (1985) believed that this “homogenous sensitivity” was due
to lack of genetic variation. They also reviewed an experiment, which would test
the genetic variability of the major histocompatibility complex (MHC), which is
usually the most polymorphic locus in vertebrates (Snell et al., 1976). The MHC
is closely linked to personal immunity and it is what causes rejection when
foreign organs or tissues are transplanted or grafted (Snell et al., 1976). The
variability of the cheetahs' immune systems was tested by measuring the time it
took for individuals to reject skin grafts from other cheetahs. A fast rejection in
humans usually occurs in 10.5 days (Snell et al., 1976) and domestic cats will
usually show rejection at 7 to 13 days (Billingham and Medawar, 1951). In 14
skin grafts (each cheetah received one "autograft" of its own tissue next to the
allograft from another cheetah), none were rejected quickly. In fact, the two
grafts looked exactly the same at 2 weeks in all cases. Only 3 of the allografts
27
were eventually rejected after 39 to 70 days, all other grafts were accepted
(O'Brien et al., 1985). Rapid rejection was only seen after a xenograft of a
domestic cat was performed on 2 of the cheetahs. These grafts were rejected
within 9 to 16 days, illustrating that the MHC reaction of the cheetah does
“work” by rejecting the foreign tissue of other species but does not reject the
tissue of other individuals of its own species (O'Brien et al., 1985).
The devastating effects of monomorphism and lack of genetic variability
in the cheetah species leaves it more vulnerable to disease, which could wipe
out the entire population. It is important to prevent monomorphism in other
endangered species, especially our captive populations.
The Effects of Inbreeding on Reproduction
The effects of inbreeding are well documented. Inbreeding effects on
laboratory animals and domestic stock were the first to be studied. Some
effects of inbreeding include: increased juvenile mortality, increased perinatal
mortality and morbidity, adverse effects on ovulation and spermatogenesis
resulting in lower numbers and lower motility of sperm along with higher
instances of morphological abnormalities (Spearow, 1988; Lewontin, 1974;
Frankel and Soule', 1981). These same problems have also been reported in
captive exotic species (Ralls et al., 1979, 1988; Ralls and Ballou, 1983; Foose,
1983; Soule', 1980). Although this subject is expansive, this review will focus
on the effects of inbreeding on sperm production, especially in the exotic
carnivores.
28
Although inbreeding has not, as yet, become a problem in most of the
captive Grey wolf populations, Laikre and Ryman (1991) published a report on
inbreeding in the captive wolves of the Scandinavian zoo system. The entire
population of this group of wolves descended from 2 full-sibling pairs who were
wild-caught and later supplemented by 2 female siblings from Russia. Analysis
of studbook data shows that juvenile weight, life span and reproductive ability
have decreased as the degree of inbreeding increases. Instances of blindness
have also increased and are linked to inbreeding. Any captive population of
wild canids that has resulted from small number of founders could suffer from
similar inbreeding effects.
The domestic dog can also suffer from the effects of inbreeding (Wildt et
al., 1992a; Olson et al., 1992). Wildt et al. (1992a) found that inbred male dogs
had significantly lower sperm volume and concentration and had poorer
reproductive performance when compared with male dogs who were not inbred.
Olson et al. (1992) investigated the causes of azoospermia in 18 dogs. In 6 of
the cases, inbreeding was considered the cause.
As previously discussed, the cheetah has suffered from the effects of a
genetic bottleneck. This reduction in numbers has left the species essentially
monozygotic and has led to increased juvenile mortality (O'Brien et al., 1985).
To add to its problems, inbreeding has also led to depressed sperm production
and high levels of morphological abnormalities. In an experiment comparing
cheetah semen to that of domestic cats, sperm concentrations in cheetah
ejaculates were found to be 10 times lower than the domestic cats. In addition,
29
71% of the ejaculated cheetah sperm were morphologically abnormal, while the
domestic cat had only 29% abnormal (Wildt et al., 1983).
Wildt et al. (1987) reported a direct correlation between genetic variability
and both abnormal sperm and circulating testosterone in three populations of
lions (Panthera leo), each with a varying degree of heterozygosity. The three
populations included the Serengeti National Park population (most genetically
diverse), the Ngorongoro Crater population (some loss of heterozygosity) and a
group of Asian lions held at the Sakkarbaug Zoo (nearly homozygous). The
mean heterozygosity for the Serengeti group was 0.038, the Ngorongoro Crater
lions was 0.014, the Asian lion group was found to be genetically monomorphic
at each of the typed loci (Wildt et al., 1987).
Semen parameters were the most optimal for the Serengeti group with
9.4 ml volume, 91% motility, 34.4 million sperm per ml with only 25% abnormal
forms. The Crater group seemed to begin to show the effects of its loss of
heterozygosity. The average volume of ejaculates fell slightly to 8.5 ml with
only 25.8 million per ml. The sperm motility was also slightly lower at 83% with
a much higher rate of abnormality at 50.5%. Also, the Gir Forest lions’ semen
parameters were greatly depressed. The average volume was 5.9 ml with 61
million sperm per ml and only 61% motile. Abnormalities also increased with an
average of 66.2% (Wildt et al., 1987). This study showed a correlation between
loss of heterozygosity and loss of proper sperm production.
A third study, Brown et al. (1989), reported on leopards isolated on the
island of Sri Lanka and discovered that the same trend of low heterozygosity,
30
low testosterone, levels, depressed sperm output and high levels of sperm
abnormalities was present.
Need for New "Founder" Animals
Considering the myriad of problems that arise from inbreeding in small
populations, the question becomes, how can this be fixed? Or if a new species
is brought into captivity, how can we prevent this from happening? The answer
in both of these cases is to infuse the populations, periodically, with the genetic
contribution of a new founder (Jones et al., 1985).
A founder is defined as "an animal with no known genetic relationship to
any other animal in the pedigree except for its own descendants" (Lacy, 1989).
Willis (1993) discussed the problem of unknown ancestry. Good records on
imported animals and even some born in captivity are often hard to come by
and because the effects of inbreeding are so detrimental to the species, the
general rule of thumb in captive breeding programs has become "when in
doubt, leave them out". This practice minimizes the chance of inbreeding, but it
also takes the chance of excluding an important and available founder.
Willis (1993) believes that when the number of original founders is low,
that it is genetically better to use these potential founders and risk some
inbreeding, than to exclude them all and miss the opportunity of utilizing a true
founder. If the original number of founders is high, then unknowns should be
excluded from breeding (Willis, 1993). Although the scientific basis for including
these unknown animals is there, the practice of "when in doubt, leave them out"
is still the norm, especially if the animals will ever be reintroduced in the wild,
31
because participating nations will often only accept "pure" animals. The use of
epididymal sperm from wild founders would allow input of new genetics that
could not be contested for genetic purity.
Problems Faced by Zoos
The main problems faced by zoos in attempting to manage captive
populations include: controlling reproduction to optimize genetic diversity,
location and shipping barriers, nutrition and health, behavioral problems,
funding and probably the most limiting, lack of space (Lasley et al., 1994;
Ballou, 1984; Knowles, 1977; Kear, 1977).
Space in America's zoos and animal parks is grossly inadequate for the
number of species housed (Conway, 1987). This situation makes it necessary
to carefully pick and choose which animals will be bred, which will be added to
and which will be given priority over others.
This lack of space in zoos is compounded by the problem of "surplus"
animals. Lindburg (1991) discussed the causes of the surplus animal problem
and suggested a solution. Surplus animals are animals that will not be used in
the captive breeding program. These animals include those resulting from the
accidental mating of two animals that were not meant to breed, those born
deformed, excess "bachelor males" (excess males not needed for breeding
purposes) and those animals who are past reproductive age or are unable to
contribute to the gene pool. He advocated retirement communities for surplus
animals and dismissed euthanasia of these animals as unethical and
unacceptable. Some disagree (Lacy, 1991) and believe that we should put the
32
survival of a species ahead of our emotionalism and euthanize any surplus
animal that simply cannot be placed anywhere else to both save much needed
funds and to open up precious space for more valuable animals.
Zoos get help with their problems of genetic planning from the American
Zoo and Aquarium Association (AZA) through their development of a program
referred to as Species Survival Plans (SSPs). This program was developed in
1980 (Merritt, 1980) and implemented in 1982 (Conway, 1982). The AZA works
in conjunction with the ISIS (International Species Inventory System) system to
develop masterplans for many animal species (Earnhardt et al., 1995). These
masterplans give suggestions for which animals should be mated (and which
should be kept from mating) to maximize genetic diversity, including
recommendations on animal relocations from one facility to another. The
masterplans would not be possible without the information contained in
studbooks. These studbooks are kept by keepers and other zoo personnel and
document the pedigrees and births and deaths of all of the captive populations
of a given species.
The recommendations made by the SSP can at times be difficult to fulfill.
If the plan calls for an animal to be moved, there are inherent dangers and
expense when transporting animals. Behavior also plays a major role in
whether or not the recommendations can be carried out. There are aspects of
captive life that affect an animals’ behavior and influences their reproductive
abilities. Some "couples" who are meant to breed simply do not have any
interest in one another. In some cases this is just a disappointment, but in
33
many cases, introductions can be quite dangerous. Especially with carnivores,
introductions are lengthy, time consuming procedures that have the potential to
end very badly with the injury or death of a valuable breeding animal.
The influence of more dominant animals, either physiological or physical,
can "shut down" the reproductive systems of subordinates leaving them unable
to reproduce (Barrett et al., 1993; Kleinman, 1980). Hand-rearing by humans
can imprint an animal to the point that it does not interact with its own species
well enough to reproduce (Mellen, 1992).
How ART Can Help Address These Problems
There have been many articles in the last several years focusing on the
potential of using ART to help preserve genetic variability and overcome some
of the problems inherent in captive breeding of exotic species (Wildt et al.,
1992a, 1992b; Thompson, 1993; Loskutoff et al., 1995; Ballou, 1984; Howard,
2000).
The first problem ART can help answer is the issue of space for existing
animals and new animals. ART allows the introduction of new genetics, without
the introduction of a new animal. It is possible to import sperm, oocytes, or
embryos instead of the animals themselves, this allows the addition of new
founders without taking up more room, costing more money, or removing more
animals from the wild. This use of imported sperm also eliminates the expense
and danger of moving animals from one facility to the next, removing the danger
of introducing animals to one another for mating and circumventing any
behavioral or physical problems that might keep an animal from breeding
34
naturally. Also, existing founders who are underrepresented because of such
problems could be added into the genetic makeup of the species.
New developments, such as sexed sperm and sexed embryos may be
used to decrease surplus bachelors. Unwanted "surplus" females could be put
to good use serving as surrogate mothers for females of their species (or even
related species) who are more genetically valuable. This would decreases the
risk to valuable females by producing their genetic offspring without the
exposing them to the risks of pregnancy and parturition. ART also allows us to
salvage genetic materials from animals who have died before their genetic
contribution has been made or who have been sterilized.
Importing epididymal sperm from "founder" or wild individuals either
collected through the scrotum during an immobilization, or collected from
animals that have been culled or killed illegally would help bring in new genes
that would decrease inbreeding and increase heterozygosity.
Using domestic animal models has already been beneficial for some
exotic species. The dog serves as a good model for exotic canids because
they are closely related, preliminary studies of dog reproduction and ART in the
dog are numerous and the supply of gametes in the domestic dog are plentiful.
Also, the birth of a live pup after AI with frozen-thawed epididymal dog sperm
collected after death (Marks et al., 1994), shows the promise of freezing
epididymal dog sperm as a model for endangered canids.
35
CHAPTER III
THE EFFECTS OF STORAGE AT 22°C AND 4°C ON THE SURVIVAL OF
EPIDIDYMAL MOUSE SPERM
Introduction
The interest in using epidiymal sperm for assisted reproductive
technology (ART) began decades ago and includes artificial insemination (AI),
in vitro fertilization (IVF), embryo transfer (ET) and intracytoplasmic sperm
injection (ICSI). There have been pregnancies and live births from ART using
epididymal sperm in the horse (Barker and Gandier, 1957), sheep (FournierDelpech et al., 1979), dog (Marks et al., 1994), Common marmoset (Morrell et
al., 1997, 1998), rat (Nakatsukasa et al., 2001) and eland (Bartels et al., 2001)
using AI. The mouse (Miyamoto and Chang, 1972; Hoppe and Pitts, 1973), rat
(Toyoda and Chang, 1974), cow (Graff et al., 1996), human (Temple-Smith et
al., 1985 and Bonduelle et al., 1996; 1998), goat (Blash et al., 2000) and rat
(Hirabayash et al., 2002) through IVF and ICSI. Epididymal sperm is the nearly
exclusive source for the production of mice through IVF (Hogan et al., 1986).
The mouse is the most commonly used laboratory animal. Using the
mouse model for research offers advantages such as: their easily manageable
size, easy accessibility to a large number of animals of the same age and size
and their relative inexpense. In addition, laboratory procedures for mouse IVF
almost exclusively involve the use of epididymal sperm. Based on these
benefits, our initial studies on the effect of storage temperatures on epididymal
sperm utilized the mouse as a model.
36
Early experiments in the culture of mouse embryos often relied on in
vivo-produced embryos that were collected from the reproductive tracts of mice
and incubated to later stages in vitro (Brinster, 1963; Whittingham, 1971).
Attempts to fertilize mouse ova in vitro often used ejaculated mouse sperm
collected from the uteri of female mice after they were mated (Whittingham
1968; Cross and Brinster, 1970).
The first use of epididymal mouse sperm for the fertilization of mouse
ova in vitro was accomplished by placing the sperm and oocytes within an
explanted oviduct for the fertilization and subsequent development (Brinster and
Biggers, 1965; Pavlok, 1967). Later, several investigators used heated bovine
follicular fluid to capacitated epididymal mouse sperm for fertilization in vitro,
however the results varied greatly (Iwamatsu and Chang, 1969, 1970, 1971).
Hoppe and Pitts (1973) developed a method of IVF with epididymal mouse
sperm that included capacitation of the sperm and fertilization of the ova in a
chemically defined medium leading to the birth of live young after transfer of the
embryos. A comparison of epididymal and ejaculated sperm (collected from the
uterus of a mated female) revealed a significantly greater fertilization rate (122
of 131) with epididymal sperm when compared with uterine sperm (39 of 122).
Since these early experiments, epididymal sperm has become the standard in
mouse IVF as referenced in “Manipulating the Mouse Embryo: A Laboratory
Manual”.
Problems with cryopreservation of mouse sperm have limited storage
capability. In their initial study, Fuller and Wittingham (1996) investigated the
37
effects of slowly cooling mouse sperm to 4°C over a 4-hour period. The sperm
were then warmed to 37°C and then used for IVF. Epididymal mouse sperm
cooled and rewarmed in PBS had the exact same 2-cell embryo rates as control
sperm (85.1%). Implantation rates were not significantly different after transfer
of embryos produced through IVF with the sperm from the three cooling media
compared with the control sperm. Transferred IVF embryos produced from all
three cooled sperm groups had significantly higher percentage of fetuses when
compared with the results from the control sperm.
A subsequent study by Fuller and Whittingham (1997) found that sperm
penetration was faster with cooled and rewarmed epididymal mouse sperm
than with fresh epididymal sperm because fresh epididymal sperm needed a 90
minute pre-incubation time before they could penetrate the ova. They found
that cooling the sperm appeared to bring on “capacitation-like” changes in the
sperm that eliminated the need for pre-incubation to induce capacitation.
Although these previous studies (Fuller and Whittingham, 1996, 1997)
offered valuable information, there was no real effort to store the mouse
epididymides at 4°C. Therefore, our studies were extended to evaluate how
longer-term storage of mouse epididymides at 4°C would affect the sperm
motility and percent membrane-intact sperm in epididymal mouse sperm.
An effort was made to also evaluate how storage at 22°C would effect
epididymal mouse sperm. A previous study (Christian et al., 1993) found that
motile sperm could be retrieved from mice after up to 24 hours of “room
temperature” storage (no refrigeration provided). IVF with sperm collected at 15
38
hours post-mortem at room temperature yielded a 25% fertilization and 20%
blastocyst development rates. We wanted to extend the time of roomtemperature storage to determine if motile sperm could be retrieved from
mouse epididymides beyond 24 hours.
Materials and Methods
Experimental Animals
The experimental animals consisted of a group of 36 male CD-1 mice
from Charles River Laboratories (Boston, MA). The mice were all 8-weeks-ofage (breeding age) and certified healthy and disease free by the supplier. Body
weights ranged from 29 to 40 g at the time of the study. The animals were
maintained for the 5-day experimental period in plastic bottomed, wire topped
rodent cages and were given water and Purina mouse chow ad libitum.
Experimental Design
The mice were sacrificed by cervical dislocation at the onset of the study
and were randomly assigned into four equal treatment groups. The first group,
sampled at 0 hour served as the control. The testicles were collected and
randomly separated into an ambient temperature group (Treatment A - 22°C)
and a refrigerated group (Treatment B - 4°C), but the testicles were not
exposed to the treatments (See Table 3.1). In this case, both testicles were
processed immediately after death and the sperm parameters for each were
recorded. This was done to establish "day 0" parameters for sperm motility and
percent membrane-intact sperm of the epididymal mouse sperm to serve as a
baseline comparison for the other treatment groups. It was also done to
39
evaluate if there was any variation between the weight, sperm motility, or
percent membrane-intact sperm of the two testicles and resulting epididymal
sperm of a single animal. Another objective for this group was to compare the
testicle weight of each treatment at each time period to show that the original 36
mice had low variability from one another.
The second group was the 24-hour group. In this case the testicles were
collected and separated into Treatment A and Treatment B. The sperm was not
collected and processed until after the testicles had been exposed to one of the
temperature treatments for 24 hours.
The third was the 48-hour group. The testicles were allocated into
Treatments A and B. The sperm was collected and tested after the testicles
had been in their treatments for 48 hours.
The fourth group was separated into a 76-hour group and a 96-hour
group and both sets of testicles were kept in Treatment B.
Sperm Extraction and Evaluation
After the mice were sacrificed by cervical dislocation the testicles were
removed from the scrotum and the tunicae were cut away. The testicles were
randomly assigned into an experimental group and then each was weighed and
the weight in grams was recorded. The testicles of both groups were kept in
separate, identical stainless steel instrument pans with nonairtight lids. The
pans were lined with sterile gauze (Johnson & Johnson, Arlington, TX) and the
testicles were covered with two additional layers of sterile gauze. Sterile saline
40
Table 3.1. Experimental design for epididymal mouse sperm stored at 22°C (Treatment A) and 4°C (Treatment B)
Treatment
No. of
testicles
A
9
Time of sampling
Testicle weight (g)
Sperm motility (%)
Membrane-intact
sperm (%)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0 hours
B
9
A
9
24 hours
B
9
A
9
48 hours
B
9
B
9
72 hours
+
+
+
B
9
96 hours
+
+
+
41
solution (Sigma, St. Louis, MO) was then added to moisten the gauze and
prevent the testicles from drying out. Tape was placed on the pan adjacent to
the position of each testicle and labeled for identification. The ambient
temperature group (Treatment A) was kept in a climate-controlled room at
~22°C and the refrigerated group (Treatment B) was kept in a standard
refrigerator at 4°C.
After the appropriate time had elapsed, the testicles were further
processed and sperm evaluated. The cauda epididymides were cut away from
the remainder of the epididymis with scissors. The piece of tissue was minced
with a sterile razor blade in a 1 ml volume of Tyrode Lactate HEPES (TLH) (BioWhittaker, Walkersville, MD) in a 60-mm plastic Petri dish (Becton Dickinson,
Franklin Lakes, NJ).
The sperm were then allowed to "swim out" of the minced tissue into the
medium during a 30-minute period at room temperature. The sperm
suspension was then transferred into a microcentrifuge tube (DOT Scientific,
Inc., Burton, MI) and placed into a humidified incubator set at 39°C with 5% CO2
for ~30 minutes to allow the sperm to equilibrate to 39°C. After this equilibration
period, a 10 ml droplet of warmed sperm solution was placed on a warmed glass
slide (Gold Seal Products, Portsmouth, NH) and a glass cover slip (Baxter
Health Care Corporation, McGaw Park, IL) was placed on top of the sample.
The slides and coverslips were pre-warmed on a 39°C slide warmer to avoid a
drop in temperature that would effect the motility of the sperm. The sample was
42
then analyzed under 200X magnification using a compound microscope.
Sperm motility (percent) was estimated by visual observation.
After the sperm motility was evaluated, an Eosin B/Fast-Green (Sigma,
St. Louis, MO) slide was prepared for each sample. A 5 ml drop of sperm
solution was placed on a labeled slide and a 5 ml drop of the stain was added
and gently mixed with the tip of the pipette. Another slide was used to smear
the sample thinly across the entire surface of both slides. The samples were
immediately dried with a standard hair dryer on low setting. This was done to
avoid slow death of the cells as they dried in air, which would have skewed the
membrane-intact:dead ratio.
The Eosin-B was used as an indicator dye, while the Fast-Green dye
acted as a background color (Aalseth and Saacke, 1986). If the cell was “alive”
(membrane-intact), it was able to exclude the Eosin-B dye and appeared white
against the green background. If the sperm cell was not “live” (if the membrane
was ruptured) the cell was not able to exclude the indicator dye and the cell
appeared pink because the dye had entered the cell. The Eosin-B/Fast Green
slides were read on the same microscope under 400X magnification. A group
of 100 sperm cells were counted on each of the two slides prepared for each
sampling using a counter to keep track of membrane-intact and dead cells. The
number of membrane-intact cell from each slide of the two slides were added
together and divided by 2 to obtain the percent membrane-intact cells of the
sample.
43
Statistical Analysis
Data for this experiment was analyzed using ANOVA with Proc GLM
(SAS, 1985). Because we were comparing two testicles from the same animal,
comparisons of treatments A and B within Time groups were made using a
paired t-tests. Comparisons of Treatments A and B between time groups were
unpaired t-tests because they were comparisons of sperm from different
animals. Differences between the treatments were considered significant at the
P<0.05 level.
Results
The tissue of the epididymides of the mice were completely degenerated
by 48 hours in Treatment A (22°C), therefore, this treatment was not extended
to an further time point. The remaining testicles were allocated into Treatment
B for 72 hours and Treatment B for 96 hours.
The mean testicle weights are shown in Figure 3.1. The smallest mean
testicle weight was 0.127±0.005 g in Treatment A for the 48-hour group, while
the largest mean testicle weight was 0.143±0.009 g in Treatment B for the
control group. There was no significant difference when the smallest mean
testicle weight was compared with the largest mean testicle weight.
There was no significant difference between the testicle weights of
Treatment A when compared with the testicle weights of Treatment B at Time 0hour (control groups). This finding illustrated the lack of variability between the
two testicles of the mice in this experiment.
44
No significant difference was found between the testicle weights of
Treatment A and Treatment B for the 24 and 48-hour groups. There was also
no significant difference when the testicle weight of Treatment B for 72 hours
was compared with the testicle weight of Treatment B for the 94-hour group.
There was no significant difference when the testicle weight of Treatment A for
the Time 0-hour group was compared with the testicle weights of Treatment A
for the 24- and 48-hour groups. There was also no significant difference
between the testicle weights of Treatment B, when the testicle weight of the
Time 0-hour group (the Treatment B control) was compared with the testicle
weights of Treatment B for the 24-, 48-, 72- and 96-hour groups. Tabular results
from this experiment are listed in Appendix Table 1.
The mean sperm motility of the epididymal sperm from the testicles of
the Treatment A control (Time 0-hour) was not significantly different from the
mean sperm motility of the Treatment B control (Time 0-hour) (48.3±2.8%
compared with 47.8±2.1%), (see Figure 3.2). The mean sperm motility of the
24-hour group in Treatment A (22°C at 24 hours) was significantly lower than
that of the 24-hour group of Treatment B (4°C at 24 hours) (22.2±3.8%
compared with 43.3±3.2%, P<0.0007). The mean sperm motility was also
significantly lower in Treatment A at 48 hours (0%) when compared with
Treatment B at the 48-hour sampling time (40.6±3.3%) (P<0.0001). Tabular
results from this study are compiled in Appendix Table 1.
The mean percent sperm motility of epididymal sperm from testicles in
Treatment A at 24 hours was 22.2±3.8% and was significantly lower than the
45
percent sperm motility of the Treatment A control at Time 0-hours (48.3±2.8%).
The percent sperm motility of Treatment A at 48 hours was significantly lower
than the percent sperm motility of Treatment A at 24 hours (0% compared with
22.2±3.8%, P<0.0001).
The percent sperm motility of epididymal mouse sperm was preserved
for 2 days in Treatment B. There was no significant difference in mean sperm
motility for Treatment B at 24 or 48 hours (43.3±3.2% and 40.6±3.3%) when
compared with the initial sperm motility for Treatment B at Time 0-hour
(48.3±2.8%). The percent sperm motility of Treatment B at 72 hours
(30.0±3.7%) was significantly lower than the percent sperm motility of the
Treatment B control (P<0.0002). The percent sperm motility of Treatment B at
96 hours (16.1±3.5%) was also significantly lower than the percent sperm
motility of the Treatment B control and Treatment B at 72 hours (P<0.0003 and
P<0.0154, respectively).
The mean percent membrane-intact sperm results from the epididymal
sperm of testicles in Treatments A and B are presented in Figure 3.3. As with
mean testicular weight and mean sperm motility, there was no significant
difference between the mean percent membrane-intact sperm of the testicles in
the Treatment A control (Time 0-hour) and the Treatment B control (Time 0hour) (54.5±3.0% compared with 53.2±3.2%). The percent membrane-intact
sperm in Treatment A was significantly greater than that of Treatment B at both
the 24- and 48-hour time points (60.0±3.8% compared with 35.2±4.1%,
P<0.0004 and 47.3±3.7% compared with 0.5±0.2%, P<0.00001, respectively).
46
The percent membrane-intact sperm of Treatment A in the 24-hour group
(35.2±4.1%) was significantly lower (P<0.0015) when compared with the control
group of Treatment A (Time 0-hour) (54.5±3.0%). The percent membraneintact sperm of Treatment A of the 48-hour group (0.5±0.2%) was also
significantly lower than the percent membrane-intact sperm of the Treatment A
control group (54.5±3.0%) and Treatment A of the 24-hour group
(35.2±4.1%)(P<0.0001 in both cases).
The percent membrane-intact sperm for Treatment B of the 24-hour and
48-hour groups (60.0±3.8% compared with 47.3±3.7%) were not significantly
different from the control percent membrane-intact sperm for Treatment B
(53.2±3.2%). Unlike the percent sperm motility, the percent membrane-intact
sperm for Treatment B at 72 hours (44.1±4.4%) was not significantly different
from the percent live sperm of Treatment B at Time 0-hours. There was a
significant decline in percent membrane-intact sperm of Treatment B at 96
hours (24.6±4.6%) when compared with the control for percent membraneintact sperm in Treatment B at Time 0-hour (53.2±3.2%) (P<0.1403).
The percent membrane-intact sperm for the 72-hour group was
significantly higher than the percent membrane intact sperm of the 96-hour
group (both in Treatment B) (44.1±4.4% compared with 24.6±4.6%, P<0.0073).
The percent membrane-intact sperm for Treatment B at 96 hours was also
significantly lower when compared with the percent membrane-intact sperm of
the Treatment B control (24.6±4.6% and 53.2±3.2%, respectively) (P<0.0003).
47
0.160
Mean (±SEM) Testicle Weight (g)
0.155
0.150
0.145
0.140
0.135
0.130
0.125
0.120
0.115
0.110
Treatment A
Treatment B
0.105
0.100
0
24
48
72
96
Treatment Sampling Time (hours)
Figure 3.1 Effects of storage of mouse testicles at 22°C (Treatment A) and 4°C
(Treatment B) on testicle weight. Due to tissue degeneration,
Treatment A could not be continued after 48 hours of sampling.
48
60
Mean (±SEM) Sperm Motility (%)
a,d
50
a,d
a,d
a,d
40
b
30
b,e
c
20
10
Treatment A
Treatment B
c,e
0
0
24
48
72
96
Treatment Sampling Time (hours)
Figure 3.2 Effects of storage of epididymal mouse sperm at 22°C (Treatment
A) and 4°C (Treatment B) on sperm motility. Due to tissue
degeneration, Treatment A could not be continued after 48 hours of
a,b,c
sampling.
Means within treatment across sampling periods with
d,e
Means
different superscripts are significantly different (P<0.05).
within sampling periods across treatments with different
superscripts are significantly different (P<0.05).
49
Mean (±SEM) Membrane-Intact Sperm
70
60
50
a,d
a,d
a,d
a,d
a
b,e
40
b
30
20
Treatment A
Treatment B
10
c,e
0
0
24
48
72
96
Treatment Sampling Time (hours)
Figure 3.3 Effects of storage of epididymal mouse sperm at 22°C (Treatment
A) and 4°C (Treatment B) on percentage of membrane-intact
sperm. Due to tissue degeneration, Treatment A could not be
a,b,c
Means within treatment
continued after 48 hours of sampling.
across sampling periods with different superscripts are significantly
d,e
different (P<0.05).
Means within sampling periods across
treatments with different superscripts are significantly different
(P<0.05).
50
Discussion
The immediate decrease in both the sperm motility and the percent of
membrane-intact sperm cells from the sperm in the testicles held at ~22°C was
an expected event. Tissues exposed to room temperatures degrade quickly,
which obviously adversely affects the sperm cells within those tissues. It is
important, however, to note that while the decline in sperm motility and percent
membrane-intact sperm cells was significant, there were still membrane-intact,
motile cells present after 24 hours of exposure to 22°C. These findings show
that it is possible to extract membrane-intact, motile sperm from a mouse that
has been dead for up to 24 hours. In most cases, the exact time of an animal’s
death is often unknown. These data indicate that every effort should be made
to collect epididymal sperm from important males, even if their time of death is
uncertain, because there may still be membrane-intact sperm up to 24 hours
post-mortem. Epididymal sperm collection should be performed as quickly as
possible after the animal dies, however, considering that sperm motility
decreased to 0% and percent membrane-intact sperm cells had decreased to
0.5% by 48 hours at 22ºC.
No significant difference in the sperm motility and percent membraneintact sperm between the 0-hour and 24- and 48-hour groups at 4ºC was also
very encouraging. The data showed that refrigerated storage for up to 2 days in
the epididymis was not different from fresh epididymal sperm for both sperm
motility and percent membrane-intact sperm. This may be beneficial if an
experimental animal dies shortly before its sperm is needed for an experiment.
51
The epididymis could be stored at 4°C until it was needed, to avoid the
damages that freezing and thawing induce in sperm cells.
In addition, cryopreservation of mouse sperm has historically been
difficult because of unique physical features of the mouse sperm cell, including
a longer tail than most domestic animals sperm cells (Noiles et al., 1995).
Epididymal mouse sperm was frozen first by Tada et al. (1990). Subsequent
experiments have led to more “reliable” methods of cryopreservation
(Songsasen et al., 1997). But even the more reliable method using a raffinoseglycerol solution with egg yolk only yields a post-thaw sperm motility of 13%
after 30 minutes of incubation at 37°C. This decline in sperm motility is much
greater than the decline seen after even 72 hours at 4°C (30.0±3.7%).
Therefore, short-term storage of epididymal mouse sperm would give an
advantage over freezing and thawing the sperm due to the higher motility rates
of cold storage vs. freezing.
Normally, the next step in the research of the preservation of epididymal
mouse sperm by refrigeration or at ambient temperatures would be fertilization
studies. Several studies have been published subsequent to our preliminary
experiment. Songsasen et al. (1998) euthanized male mice and stored their
bodies at 22°C for up to 24 hours. The epididymal sperm was collected after 6,
12, 18 and 24 hours of 22°C storage. The sperm was then use to fertilize
mouse ova in vitro. The cleavage rates for these sperm were 81% for control
epididymal mouse sperm, 70% for sperm subjected to 6 hours of storage at
22°C, 64% after 12 hours of storage, 34% for sperm stored for 18 hours and
52
19% for those stored for 24 hours at 22°C. The development to morulae and
blastocysts for each of these groups was 65%. Live pups were derived from
the transfer of embryos from each of these groups with 3 live pups from 11
transferred embryos derived from IVF with epididymal mouse stored for 24
hours at 22°C. This experiment illustrated that epididymal sperm from an
animal left at “room” temperature after death for up to 24 hours can be used to
fertilize ova in vitro and result in live offspring from the transfer of those
embryos.
A subsequent experiment (An et al., 1999) investigated the fertilizing
capability of epididymal mouse sperm stored at 0°C, 4 to 6°C or 8 to 10°C.
Male mice were euthanized and refrigerated whole with epididymal sperm
collected as a test case for future use in the rescue of epididymal sperm from
valuable laboratory and domestic animals and exotic and endangered animals.
In this study, storage at 0°C for 3 days had a detrimental effect on the
epididymal mouse sperm. Only 2% of ova were fertilized when this sperm was
used for conventional IVF and 19% fertilized when partial zona dissection (PZD)
was performed before introduction of the sperm (An et al., 1999). Epididymal
mouse sperm stored for 3 days at 4 to 6°C had the best fertilization rates when
used for conventional IVF (28%) and IVF with PZD (64%). Fertilization rates
using epididymal mouse sperm stored at 8 to 10°C were lower than fertilization
rates for sperm stored at 4 to 6°C but higher than the fertilization rates of
epididymal sperm stored at 0°C (16% for conventional IVF and 58% for IVF with
PZD (An et al., 1999).
53
An et al. (1999) continued their research by comparing the fertilizing
capability of the epididymal sperm of two strains of mice (ICR and BDF1) after
storage for 0, 3, 5 and 7 days at 4 to 6°C. Fertilization was achieved during IVF
for all of these groups of epididymal sperm and embryos continued in their
development. Further studies on the embryos derived from IVF with epididymal
sperm of the BDF1 mice stored at 4 to 6°C for 5 and 7 days lead to the live birth
of pups with and without the use of PZD. The IVF embryos derived from the
epididymal sperm stored at 4 to 6°C for 5 days yielded a 47% live birth rate
without PZD and 38% live birth rate with PZD. The transfer of embryos from
the epididymal sperm of mice stored at 4 to 6°C for 7 days led to a 42% live
birth rate for embryos derived from conventional IVF and 34% live birth rate for
embryos derived after PZD was performed. These results are encouraging for
the use of epididymal sperm stored within whole testicles (in this case the whole
animal) under refrigerated conditions.
Kishikawa et al. (1999) took an important step in the investigation of IVF
with epididymal mouse sperm by performing ICSI with the sperm. Euthanized
male mice were refrigerated at 4°C. Epididymal sperm was sampled and used
for IVF after 0, 5, 10 and 15 days of storage within the epididymis at 4°C (sperm
motility and viable sperm had both declined to 0% after 20 days of storage at
4°C). Fertilization using conventional IVF led to an 81.5% fertilization rate when
control (freshly collected) epididymal sperm were used (0 days at 4°C). The
fertilization rate declined to 26.4% when epididymal sperm stored at 4°C for 5
days was used, a further decline to 2.8% for sperm stored for 10 days and 2.7%
54
for sperm stored for 15 days. ICSI using epididymal mouse sperm stored at
4°C for 5 days fertilized 85.2% of injected ova and the implantation rate after
embryo transfer was not different from the implantation rate of the control ICSI
embryos. The live birth of 18 pups resulted from the ICSI control group and the
day-5 group. Fertilization rates did decline with the use of epididymal mouse
sperm stored at 4°C for 10, 15 and 20 days, but live pups were born from
embryos produced from each of these groups. Even the immotile epididymal
sperm retrieved after 20 days of refrigerated storage had a 14% implantation
rate resulting in the birth of 6 live pups. These results are encouraging for ICSI
using cool-stored epididymal sperm.
The apparent fertilization capabilities of cooled epididymal mouse sperm
and the lack of decline in sperm motility and percent membrane-intact sperm for
2 and 3 days of storage, respectively, makes the short-term storage of
epididymal mouse sperm a viable option for use in IVF. The objective of a
genome resource bank is to successfully cryopreserve epididymal sperm after
post-mortem collection for the salvage of genetic information from endangered
species.
Several studies on cooling epididymal sperm collected from exotic and
endangered animals have been performed in the African buffalo, eland, Red
hartebeest and Burchelli's zebra (Bezuidenhout et al., 1995), the White
rhinoceros (Williams et al., 1995), the impala (Rush et al., 1997), the African
buffalo (Friedmann et al., 2000), the Roan Antelope, gemsbok and African
buffalo (Kilian et al., 2000) and the White rhinoceros and Burchelli's zebra
55
(Lubbe et al., 2000). The majority of these studies are performed on animals
who were culled at large game preserves in Africa.
Bezuidenhout et al. (1995) simply observed progressive motility in 4
hoofstock species at 8-hour intervals. The results in this study followed the
same general pattern as the decline in motility in our study. Each species had a
different starting motility and different rate of motility decline but the pattern was
the same. The pattern of decline in motility illustrated in a similar manner in the
impala (Kilian et al., 2000) and the zebra and rhinorceros (Lubbe et al., 2000).
Williams et al. (1995) compared refrigerated epididymal White rhinoceros
sperm to room temperature and physiological temperature. The initial motility of
85% was preserved for 12 hours at 4ºC, the motility declined to 80% at 22 to
25ºC by 12 hours and to 0% by 12 hours at 38ºC each in sperm TALP. Percent
live sperm was 95% initially, declined to 83% after 12 hours at 4ºC, 92% at 22
to 25ºC and 12% at 38ºC. Although the time period of this study was much
shorter than in the present study, the pattern of decline in motility and percent
live sperm is similar between the two studies.
Rush et al. (1997) cultured epididymal impala sperm at 4-7ºC and 2832ºC for 16 hours in five different media with the purpose of choosing the least
toxic cryoprotectant. In that study, glycerol was found to be the least toxic
cryoprotectant for impala sperm.
The exotic animal sperm study that was the most like ours was
performed by Friedmann et al. (2000). In this study, African buffalo testicles
were stored at 4ºC and at 18 to 28ºC. Initial progressive sperm motility of the
56
epididymal sperm was ~37% (slightly lower than the initial motility of epididymal
mouse sperm in this study of ~48%). The progressive motility fell of the buffalo
sperm only slightly to ~33% after 24 hours at 4ºC, the decline was similar to the
decline in motility of the epididymal mouse sperm in this study (~43%). The
motility of the buffalo sperm had decreased to only ~5% in the ambient
temperature testicle during the 24 hour storage, compared with a decline to only
~22% for the mouse. Both the sperm of the buffalo and the mouse had
decreased to 0% by 48 hours of storage at room temperature.
Gamete ”rescue” has also been performed on the female of the mouse
species. Schroeder et al. (1991) collected oocytes from euthanized female
mice 3, 6, 9 and 12 hours post-mortem. The number of oocytes collected with
intact cumulus culls decreased as the amount of time after death increased, but
maturation was the same for all groups, including the control. Development of
2-cell embryos was the same for the control group and the 3-hour and 6-hour
groups, but there was a significant decrease in 2-cell embryo development for
the 9-hour and 12-hour groups. Blastocyst rates were the same for the control
group and the 3-, 6- and 9-hour groups, however, no blastocysts developed
from the 12-hour group. Live young were achieved from 36% of transferred 2cell embryos derived from oocytes collected from a mouse 6 hours after
euthanasia.
Tada et al. (1997) achieved the ultimate in gamete rescue technology
when they produced live mice using frozen-thawed oocytes and frozen-thawed
57
epididymal sperm. This model is the reason for the modern genome resource
bank and the goal of endangered species research.
58
CHAPTER IV
THE EFFECTS OF STORAGE AT 22°C AND 4°C ON THE SURVIVAL OF
EPIDIDYMAL CANINE SPERM
Introduction
Epididymal sperm can produce pregnancies and live births from assisted
reproductive technology (ART), such as artificial insemination (AI), in vitro
fertilization (IVF) and intracytoplasmic sperm injection (ICSI). There are several
species in which epididymal sperm has produced pregnancies and live births,
these include: the horse (Barker and Gandier, 1957), sheep (Fournier-Delpech
et al., 1979), dog (Marks et al., 1994), Common marmoset (Morrell et al., 1997
and 1998), rat (Nakatsukasa et al., 2001) and eland (Bartels et al., 2001)
through AI. The mouse (Miyamoto and Chang, 1972; Hoppe and Pitts, 1973),
rat (Toyoda and Chang, 1974), cow (Graff et al., 1996), human (Temple-Smith
et al., 1985; Bonduelle et al., 1996; 1998) and goat (Blash et al., 2000)
employing IVF and human (Temple-Smith et al., 1985; Bonduelle et al., 1998)
and rat (Hirabayash et al., 2002) using ICSI. These offspring illustrate that the
use of epididymal sperm is a worthwhile avenue for further study.
The previous studies used the mouse as a model for the study of the
effects of storage temperature on epididymal sperm. The objective in this study
was to use a larger species, with testicles large enough to sample repeatedly
over consecutive days, to establish the effect of temperature on the motility,
percentage of membrane-intact sperm and longevity of epididymal dog sperm
stored at 4ºC.
59
The domestic dog is a good species on which to perform epididymal
sperm experiments because of the availability of tissue from animal control
centers and the abundance and similarity of the epididymal sperm. The dog is
also an excellent model for endangered canine species. The dog has promise
in the area of ART with epididymal sperm, as evidenced by the birth of a live
pup after AI with frozen epididymal sperm collected after the death of the male
dog (Marks et al., 1994). Epididymal dog sperm has also been used to fertilize
dog ova in vitro at a rate of 57% (4 of 7) (Williams et al., 2001).
Advances with the use of epididymal sperm could be especially
important to exotic and endangered animal conservation efforts. There are
many instances when epididymal sperm could be the only sperm available. In
some cases, animals in a captive population die before they have made a
genetic contribution to the population. If that genetic contribution could be
salvaged by collecting the sperm cells from the epididymis for use in ART
procedures, then the gene pool of the species could be better preserved. Also,
the ability to use epididymal sperm from animals frequently culled by wildlife
parks, due to space limitations, could preserve their genetic material for use in
bringing in “wild” DNA into captive populations. This introduction of wild DNA is
very important in many captive species whose progeny all came from just a few
captive founder animals.
African buffalo embryos have been produced using oocytes collected
from post-mortem ovaries and sperm from the epididymides of bulls postmortem (Shaw et al., 1995). Following tissue harvest during a cull at a wildlife
60
park in South Africa, oocytes were removed by mincing the ovaries. The
testicles were refrigerated for up to 24 hours before the sperm was collected for
IVF. The best IVF treatment group (CR1 medium with no somatic cell coculture) resulted in a 50% cleavage rate, with 76% developing to the 8- to 16cell stage and 65% to the morula stage. This kind of gamete rescue and
resultant embryo production could lead to the infusion of new “wild” DNA if the
embryos are transferred and produce live births. Bartels et al. (2001) used
frozen-thawed eland epididymal sperm to perform AI on eland cows, which
resulted in the birth to a healthy calf.
Another important aspect of collecting epididymal sperm is knowing how
long viable sperm cells can be collected from testes that are stored under
refrigeration. Bezuidenhout et al. (1995) evaluated progressive motility of
epididymal sperm collected from the African buffalo, Red hartebeest, eland and
Burchell’s zebra over a 2-day period of storage at 4ºC. These testicles were
also harvested during a culling at a wildlife park. The motility of the epididymal
sperm decreased slowly over the 2-day period (the epididymal sperm was
sampled every 8 hours). The African buffalo epididymal sperm had an initial
motility of ~65% and then declined to ~10% by 48 hours.
Viable sperm cells were recovered from the epididymides up to 5 days
after the death of the animal. Similar studies have been performed on other
African species during culling procedures: the White rhinoceros (Williams et al.,
1995; Lubbe et al., 2000), the impala (Rush et al., 1997), the African buffalo
61
(Friedmann et al., 2000; Kilian et al., 2000), the Roan antelope and gemsbok
(Kilian et al., 2000) and the Burchelli's zebra (Lubbe et al., 2000).
Pangestu (1997) evaluated goat epididymal sperm parameters including
motility, concentration, percentage of membrane-intact sperm and percentage
of normal sperm at 0, 1, 2 and 3 days of cold storage after the death of the
animal. These values were compared with those of freshly ejaculated semen
from the same animals before they were euthanized. The percentage of normal
sperm and sperm concentration were not reduced after the cold storage, but
motility and percentage of membrane-intact sperm did decrease over the 3-day
period. However, even after 3 days of refrigerated storage there were still
membrane-intact cells within the goat epididymides, leaving open the option of
using the ICSI procedure for fertilization.
Cryopreservation associated with ART procedures involves the use of
cryoprotectants that are toxic to sperm cells and coupled with the damage of
the freezing process itself (sperm are usually less motile post-thaw). One way
to avoid this damage, at least for short-term use of the sperm, is to refrigerate
the testicle rather than collection and cryopreservation of the sperm. Kikuchi et
al. (1998) have shown that the motility of sperm cells in cold stored (4ºC) boar
epididymides (stored for 2 days) is significantly greater than frozen-thawed
sperm. They also found that the cold-stored epididymal boar sperm could
fertilize pig ova, although at a lower rate than control boar epididymal sperm.
This illustration of the fertilizing ability of epididymal sperm stored for 2 days at
4ºC in the pig was encouraging for research on other species.
62
Materials and Methods
Experimental Design
Dog testicles were collected and randomly assigned to two storage
temperature groups: room temperature (22ºC, Treatment A) and refrigerated
(4ºC, Treatment B). Motility, percentage of membrane-intact sperm and
concentration of the epididymal sperm were taken for each testicle at time of
collection (day 0) and again at 24 hours and 48 hours post-treatment (see Table
4.1).
After the 48-hour parameters were recorded, a longevity study was
begun on the epididymal sperm of the refrigerated testicles. The testicles were
maintained under refrigerated storage. Sperm motility, percent membraneintact sperm and concentration of sperm were evaluated at 24-hour intervals
until sperm motility decreased to 0%.
The tissues used in this study were obtained from the East Baton Rouge
Parish Animal Control Center from euthanized animals. Initial estimates of age,
weight and breed were recorded for each dog. Scrotal circumference was also
measured in cm and recorded. The testicles of the euthanized dogs were
collected by cutting them away from the body using scissors. The testicles
were left within the scrotum and placed in a plastic ziplock bag for transport in
an air-conditioned vehicle to the St. Gabriel Research Station. The testicles
were not refrigerated during the ~30 minute transport.
63
Table 4.1. Experimental design of epididymal dog sperm at 22°C (Treatment A) and 4°C (Treatment B)
Time
Treatment
(n)
Sperm motility (%)
Membrane-intact
sperm (%)
Concentration
(million/ml)
0 hours
(Control)
A
22
+
+
+
B
22
+
+
+
A
22
+
+
+
B
22
+
+
+
A
22
+
+
+
B
22
+
+
+
24 hours
48 hours
64
Tissue Processing
Upon arrival at the laboratory, the testicles were washed and removed
from the scrotum. The tunica albuginea were also cut away being careful not to
damage the epididymis where it was attached to the tunica. The blood vessel
leading to the testicle and the vas deferens was trimmed along the curve of the
testicle using curved scissors, so that no connective tissues protruded from
their point of connection to the testicle.
The circumference of each testicle was measured by using a piece of
rubber tubing to encircle the testicle at its largest point. The tubing was then
laid flat against a metric ruler and the measurement in centimeters was
recorded. The length of each testicle was measured and recorded in a similar
manner, with the tubing laid along the side of the testicle from bottom edge of
the cauda epididymidis to the ventral point of the testicle. Each testicle was
weighed on an electronic balance and the weight in grams recorded. The
testicles from each dog were then assigned randomly to either the room
temperature treatment group (Treatment A) or the refrigerated treatment group
(Treatment B).
Sperm Analysis
An initial sperm sample from the cauda epididymidis was taken from
each testicle. Only sperm cells form the cauda epididymidis were used
because they are the most mature cells within the epididymis and are stored in
the cauda epididymidis for ejaculation. A 16-gauge hypodermic needle (Becton
Dickinson, Franklin Lakes, NJ) was used to puncture the cauda epididymidis to
65
allow the concentrated sperm solution to well up out of the epididymal tubule. A
5-ml Drummond pippettor (Drummond Scientific Corporation, Broomall, PA) was
used to aspirate exactly 5 ml of the epididymal sperm. The first 5 ml sample was
added to 95 ml of water to immobilize the sperm cells so that a concentration
could be counted by placing a 10 ml aliquot onto a each side of a
hemacytometer (Cambridge Instruments, Inc., Buffalo, NY). The number of
sperm cells in five of the hemacytometer squares was counted on each side.
The total from both sides was divided by 2 to get an average, which
represented the number of million cells/ml of fluid in the sample.
Another 5 ml sample was added to a 0.5-ml microcentrifuge tube (DOT
Scientific, Inc., Burton, MI) containing 95 ml of Tyrode Lactate HEPES solution
(TLH) (Bio-Whittaker, Walkerville, MD). A 5 ml portion of the extended sperm
solution was then placed on a glass slide (Gold Seal Products, Portsmouth,
NH). A 5 ml drop of Eosin-B/Fast Green dye (Sigma, St. Louis, MO) was added
to the sperm solution and thoroughly mixed. A second glass slide was then
used to smear the sperm and dye solution along the length of the slide. A hair
dryer was used to quickly dry the smear to prevent live sperm from dying as the
slide dried in air. The death of these sperm cells would cause more of them to
take up the indicator dye and cause the reading of dead sperm on the slide to
be an inaccurate representation of the initial sample. The samples in the
microcentrifuge tubes were then placed in a humidified incubator set at 39°C
5% CO2 for ~30 minutes to allow the sperm to warm back up to body
temperature.
66
Glass slides were pre-warmed on a slide warmer set at 39°C. This was
done to keep the sperm samples warmed after they left the incubator and
before they were placed under the microscope. A 10 ml drop of warmed sperm
solution was placed on the glass slide and a glass cover slip (Baxter Health
Care Corporation, McGaw Park, IL) was placed on top of the sample. The
sample was then evaluated at 200X magnification using a compound
microscope. Percent sperm motility was estimated by visual observation.
The Eosin-B/Fast Green slides were read on the same microscope under
400X magnification. The Fast Green dye acted as a background color and the
Eosin-B was the indicator dye. If the cell had intact membranes, it was able to
exclude the Eosin-B dye and appeared white against the green background. If
the membrane of the sperm cell became ruptured or torn the cell was not able
to exclude the indicator dye and the cells appeared pink (Aalseth and Saacke,
1986). A total of 200 sperm cells were counted on each slide using a counter to
keep track of membrane-intact and dead sperm and the two counts were
averaged. The “percentage of membrane-intact sperm” of the sample was
obtained by dividing the average number of membrane-intact sperm counted by
2.
Testicle Storage
The testicles were placed in their respective treatments as soon as the
initial sperm sample had been taken. The testicles were placed in either of two
identical rectangular stainless steel surgical pans each with a non air-tight
stainless steel lid. The pans were lined with two layers of sterile gauze
67
(Johnson & Johnson, Arlington, TX) the testicles were placed on top and
covered with two layers of sterile gauze. Sterile saline (Sigma, St. Louis, MO)
was added to the pan until the gauze was completely wet to keep the sample
from drying out. The pan of testicles allotted to the room temperature group
(Treatment A) was placed in a climate-controlled room (~22°C). The pan of
testicles allotted to the refrigerated group (Treatment B) was placed in a
standard refrigerator (4°C). The testicles were sampled and allocated into their
treatment within 3 hours of euthanization.
The testicles were sampled in the same manner after 24 hours and 48
hours of treatment. The sperm was aspirated from the initial puncture of the
epididymis if possible, otherwise additional punctures were made. The
longevity study on the refrigerated testicle continued the same sampling at 24hour intervals until the motility of the sample reached 0%.
Statistical Analysis
The parameters of testicular weight, testicular length, testicular
circumference, motility, percentage of membrane-intact sperm and
concentration of sperm were analyzed using ANOVA with repeated measures.
The main effects of treatment (22ºC vs. 4ºC), sampling time (0, 24 and 48
hours) and treatment by sampling time interactions were compared. No
statistical analysis of the longevity study on the epididymal sperm of the
refrigerated testicles was executed because there were no corresponding
readings at room temperature to compare with the refrigerated values.
68
Results
The first analysis was on the initial parameters of the two testicle groups
of Treatment A and Treatment B to test for any differences between the
testicles from the same dog. There were no significant differences between the
two groups of testicles of the experimental dogs for testicular length (4.6±0.1
compared with 4.6±0.1 cm), testicular circumference (7.8±0.2 and 7.7±0.2 cm),
testicular weight (16.5±1.2 and 16.2±1.2 g) and initial sperm concentration
(394.0±32.5 and 390.0±32.5 million/ml) for Treatment A at Time 0-hour
compared with Treatment B at Time 0-hour.
The main parameters of interest (sperm motility and percentage of
membrane-intact sperm) were expected to change over time due to the
influence of the treatments. Therefore, it was important to establish the fact that
there was no significant difference between the initial measurements of these
parameters when the testicles allotted to Treatment A was compared with the
testicles allotted to Treatment B.
The mean sperm motility (±SEM) and mean percentage of membraneintact sperm (±SEM) of the epididymal sperm from the testicles in Treatment A
at 0 hour were compared with the motility and percentage of membrane-intact
sperm of the epididymal sperm from the testicles in Treatment B at 0 hour (see
Table 4.2). The initial sperm motility for Treatment A was 86.1±1.8% and
86.6±1.8% for Treatment B. The initial percentage of membrane-intact sperm
for Treatment A was 90.6±1.5% and 90.3±1.2% for Treatment B. No significant
difference was detected for either of these two parameters evaluated. These
69
results established that the two groups of testicles were not different at the
onset of the experiment, thus any future difference in these parameters would
likely be due to the treatment effects.
The initial parameters of the epididymal dog sperm, including initial
concentration are presented in Table 4.2. The subsequent concentration
measurements taken after 24 and 48 hours of storage are listed in Appendix
Table 2. These data were not subjected to statistical analysis. The initial
concentration of the epididymal sperm was obtained by puncturing the cauda
epididymidis with a needle and removing a portion of the sperm. Degeneration
of the tissues in the 22°C group and leakage from the initial puncture in both
treatments also effected the subsequent concentration readings, therefore we
felt that any analysis of these data would be misleading.
One of the main variables of interest in this study was the change in the
motility of the epididymal sperm over time. As indicated previously, there was
no significant difference when the sperm motility of the Treatment A control (at
Time 0-hour at 86.1±1.8%) was compared with the sperm motility of the
Treatment B control (at Time 0-hour) 86.6±1.8%. The motility results for these
comparisons are presented in Figure 4.1A.
In this study, it was found that the temperature as a treatment caused a
significant decrease in sperm motility (P<0.0001) as did the repeated measure
over time (day) and the interaction of day by treatment (P<0.0001 in both
cases). The treatments, the effect of time and the interaction of the two were all
significantly different effects when sperm motility of Treatment A was compared
70
with the motility of Treatment B at both 24 hours (55.7±2.4% vs. 79.3±1.7%)
and 48 hours (1.0±0.7% vs. 69.5±2.3%) (P<0.0001 for both).
The comparisons of the sperm motility of Treatment A among time
periods and the comparison of the sperm motility of Treatment B among time
periods are presented in Figure 4.1B. The sperm motility of Treatment A at
Time 0-hour (86.1±1.8%) was significantly higher (P<0.0001) when compared
with the sperm motility of Treatment A at 24 hours (55.7±2.4%). The sperm
motility of Treatment A at 48 hours (1.0±0.7%) decreased significantly
(P<0.0001) from the sperm motility of Treatment A at 24 hours (55.7±2.4%) and
from the sperm motility of Treatment A at Time 0-hour (86.1±1.8%, P<0.0001).
The sperm motility of Treatment B at time 24 hours (79.3±1.7%) was
significantly lower than the sperm motility of Treatment B at Time 0-hour
(86.6±1.8%, P<0.0071). The sperm motility of Treatment B at 48 hours
(69.5±2.3%) was significantly lower than both the sperm motility of Treatment B
at Time 0-hours (86.6±1.8%) and at 24 hours (79.3± 1.7%) (P<0.0001 for both).
The same statistical comparisons made for sperm motility were also
made for the percentage of membrane-intact sperm and had similar results.
The percentage of membrane-intact sperm of Treatment A at Time 0-hour
(90.6±1.5%) was not significantly different when compared with the percentage
of membrane-intact sperm for Treatment B at Time 0-hour (90.3±1.2%) (Figure
4.2A). The percentage of membrane-intact sperm for Treatment A at 24 hours
of storage (67.5±2.5%) was significantly lower than (P<0.0001) the percentage
of membrane-intact sperm for Treatment B at 24 hours (86.5±1.5%). The
71
percentage of membrane-intact sperm of Treatment A at 48 hours (7.4±2.7%)
was also significantly lower than the percentage of membrane-intact sperm of
Treatment B at 48 hours (80.8±1.9%) (P<0.0001).
The comparison of the percentage of membrane-intact sperm of
Treatment A among time periods and the comparison of the percentage of
membrane-intact sperm of Treatment B between time-periods is illustrated in
Figure 4.2B. The percentage of membrane-intact sperm for Treatment A at
Time 0-hour (90.6±1.5%) was significantly greater than the percentage of
membrane-intact sperm of Treatment A at 24 hours (67.5±2.5%, P<0.0001).
When the percentage of membrane-intact sperm for Treatment A at 48 hours
(7.4±2.7%) was compared with the percentage of membrane-intact sperm for
Treatment A at both 0 hour (90.6±1.5%) and 24 hours (67.5±2.5%), it had
significantly decreased in each case (P<0.0001).
When the percentage of membrane-intact sperm of Treatment B at Time
0-hour (90.3±1.2%) was compared with the percentage of membrane-intact
sperm for Treatment B at 24 hours (86.5±1.5%), unlike with the parameter for
motility, there was no significant difference (P<0.1730). The percentage of
membrane-intact sperm for Treatment B at 48 hours of storage (80.8±1.9%)
was significantly lower (P<0.0009) when compared with the percentage of
membrane-intact sperm of Treatment B at 0 hour (90.3±1.2%).
72
Table 4.2. Initial testicular measurements and epididymal sperm parameters of canine testicles for Treatment A (22ºC)
and Treatment B (4ºC). Values are presented as mean±standard error of the mean.
Treatment
Testicular Testicular
weight (g) length (cm)
Testicular
circumference(cm)
Initial sperm
motility (%)
Initial
membrane-intact
sperm (%)
Initial
concentration
(million/ml)
A
16.5±1.2
4.6±0.1
7.8±0.2
86.1±1.8
90.6±1.5
394.0±32.5
B
16.2±1.2
4.6±0.1
7.7±0.2
86.6±1.8
90.3±1.2
390.0±32.5
73
Mean (±SEM) Sperm Motility (%)
100
A.
a
a
b
80
b
a
60
40
20
a
0
0
24
48
Mean (±SEM) Sperm Motility (%)
100
B.
a
a
b
80
c
b
60
40
20
c
0
0
24
0
48
24
48
Treatment Sampling Time (hours)
Figure 4.1.
Effects of storage at 22ºC (Treatment A; open bars) and 4ºC
(Treatment B; shaded bars) on motility of epididymal dog sperm.
A. Comparison of Treatment A with Treatment B. ab P<0.0001
B. Comparison between time periods of Treatment A and
comparisons between time periods of Treatment B.
abc
denotes significant difference between the time periods.
74
Mean (±SEM) Membrane-Intact Sperm
100
a
a
b
80
A.
A.
a
60
40
20
a
0
0
Mean (±SEM) Membrane-Intact Sperm
b
100
24
a
80
48
a
a
B.
b
b
60
40
20
c
0
0
24
48
0
24
48
Treatment Sampling Time (hours)
Figure 4.2.
Effects of storage at 22ºC (Treatment A; open bars) and 4ºC
(Treatment B; shaded bars) on the percentage of membraneintact sperm of epididymal dog sperm.
A. Comparison of Treatment A with Treatment B. abP<0.0001.
B. Comparison between time periods of Treatment A and
comparison between time periods of Treatment B.
abc
denotes significant difference between time periods.
75
100
90
80
Percent
70
60
Sperm motility
50
Live sperm
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (Days)
Figure 4.3. Mean motility and mean membrane-intact sperm (%) of canine epididymal sperm stored within the
testicle at 4ºC.
76
The percentage of membrane-intact sperm for Treatment B at 48 hours
(80.8±1.9%) was significantly lower (P<0.0444) when compared with the
percentage of membrane-intact sperm for Treatment B at 24 hours
(86.5±1.5%).
To exclude any interactions from dog breeds and animal size on motility,
the dogs were grouped by breed and size and the sperm motility of Time 0hour, 24-hour and 48-hour were evaluated and compared. In the present
study, known aggressive breeds (e.g., Chow, Rottweiler, Pitt bull) were
compared with more non-aggressive breed-types (all other breeds). There
were 11 dogs in each of the two breed-type categories in this study. Values for
breed-type and the day by breed interaction were both not significantly different
in the present study. The effect of the dogs body size on sperm motility was
also tested for the same 3 time points (0, 24 and 48 hours). When small dogs
(18 kg or less) were compared with large dogs (over 18 kg), the values from
size and day by size were not significantly different.
Discussion
In this study sperm motility and percentage of membrane-intact sperm of
epididymal dog sperm was greatly impaired by extended maintenance at
standard room temperature (~22°C). Sperm motility and percentage of
membrane-intact sperm were higher when the testicles were held at 4°C when
compared with those stored at room temperature. The lack of breed-type or
body size effect on sperm motility suggests that this procedure could be applied
77
to non-domestic canids (regardless of size) to preserve epididymal sperm from
wolves, wild dogs and foxes.
Pangestu (1997) collected epididymal sperm up to 3 days after death
from goats. The initial motility of 80% declined to 40% by 24 hours, 18% by 48
hours and 15% by 72 hours. The decrease in motility by 24 hours was greater
that the loss of motility by epididymal dog sperm at room temperature in our
study, however, the motility at 48 and 72 hours were greater for the goat
(Pangestu, 1997) than for the dog. Songsasen et al. (1998) were able to use
epididymal mouse sperm retrieved 24 hours after the death of the animal to
produce live pups through IVF and ET.
Kikuchi et al. (1998) stored boar epididymides at 4ºC for 1, 2 and 3 days.
They found, as we did, that the motility declined over the storage period,
eventually equaling the motility of frozen-thawed epididymal sperm by day 3 of
storage. The epididymal sperm collected after 1, 2 and 3 days of storage at 4ºC
was able to penetrate ova at 12%, 13% and 2%, respectively.
Researchers have begun collecting epididymal sperm from exotic and
endangered animals. Some of these studies are investigating the longevity of
epididymal sperm at 4ºC (Kilian et al., 2000; Lubbe et al., 2000). Kilian et al.
(2000) collected epididymal sperm from a cull of Roan antelope, gemsbok and
African buffalo and stored the testicles at 4ºC. The epididymal sperm of the
stored testicles was sampled every 12 hours by puncturing the cauda
epididymidis. The Roan antelope epididymal sperm motility was initially ~80%
and slowly decreased until reaching 0% by 96 hours of storage at 4ºC. The
78
gemsbok epididymal sperm motility was initially ~70% and also decreased to
0% by 96 hours of storage at 4ºC. The African buffalo epididymal sperm
motility was also ~70% initially, but its decline was slower and had reached only
~10% at the end of the experiment (108 hours of storage at 4ºC). In all three
cases, the general pattern of sperm motility decline was similar to that of the
dog, however, the epididymal sperm remained motile for slightly longer in the
dog (up to 7 days).
Lubbe et al. (2000) compared the motility of epididymal sperm of the
horse, the zebra and the White rhinoceros when stored at 4ºC. The epididymal
horse sperm was the hardiest, with an initial motility of ~65% and a lowest
motility of ~10% at 132 hours of storage. The epididymal sperm of the White
rhinoceros was more sensitive to the cold storage and its initial motility of ~60%
decreased to 0% by 108 hours. The Burchell’s zebra epididymal sperm had the
lowest initial motility of only ~45% and was the most sensitive to the cold
temperature becoming completely immotile by 60 hours.
In the present study, the motility of epididymal dog sperm remained
motile longer than reported for the exotic species. The pattern of motility of
epididymal sperm for the dog was, interestingly very similar to the horse, which
served as the domestic model for the zebra and the White rhinoceros in the
study by Lubbe et al. (2000). This similarity of domestic “model” animals
epididymal sperm remaining motile at 4ºC for a longer period that their exotic
counterparts might suggest that the epididymal sperm of most exotic species
79
are more sensitive than that of its domestic animal model. Certainly, more
research is necessary in this area.
Friedmann et al. (2000) performed an experiment on the African buffalo
that closely resembled the design of the present study. Testicles were collected
from culled buffalo and stored the testicles at 18 to 28ºC or at 4ºC. The initial
sperm motility of ~37% was lower than what we found for the dog at ~86%. The
decline in motility by 24 hours at 4ºC to 33% was only 4%, which was a slower
decline than that of the dog (a 7% drop to ~79%). The decline by 24 hours at
18 to 28ºC, however, was more dramatic than the dog, falling to ~5% vs. ~56%.
Longevity was an important aspect of our study. The measurements of
sperm motility and percentage of membrane-intact sperm of the epididymal dog
sperm stored at 4ºC could not be compared with 22ºC readings because tissue
degeneration prevented any further sampling after 48 hours. Motile epididymal
sperm cells were still present, although rare in dog epididymides for up to 6 and
7 days of storage at 4ºC, also the percentage of membrane-intact sperm was
still at 29% after 7 days. This pattern has also been reported in the African
buffalo (Bezuidenhout et al., 1995) in which live sperm cells were recovered
from epididymides stored at 4ºC up to 5 days after collecting the testicles.
More recently, dog ova have been fertilized by ICSI (using ejaculated
sperm that had been held at 5ºC overnight) (Fulton et al., 1998), therefore, it
could be possible to isolate the few remaining motile sperm in a sample to use
for sperm injection. It could also be possible to use an immotile sperm cell for
ICSI as Goto et al. (1990) have done to fertilize bovine ova. Another option
80
might be to revive the motility of the immotile, membrane-intact sperm through
the addition of caffeine or pentoxifylline. Caffeine has been used to stimulate
sperm motility in the bull (Milkowski et al., 1976) and pentoxifylline to increase
motility in dog sperm (Koutsarova et al., 1997).
Terriou et al. (2000) found that pentoxifylline initiated motility in immotile
epididymal and testicular human sperm. These “revived” sperm was then used
for ICSI with a 42.5% fertilization rate and a 30% pregnancy rate, with 4
ongoing pregnancies and 2 healthy babies born at the time of this report.
It is important to note that like the dog, motile sperm has been found
after several days of storage at 4ºC in other species. Live sperm cells have
been found in epididymides stored at 4ºC in the African buffalo (Bezuidenhout
et al., 1995) and live young have been produced from epididymal mouse sperm
after 7 days of storage at 4ºC (An et al., 1999).
The domestic cat is an excellent model for endangered exotic felids and
research on the cat has increased in the last several years. Domestic cat
epididymal sperm is often used in IVF because it is also plentiful from veterinary
clinics and animal control centers. Goodrowe and Hay (1993) performed an
experiment on epididymal cat sperm as a model for nondomestic felids. They
determined some of the basic characteristics of epididymal cat sperm, timed
capacitation of the sperm and investigated if and how storage temperature
affected the fertilizing ability of the sperm. They reported that epididymal cat
sperm is ~52% motile, has a concentration of ~80 million cells/ml. These
81
findings were lower than the percentage of motility and concentration (~86%
motility and ~ 390 million cells/ml) in the present study of epididymal dog sperm.
Goodrowe and Hay (1993) also reported that ~51% of epididymal cat
sperm cells were abnormal, with the cytoplasmic droplet as the most common
form of abnormality. Optimal capacitation of the epididymal cat sperm was
reached after 4 hours of co-culture with feline zonae pellucidae.
To determine “fertilizing ability”, Goodrowe and Hay (1993) performed a
zona binding study and a hamster penetration assay comparing fresh cat
epididymal sperm with cooled epididymal sperm (stored at 5ºC in the epididymis
overnight). The motility of the epididymal sperm decreased from 71% for fresh
to 57% after overnight cold storage. But both zona binding and hamster ova
penetration were significantly higher with cooled epididymal cat sperm than with
fresh epididymal cat sperm.
Studies on cooled ejaculated sperm have also been performed by Pope
et al. (1997; 1998). Pope et al. (1997) used cooled ejaculated cat semen for
IVF. The semen from one tomcat was either extended in Test-Tris egg-yolk
buffer and stored at 2ºC for 18 to 22 hours or was used immediately for IVF.
The fertilization rate of the cooled cat sperm was significantly greater than the
fertilization rate from the fresh cat sperm (74% vs. 28%). Embryos derived from
IVF with cooled ejaculated cat sperm were transferred and produced two live
kittens.
Pope et al. (1998) used cooled ejaculated cat sperm to produce IVF
embryos through ICSI for comparison with conventional IVF. Non-domestic
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felids often have poor quality sperm therefore, the ability to use ICSI would be
advantageous with these animals. The fertilization rate for conventional IVF
with cooled cat semen was reported to be 68%, while the fertilization rate was
58% for the ICSI group (Pope et al. 1998). Transfer of embryos that developed
after ICSI using cooled cat semen resulted in the birth of three kittens.
Cold storage of dog sperm for AI is commonplace, but it is usually with
ejaculated sperm, not epididymal sperm. Bouchard et al. (1990) reported the
effects of storage temperature, cooling rate and semen extenders on the
motility of ejaculated canine sperm. They found that a nonfat dried milk solidglucose diluent when paired with a medium or fast (-1ºC or –3ºC) cooling rate to
4ºC led to the best motility preservation. Rota et al. (1995) also performed a
study on extenders of ejaculated, cooled dog sperm. It was found that an initial
ejaculated sperm motility of ~75% declined over the 4-day test but had only
declined to ~55% when used with a Tris/egg-yolk diluent.
Pinto et al. (1999) performed an AI study with chilled dog semen, which
investigated the fertility of cold stored dog sperm. It was found that sperm
motility decreased after 24 and 48 hours of storage at 4ºC (from 84% for fresh
semen to 76% after 24 hours and 63% after 48 hours). However, pregnancy
rates were 94% following AI with fresh dog semen and were not significantly
different from pregnancy rates from AI with dog semen that had been extended
and chilled for 24 hours (90%) or 48 hours (100%). Although these data are for
ejaculated, rather than epididymal sperm, they are encouraging for the use of
cooled sperm for assisted reproductive technologies. It is important to note that
83
the fertilized dog ova produced from ICSI were injected with ejaculated sperm
stored at 5ºC overnight (Fulton et al., 1998) and the dog embryos produced by
IVF with epididymal sperm were also produced from sample refrigerated
overnight at 4ºC (Williams et al., 2001).
Fuller and Wittingham (1996) used epididymal mouse sperm that had
been cooled and rewarmed for IVF. When the resultant embryos were
transferred, a significantly higher number of fetuses resulted from the cooled
epididymal sperm when compared with fresh epididymal sperm. In a later study
Fuller and Wittingham (1997) revealed that sperm penetration was faster when
cooled and rewarmed mouse epididymal sperm was used for IVF. They
proposed that cooling of the sperm caused “capacitation-like” changes in the
sperm.
Another option to consider would be to save a portion of epididymal
sperm for cooled storage for immediate use, while freezing the rest for future
use. Vishwanath and Shannon (2000) reviewed the practice of storing semen
in a cooled liquid state compared with frozen storage for bull semen. Storage in
a cooled liquid state is common for the dog, but is usually performed on
ejaculated sperm instead of epididymal sperm. Refrigeration slows metabolic
functions, which aids in preserving the cells. Certainly it does not cause as
much damage to the sperm as does freezing. Refrigerated storage could be
useful if sperm for an insemination was needed within a few days after
collection. A portion of a sperm collection could easily be put aside in the
refrigerator for the insemination, while freezing the rest.
84
Cold storage of canine testicles is a promising new approach for sperm
preservation and or storage. This technique could also be employed for
transporting whole testicles across countries or continents for processing and
use/storage at a separate facility (such as a genome resource bank), especially
if collection is not feasible where the testicles were obtained.
More research on the fertilizing capability of cooled epididymal sperm as well as
tests on freezing sperm after they have been cooled should be done to enhance
usability of these sperm cells.
85
CHAPTER V
COMPARISON OF FRESH, REFRIGERATED AND FROZEN-THAWED
CANINE EPIDIDYMAL SPERM
Introduction
Domestic animals are often used as a model for their exotic relatives
(Wildt et al., 1986). Some examples are the cow as a model for the gaur
(Loskutoff et al., 1995) and the domestic cat as a model for wild felids (Pope et
al., 1998; Pushett et al., 2000). The dog can be used as a domestic model for
wolves, foxes and other wild canids and has even been used as a model for
bears (Kojima et al., 2001).
Epididymal sperm has been proven to be effective when used for
assisted reproductive technology (ART) such as artificial insemination (AI), in
vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Pregnancies
and live births from ART using epididymal sperm have been accomplished in
several species utilizing fresh epididymal sperm in the mouse (Miyamoto and
Chang, 1972; Hoppe and Pitts, 1973), rat (Toyoda and Chang, 1974), sheep
(Fournier-Delpech et al., 1979), Common marmoset (Morrell et al., 1997) and
the cow (Graff et al., 1996). Others utilized frozen-thawed epididymal sperm to
produce pregnancies and live young in the horse (Barker and Gandier, 1957),
dog (Marks et al., 1994), Common marmoset (Morrell et al., 1998), goat (Blash
et al., 2000), eland (Bartels et al., 2001) and rat (Nakatsukasa et al., 2001;
Hirabayash et al., 2002). Both fresh and frozen-thawed epididymal sperm have
been used to produce human offspring (Temple-Smith et al., 1985; Bonduelle et
al., 1996; 1998; Cayan et al., 2001).
86
Epididymal sperm cells have long been the common source of sperm for
mouse and rat IVF (Hogan et al., 1986). Bowen (1977) used sperm from the
ductus deferens in early cat IVF in which 79% of inseminated ova cleaved.
Epididymal cat sperm was first used in the mid-1980s (Niwa et al., 1985) and
again recently to produce ICSI embryos in the domestic cat (Pushett et al.,
2000; Boglioli et al., 2001). Rath and Niemann (1977) found higher fertilization
rates using frozen-thawed epididymal sperm compared with frozen thawed
ejaculated sperm from genetically identical boars (59.7 vs. 16%, respectively).
Blash et al., (2000) collected epididymal sperm from goats at necropsy for
cryopreservation. The samples were then thawed and used to fertilize ova in
vitro (37% fertilization rate). One live kid was produced by AI with the frozenthawed epididymal goat sperm.
Epididymal dog sperm has been used to fertilize dog ova in vitro at a rate
of 57% (4 of 7) (Williams et al., 2001). Epididymal dog sperm has also been
used to produce a live pup through AI with a frozen-thawed sample collected
after death (Marks et al., 1994).
Exotic animals have been born after AI with epididymal sperm, using
fresh (Common marmoset, Morrell et al., 1997) and frozen-thawed samples
(Common marmoset, Morrell et al., 1998; eland, Bartels et al., 2001).
IVF with epididymal sperm has been attempted with several exotic
species including the zebra (Meintjes et al., 1995), the African buffalo (Shaw et
al., 1995), the blesbok (Winger et al., 1997), the impala (Rush et al., 1997), the
87
African lion (Bartels et al., 2000) and the Red deer (Comizzili et al., 2001) and
fertilization was achieved in each case.
For epididymal sperm to be useful for the long-term production of
endangered species, the ability to cryopreserve the sperm for future use is
essential. Several recent studies have attempted to find the best cryoprotectant
and diluent for cryopreserving the epididymal sperm of exotic species including:
the blesbok (Winger et al., 1997), the waterbuck and the warthog (Watson et
al., 1997), the impala (Rush et al., 1997) and the Greater kudu (Schmid et al.,
1997; Watson et al., 1997). Others have evaluated the toxicity of glycerol on
epididymal sperm of the White rhinoceros (Williams et al., 1995). (See Table
2.1)
A “genome resource bank” for the gametes of endangered animals
implies a few or even one centralized location where these materials are stored.
However, most exotic animals would not be housed at this facility and
preserving any genetic material would involve the logistics of shipping a
sample. If an animal is at a highly developed zoo with a reproductive
physiology research facility, the animal’s genetic material could be collected
and frozen and then shipped in a frozen state to the genome resource bank.
Unfortunately, there are very few of these specialized facilities throughout the
world. Realistically, most of the collections would be at facilities without the
capabilities to collect and preserve the genetic material or in field conditions
without a facility within close proximity. This would make shipping a refrigerated
88
sample necessary, with subsequent cryopreservation at the site of the genome
resource bank.
Due to these collection circumstances, our objective was to investigate
not only if epididymal canine sperm could be harvested and frozen, but also if it
could be harvested and frozen after a period of refrigerated storage. This
period of storage was meant to simulate shipment on ice of whole testicles or
processed sperm in liquid form. Because almost any site in the world can be
reached by 2-day shipment, it was decided that a 2-day refrigerated storage
interval would simulate shipment.
Kikuchi et al. (1998) performed an experiment on epididymal boar sperm
that investigated cryopreservation of epididymal sperm after storage at 4ºC.
The epididymides of 22 boars where stored at 4ºC for 1, 2 and 3 days. The
epididymal sperm was then collected and cryopreserved. The sperm motility
decreased during refrigerated storage and after thawing, but the sperm was still
able to fertilize pig ova in vitro (67 to 71% fertilization).
Materials and Methods
Experimental Design
The testicles of 20 mixed-breed dogs were separated into two treatment
groups. The testicles of the first group were processed immediately upon
collection; sperm motility, percentage of membrane-intact sperm and sperm
concentration parameters were taken and then a portion of the sperm was
cryopreserved (Treatment A). The remaining sperm solution from the initial
processing was place under refrigeration (4ºC) for 2 days (Treatment B). The
89
second group of testicles were left whole and placed under refrigeration for 2
days (4ºC, Treatment C). After the 2-day period, the epididymides of the whole
testicles were processed and sperm motility, percentage of membrane-intact
sperm and sperm concentration were assessed and a portion of the sperm was
cryopreserved. Sperm motility and percentage of membrane-intact sperm
measurements were repeated on the extended sperm that had been stored as
an extended liquid (Treatment B) and then a portion was frozen. These three
sample types were then thawed and their motility and percentage of membraneintact sperm were compared (see Table 5.1).
The tissues used in this study were obtained from euthanized animals at
the East Baton Rouge Parish Animal Control Center. The testicles were
removed and placed in a plastic ziplock bag (the testicles were left in the scrotal
sac) and taken to the St. Gabriel Research Station. The travel time was ~30
minutes. The testicles were maintained at room temperature during transport.
Testicle Processing
Upon arrival at the laboratory, the testicles were washed and removed
from the scrotal sac. The tunica albuginea was cut away from each testicle,
with special care taken not to damage the epididymis where it is attached to the
tunica. The blood vessel leading to the testicle and the vas deferens were
trimmed away along the natural curve of the testicle using curved scissors. The
circumference (in centimeters) of each testicle was measured. A piece of
rubber tubing was used to encircle the testicle at its largest point. The tubing
was then used to obtain the measurement. The length of the testicles were
90
Table 5.1. Experimental design for storage of canine epididymal sperm in cooled and frozen conditions
Treatment
group
(n)
Processed Testicle
Concentration
(million/ml)
Sperm motility (%)
+
Membrane-intact
sperm (%)
Pre-freeze
Post-thaw
Pre-freeze
Post-thaw
Control
A
20
+
+
+
+
48 hours at 4ºC
B
20
+
+
+
+
+
+
+
+
Whole Testicle
48 hours at 4ºC
+
C
20
91
measured in a similar manner, with the tubing laid along the side of the testicle.
The end of the tubing was abutted against the bottom of the epididymal mass
and the tubing was extended to the bottom point of the testicle. The weight of
each individual testicle was measured in grams on an electronic balance and
recorded. One testicle from each set was randomly assigned to Treatment A
and was processed immediately. The second testicle from each set was
allotted to Treatment C and was kept whole and placed under refrigeration at
4°C for 2 days.
Testicle Storage
The testicles were placed in a rectangular stainless steel surgical
instrument pan with a non air-tight stainless steel lid. The pan was lined with
two layers of sterile gauze (Johnson & Johnson, Arlington, TX) and the testicles
were placed on top and then covered with two additional layers of sterile gauze.
Sterile saline (Sigma, St. Louis, MO) was poured onto the gauze until it was
completely wet to keep the tissue from drying out and the pan was placed in a
standard refrigerator (4°C).
Sperm Evaluation
Processing of testicles in Treatment A began by removing the cauda
epididymidis with scissors, cutting at the junction of the larger tubule of the
corpus and the smaller tubule of the cauda. The excised cauda epididymidis
was then placed into a 60-mm plastic Petri dish (Becton Dickinson, Franklin
Lakes, NJ) containing 3 ml of Tyrode Lactate HEPES medium (TLH) (Bio-
92
Whittaker, Walkersville, MD), which had been allowed to reach room
temperature. The cauda epididymidis was finely minced with a scalpel (BectonDickinson, Franklin Lakes, NJ) while being held with forceps to allow the sperm
cells to escape the tubule. The dish of minced tissue was placed on a 39ºC
slide warmer where it was left for 1 hour to allow the solution to warm and the
sperm to swim from the epididymal tissue. At the end of 1 hour the fluid was
aspirated from the dish and placed into a labeled 15-ml conical tube (Corning
Inc., Corning, NY).
A 10 ml drop of warmed sperm solution was placed on a warmed glass
slide (Gold Seal Products, Portsmouth, NH) and a glass cover slip (Baxter
Health Care Corp., McGaw Park, IL) was placed on top of the sample. The
sample was observed under 200X magnification using a compound
microscope. Progressive motility was estimated by visual observation.
A 5 ml portion of the extended sperm solution was placed on a glass
slide. To this drop, a 5 ml drop of Eosin-B/Fast Green dye (Sigma, St. Louis,
MO) was added and the two were thoroughly mixed. A second glass slide was
inverted and used to smear the sperm and dye solution along the entire length
of both slides. The smears were then quickly dried with a standard hair dryer
on the lowest setting. This was done to prevent the sperm from dying slowly in
air, which could cause damage to previously membrane-intact sperm causing
more of them to take up the indicator dye, making the reading of dead sperm on
the slide inaccurate.
93
The Eosin-B/Fast Green slides were read under 400X magnification on
the same microscope used to analyze sperm motility. The Fast Green dye
created a dark background color on which the light color of the sperm cells
would stand out. The Eosin-B acted as an indicator dye for membrane integrity
(Aalseth and Saacke, 1986). If the cell had intact membranes it was able to
exclude the Eosin-B dye and appeared white against the green background,
these cells were counted as “membrane-intact”. If the sperm cell membrane
was ruptured or torn the cell was not able to exclude the indicator dye and the
cells appeared pink. These cells were counted as nonliving cells. A total of 100
sperm cells from each of the two prepared slides were counted using a counter
to keep track of membrane-intact and dead cells. The percentage of
membrane-intact sperm was determined by averaging the number of
membrane-intact sperm cells counted.
The concentration of each sample was determined using a
hemacytometer (Cambridge Instruments, Buffalo, NY). The original sperm
solution was diluted 1:100 with water to immobilize the sperm cells. All of the
cells in the 5 of the small inner squares of the hemacytometer were counted.
That number was equivalent to the number of millions of sperm cells there were
in each ml of the original solution. The concentration of a 500 µl sample was
adjusted to allow a final concentration of 80 x 106 sperm cells/ml (the addition of
500 µl of freezing medium would bring the volume up to 1 ml). This was
accomplished by determining how many microliters of the original sample would
contain 80 million sperm cells. That amount of the solution was placed into a
94
microcentrifuge tube (DOT Scientific, Burton, MI) and centrifuged at 1,500 g for
10 minutes. The supernatant of TLH medium was removed and the pellet was
resuspended in 500 µl of Test Yolk Buffer (TYB) diluent (Irvine Scientific, Irvine,
CA). A volume of 500 µl of freezing medium was added later to bring the final
volume to 1 ml.
Cooling, Freezing and Thawing of Sperm
The remaining TLH sperm solution for each sample was placed into a
600-ml beaker of room temperature water and placed into a standard
refrigerator where they remained for 2 days (Treatment B). The extended
samples were also placed in a 600-ml beaker of water for slow cooling. The
microcentrifuge tubes were placed in floating foam holder to keep the tops
above the level of the water. A digital thermometer probe was placed in the
water to monitor the water temperature. The samples were allowed to cool until
the water reached 4ºC (3 to 4 hours).
When the water reached 4ºC the samples were removed from the foam
holder and placed on ice. The freezing medium used was from Irvine Scientific
(Irvine, CA) and consisted of 12% glycerol in TYB. The freezing medium was
diluted 1:1 with TYB to obtain a 6% glycerol solution, which was also kept on
ice. The 6% glycerol was added to the extended sperm samples in a stepwise
fashion of increasing increments to minimize the osmotic damages of glycerol.
The increments began at 5 µl, then 10, 15, 20, 30, 40, 50 µl and continued until
500 µl had been added. Each increment was added with ~1 minute
95
equilibration time in between increments. The final concentration of the
samples was 80 million cells/ml in 3% glycerol in TYB.
The sperm and glycerol solution was then loaded into labeled 0.25 ml
cryopreservation straws (IMV International, L’Aigle, France). An initial column
of ~50 µl of TYB was drawn into the straw and this was followed by a small
bubble of air placed there to separate the TYB from the column of sperm. The
sperm column contained 120 to 150 µl and was followed by another air bubble
and another 50 µl column of TYB. The ends of the straws were sealed by
tapping them into polyvinyl alcohol (PVA) powder (Sigma, St. Louis, MO, USA)
and dipping the end into TYB to wet the powder and cause it to plug the straw.
The straws were then placed onto a styrofoam raft floating in liquid
nitrogen. The straws were allowed to freeze in the liquid nitrogen vapor for 10
minutes and then plunged into the liquid nitrogen. They were then transferred
into a liquid nitrogen storage tank.
After 48 hours of refrigeration, the extended sperm in Treatment B were
removed from the refrigerator and sperm motility and percentage of membraneintact sperm were taken in the same manner as at the initial time period. The
sperm was then centrifuged and extended in cooled TYB. The glycerol solution
was added and the sample was frozen in the exact same manner as Treatment
A.
The testicles in Treatment C were processed in the same manner as
those in Treatment A. Sperm motility, percentage of membrane-intact sperm
96
and concentration were determined by the same methods. The sperm were
extended and cooled and frozen in the same manner as Treatment A.
All frozen samples, from each treatment were thawed at 50ºC for 10
seconds. Sperm motility was immediately evaluated and an Eosin-B/Fast
Green slide was prepared. Sperm motility was judged by at least two
examiners and a separate person thawed the straws and recorded the code on
the straw in a thaw log without the examiners knowing the code. The thaw log
was used to be sure that the readings were not influenced by the examiner
knowing which treatment he or she was evaluating. The Eosin-B/Fast Green
slides were numbered and read and the percentage of membrane-intact
recorded before the thawing log book was used to determine the identity of the
sample.
Statistical Analysis
Testicular parameters of weight, length and circumference were
analyzed using a series of Paired t-tests. Sperm parameters of motility,
percentage of membrane-intact sperm and sperm concentration were also
analyzed with a series of Paired t-tests. A P value of <0.05 was considered a
significant difference in this study.
Results
The parameters of testicular weight (16.8±7.7 g compared with 16.4±7.4
g), length (4.8±0.8 cm and 4.8±0.9 cm) and circumference (7.8±1.1 cm and
7.8±1.2 cm) were not statistically different between Treatment A and Treatment
C. The mean sperm concentration of Treatment A (395±48 million/ml) was not
97
significantly different when compared with the mean sperm concentration of the
paired testicles in Treatment C (396±39 million/ml).
The mean sperm motility comparisons are presented in Table 5.2. The
decline in sperm motility from the initial motility of (85.8±1.3%) after 2 days of
refrigeration was statistically different (P<0.0001) both for the sperm
refrigerated in medium (Treatment B, 72.3±2.5%) and for the sperm refrigerated
within the whole testicle (Treatment C, 68.8±2.4%). The sperm motility of
Treatment B was not significantly different when compared with Treatment C.
After the samples were frozen and thawed, there was a significant
decrease in sperm motility for each of the treatments. Treatments A, B and C
post-thaw (48.0±3.7%, 0.4±0.3% and 35.0±3.6%, respectively) where
significantly lower than their pre-freeze values (85.8±1.3%, 72.3±2.5% and
68.8±2.4%) (P<0.0001 for each). The percentage of sperm motility for
Treatments B (0.4±0.3%) and C (35.0±3.6%) post-thaw were significantly lower
than the post-thaw sperm motility of Treatment A (48.0±3.7%) (P<0.0001 for
Treatment B and P<0.016 for Treatment C). It should be noted that Treatment
B had a post-thaw motility of nearly zero (0.4±0.3%), while the post-thaw
motility of Treatment C was significantly higher when compared with Treatment
B (35.0±3.6%, P<0.0001).
The percentage of membrane-intact sperm results are shown in Figure
5.3. The pattern of differences in these treatment groups had similarities to
those of sperm motility. The refrigeration of the testicles and liquid sperm
caused the pre-freeze percentage of membrane-intact sperm in Treatments B
98
Table 5.2. Effects of storage of canine epididymal sperm in cooled and frozen conditions on sperm motility
Concentration
(million/ml)
Treatment
group
(n)
Processed Testicle
395±48
Sperm motility (%)
Pre-freeze
Post-thaw
Control
A
20
85.8±1.3a,c
48.0±3.7d,e
48 hours at 4ºC
B
20
72.3±2.5b,c
0.40±0.3d,g
68.8±2.4b,c
35.0±3.6d,f
Whole Testicle
48 hours at 4ºC
396±39
C
20
a,b
Means within column with different superscripts are significantly different (P<0.05).
Means within rows across columns with different superscripts are significantly different (P<0.05).
e,f,g
Means within column with different superscripts are significantly different (P<0.05).
c,d
99
Table 5.3. Effects storage of canine epididymal sperm in cooled and frozen conditions on percentage of membraneintact sperm
Concentration
(million/ml)
Treatment
group
(n)
Processed Testicle
395±48
Membrane-intact sperm (%)
Pre-freeze
Post-thaw
Control
A
20
88.8±1.1a,c
71.5±1.8d,e
48 hours at 4ºC
B
20
77.9±2.6b,c
52.5±4.7d,f
74.8±3.2b,c
61.9±2.6d,f
Whole Testicle
48 hours at 4ºC
396±39
C
20
a,b
Means within column with different superscripts are significantly different (P<0.05).
Means within rows across column with different superscripts are significantly different (P<0.05).
e,f,
Means within column with different superscripts are significantly different (P<0.05).
c,d
100
(77.9±2.6%) and C (74.8±3.2%) to decline significantly when compared with the
pre-freeze percentage of membrane-intact sperm for Treatment A (88.8±1.1%)
(P<0.0005 and P<0.0002, respectively). While the pre-freeze percentage of
membrane-intact sperm in Treatments B and C were not significantly different
from each other. The post-thaw percentage of membrane-intact sperm in
Treatment A (71.5±1.8%) was significantly lower (P<0.0001) than the pre-freeze
value of the same group (88.8±1.1%). The post-thaw membrane-intact sperm
for Treatments B and C (52.5±4.7% and 61.9±2.6%) were significantly lower
than their pre-freeze values (77.9±2.6% and 74.8±3.2%, P<0.0001 for both,
respectively). The post-thaw percentage of membrane-intact sperm in
Treatments B (52.5±4.7%) and C (61.9±2.6%) were also significantly lower than
the post-thaw percentage of membrane-intact sperm in Treatment A
(71.5±1.8%) (P<0.000l and P<0.0038, respectively). Unlike the sperm motility,
there was no significant difference in the post-thaw percentage of membraneintact sperm between Treatments B and C.
Discussion
Results from this study suggest that immediate harvesting and
cryopreservation of canine epididymal sperm provides the optimal sperm
survival for sperm cell banking. However, if immediate collection and freezing
is not possible, whole testicles can be refrigerated for up to 2 days and still yield
viable sperm cells that can be frozen and thawed for future use.
The marked drop in motility after freezing and thawing in each treatment
is a common occurrence observed in the sperm of both small and large
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animals. An initial motility of ~86% declined to 48% after freezing and thawing
in the present study. The motility of epididymal cat sperm was initially 75% and
then declined to 53% after freezing and thawing (Lengwinat and Blottner, 1994).
In one study, the initial motility of kudu epididymal sperm was reported as ~71%
pre-freeze and 0 to 65% post-thaw (Schmid et al., 1997). In another study, prefreeze and post-thaw motility for Greater kudu were 61% and 15 to 30%,
waterbuck were 84% and 60 to 72% and the warthog were 78% and 0 to 35%
(Watson et al., 1997). In one study, the Cantabric Brown bear was found to
have an initial epididymal sperm motility of 45% and post-thaw motility of 25%
(Anel et al., 1999). The White rhinoceros was reported to have a pre-freeze
motility of 70% that declined to 43% after freezing and thawing in one study
(Lubbe et al., 1999) and 50 to 60% declining to 40 to 50% in a subsequent
study (Lubbe et al., 2000). Lubbe et al. (2000) also reported the pre-freeze and
post-thaw motility of horse and zebra epididymal sperm as 60 to 65% declining
to 40% and 40 to 45% declining to 15 to 25%, respectively. This pattern of the
decline in motility of epididymal sperm after freezing and thawing has also been
noted in the Black wildebeest, blesbok, Roan antelope, gemsbok, nyala, eland
and African buffalo (Kilian et al., 2000).
A report by Rath and Niemann (1997) on the pig reported that the prefreeze motility and post-thaw motility of epididymal boar sperm were not
significantly different. They reported the pre-freeze motility of epididymal boar
sperm as 76% and the post-thaw motility as 72%. These results suggest that
with more research into the best cryoprotectant, freezing protocol and thawing
102
protocol, the post-thaw motility of epididymal sperm of other species could be
improved.
An interesting observation in the present study is that the number of
membrane-intact sperm cells is consistently higher than the sperm motility.
Further testing is needed to determine if these membrane-intact but immotile
sperm could be “revived”, possibly with caffeine, pentoxifylline, or some other
stimulatory agent. Koutsarova et al. (1997) used pentoxifylline on fresh and
frozen-thawed ejaculated canine sperm and found that it significantly increased
the motility of fresh ejaculated canine sperm and was “most beneficial” to
thawed, ejaculated canine sperm when it was added during the thawing
process. Milkowski et al. (1976) used the addition of caffeine to stimulate the
motility of epididymal bull sperm during a cell respiration study. Terriou et al.
(2000) reported that pentoxifylline caused immotile human frozen-thawed
epididymal sperm to resume motility. They were able to use the re-animated
sperm cells to perform ICSI with no significant difference in fertilization or
pregnancy rates when compared with that of fresh epididymal sperm.
In addition, the development of ICSI techniques in the dog (Farstad et
al., 1998), would allow injection of ova with immotile sperm cells. Earlier, Goto
et al. (1990) used immotile bull sperm to fertilize cow ova using ICSI.
Results from the present study also show that epididymal dog sperm that
is stored in the refrigerator in medium (Tyrode Lactate HEPES) was not motile
after cryopreservation in 3% glycerol and thawing at 50°C for 10 seconds. The
percentage of membrane-intact sperm, however, was not lower in this liquid
103
storage group when compared with the sperm stored in the epididymis. This
may be another example where a stimulant may increase the motility of the
sperm, or a different diluent might be more successful in preserving the postthaw motility of this sperm.
Another factor to consider is that most places in the continental United
States can be reached by overnight shipments. Therefore, evaluating postthaw sperm motility and percentage of membrane-intact sperm after 1 day of
refrigeration and subsequent cryopreservation might yield better results.
Graff et al. (2000) froze whole dog testicles in an attempt to preserve
epididymal dog sperm. The testicles were put on ice for 12 hours and then
frozen at -20ºC for 60 hours. The testicles were then thawed and the
epididymal sperm was aliquotted into several different media. Progressive
motility was assessed at 5 minutes at 24ºC, 2 hours at 39ºC and 24 hours at
39ºC. The control sperm from fresh epididymides was 74% motile after 5
minutes at 24ºC, 62% motile after 2 hours at 39ºC and <1% motile after 24
hours at 39ºC. The most successful aliquots of the frozen-thawed epididymal
dog sperm at the 5 minutes at 24ºC sampling were diluted in Human Tubal
Fluid (HTF) medium and P-1 medium (both had 35% motility).
The post-thaw motility was significantly lower than the pre-freeze sperm
motility and was also lower than the post-thaw motility of fresh epididymal dog
sperm noted in the present study (~48%). However, it was the same as the
post-thaw sperm motility of the epididymal dog sperm in the present study that
was left in the whole testicle under refrigeration for 2 days before
104
cryopreservation (35%). This method of cryopreservation of epididymal dog
sperm would be useful if shipment of the specimen had to be delayed, but it
does not allow for the sperm to be aliquotted into several straws before
cryopreservation. Therefore, the entire population of the thawed epididymal
sperm would likely have to be used at one time for an ART procedure.
Various studies have proven the fertilizing capacity of frozen-thawed
epididymal dog sperm. Epididymal dog sperm has successfully fertilized dog
ova in vitro at a rate of 57% (4 of 7) (Williams et al., 2001). Epididymal dog
sperm collected after death and frozen has also been used to produce a live
pup after AI (Marks et al., 1994). The post-thaw motility in the latter study was
only ~ 5% compared with 48% motility in our study. In addition, AI with frozenthawed ejaculated wolf semen has been successful with the birth of 6 live pups
(Seager et al., 1974).
Lengwinat and Blottner (1994) performed IVF with fresh and
cryopreserved epididymal cat sperm. As with the frozen-thawed epididymal
dog sperm, the post-thaw sperm motility was significantly lower than the sperm
motility of the fresh epididymal samples. A swim-up technique was used to
obtain a post-thaw sample with sperm motility as high as the pre-thaw value
(~74%). Fertilization rates were higher when fresh epididymal cat sperm was
used for IVF (~41%) than when frozen-thawed epididymal cat sperm was used
(~25%), but the fact that it was capable of fertilization makes the
cryopreservation of cat epididymal sperm a viable option.
105
Pushett et al. (2000) evaluated the ability of cryopreserved epididymal
cat sperm to fertilize ova following ICSI of in vitro matured ova. The ova
subjected to ICSI with frozen-thawed epididymal cat sperm had a higher
fertilization rate (57%) than ova undergoing conventional IVF with frozenthawed epididymal cat sperm (40%). Bogliolo et al. (2001) also used frozenthawed epididymal cat sperm to perform ICSI and reported a cleavage rate of
82%, with a 35% development to morulae.
Live mice have been produced from frozen-thawed epididymal mouse
sperm. The mouse ova were fertilized in vitro and 167 resulting embryos were
transferred into 15 recipients, 12 became pregnant and 57 pups were born
(Songsasen et al., 1997).
Rath and Niemann (1997) performed IVF with fresh and frozen-thawed
ejaculated boar semen and fresh and frozen-thawed epididymal boar sperm
from genetically identical boars. As previously mentioned, pre-freeze motility
and post-thaw motility (76% and 72%, respectively) for the epididymal sperm
were not different, while the pre-freeze motility of ejaculated sperm was ~77%
and decreased to 40% after freezing and thawing. Also, the frozen-thawed
epididymal sperm had the highest embryo development rates for the four types
of sperm preparations (fresh ejaculated, frozen-thawed ejaculated, fresh
epididymal or frozen-thawed epididymal).
Kikuchi et al. (1998) performed an experiment that had similarities to that
of the present study. Boar epididymides were refrigerated for 1, 2 and 3 days,
freezing a sample on each day. Later, these samples were thawed and IVF
106
was performed with each. The patterns of motility in the epididymal boar sperm
were similar to those of the dog in the present study. The initial motility of the
boar epididymal sperm was 79% and decreased to 44% after cryopreservation,
while the dog was 86% and 48%, respectively. The pre-freeze and post-thaw
motility for the boar sperm after 48 hours of storage at 4ºC was 58% and 33%
and for the dog they were 69% and 35% in the present study. The results of
IVF with frozen-thawed epididymal boar sperm frozen immediately or
refrigerated for 1, 2, or 3 days before freezing were not different with the
percentages of sperm-exposed ova with male and female pronuclei of 71, 74,
68 and 71%, respectively.
Blash et al. (2000) reported that frozen-thawed epididymal goat sperm
collected at the time of death could both fertilize ova in vitro and produce a live
kid when used for AI. Fertilization and blastocyst development rates using
frozen-thawed epididymal sperm (40% and 6%) and frozen-thawed ejaculated
(37% and 4%) were not significantly different. Cayan et al. (2001) have also
reported that fertilization (58.4% and 62%) and pregnancy rates (32% and 37%)
were not significantly different for ICSI with either fresh or frozen-thawed
epididymal sperm from the same couples used for ICSI over successive cycles.
Jewgenow et al. (1997) used cryopreserved epididymal sperm from two
lions and one puma for IVF. A fertilization rate of 77% resulted from IVF of lion
ova with frozen-thawed epididymal lion sperm with a 53% cleavage rate. IVF of
puma ova with frozen-thaw epididymal puma sperm resulted in 75% of the ova
fertilizing and 25% cleaving.
107
Meintjes et al. (1997) used fresh epididymal zebra sperm to fertilize
zebra ova, which had undergone zona drilling. They achieved a 38% cleavage
rate and a 16% morula/blastocyst development rate. Shaw et al. (1995) used
refrigerated epididymal sperm from African buffalo to achieve a 50% fertilization
rate and 33% blastocyst rate from IVF with African buffalo ova.
Similar to dog epididymal sperm, Bartels et al. (2000) found that
epididymal lion sperm was 85% motile upon collection and 55 to 65% motile
after freezing and thawing. When this frozen-thawed sperm was used for IVF a
fertilization rate of 12 to 13% was achieved.
Bartels et al. (2001) collected epididymal sperm from an eland killed on a
hunt and cryopreserved the sperm. The post-thaw motility of the epididymal
eland sperm was similar to the dog at 55%. Four eland heifers were
inseminated with the frozen-thawed epididymal eland sperm and one of these
became pregnant and delivered a live calf.
Morrell et al. (1997) used fresh epididymal sperm from a Common
marmoset for AI with 6 of 18 females conceiving. The same group later
performed AI with frozen-thawed epididymal sperm (Morrell et al., 1998) with 1
of 6 females conceiving and giving birth to triplets.
These studies of cryopreserved epididymal sperm from various species
leading to fertilization of ova in vitro and a live birth following AI is evidence that
the retrieval and cryopreservation of epididymal sperm from exotic species is a
worthy effort that could lead to greater genetic variability for captive populations
of exotic animals.
108
CHAPTER VI
SUMMARY AND CONCLUSIONS
The main objectives of these studies were to determine how long
refrigeration could preserve the sperm motility and sperm membrane integrity of
epididymal mouse and dog sperm and to show that epididymal dog sperm could
be cryopreserved after storage at 4ºC for 2 days and yield membrane-intact,
motile sperm cells upon thawing. The results of these experiments are
important not only for future research in exotic canids, but also for transferring
technology to other exotic species for germ plasm banking and developing
strategic plans for the preservation of species and genetic variability in captive
populations.
Research into the exotic canids has been limited by both the difficulties
of canine reproduction in general and in some cases by the politics of
endangered species captive breeding programs. For example, the U.S. Fish
and Wildlife recovery plan for the Mexican gray wolf (Canis lupus baileyi)
specifies semen banking as the only approved assisted reproduction technique
(Asa, 2001). Wolf reproduction and subsequent reintroduction into parts of its
former range have also met with resistance from local ranchers and many
reintroduced wolves have been killed (Naess and Mysterud, 1987).
Reproductive research into one of the most endangered wild canids, the
African wild dog (Lycaon pictus) is still in the stages of fecal steroid analysis
and their reproductive mechanisms are poorly understood (Monfort et al.,
1998). The current status of reproductive research in exotic canids makes the
109
banking of their gametes and genetic information very important to the future of
ART in these species. Having these specimens stored will allow researchers to
draw from a more varied genetic pool when ART in canids becomes better
understood and more successful.
Research in reproductive physiology in general and in exotic animals in
particular continues to develop and incredible breakthroughs are being made.
The utilization of a genome resource bank for endangered species insures that
the necessary materials will be available as technology progresses. Storage of
epididymal sperm could be used for AI, IVF, or ICSI. Cryopreserved oocytes or
ovarian tissue (Harp et al., 1994) might one day be utilized to produce embryos
through IVF or through the use of that oocyte as a vessel for cloning of another
animal. Frozen ovarian tissue might also one day be thawed and either
cultured to mature the oocytes within it, or even grafted into a mouse (Parrott,
1960) for oocyte harvest, as has been reported with cryopreserved elephant
ovarian tissue (Gunasena et al., 1998).
Sexing of sperm is under development for domestic animals (Cran et al.,
1993; Johnson, 2000) and would be beneficial in exotic animal reproduction to
avoid excess “bachelor” males. Frozen embryos might also be sexed for the
same reason (Kunieda et al., 1991; Bondioli et al., 1989).
Cloning an animal by transferring the nucleus of a somatic cell has been
successful in the sheep (Wilmut et al., 1997), Rhesus monkey (Wolf et al.,
1999), the cow (Wells et al., 1999) and the goat (Baguisi et al., 1999), with
research continuing on other animals. Frozen somatic cells from an exotic
110
animal might one day be used to clone that animal so that it might have a
second chance to make a genetic contribution to its species.
The potential for development of any or all of these techniques for use
with exotic animals remains to be seen, but any ART will require the availability
of sperm and oocytes and possibly somatic cells. A bank of these gametes and
genetic information should be a high priority in the conservation of endangered
species.
111
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APPENDIX: SPERM STORAGE DATA
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Table 1. Results for epididymal mouse sperm stored at 22°C (Treatment A) and 4°C (Treatment B)
(mean values ± standard error of the mean)
Time of
sampling
Treatment
Testicle weight (g)
Sperm motility (%)
Membrane-intact
sperm (%)
0 hours
(Control)
A
0.137±0.007
48.3±2.8
54.5±3.0
B
0.143±0.009
47.8±2.1
53.2±3.2
A
0.136±0.005
22.2±3.8
35.2±4.1
B
0.136±0.003
43.3±3.2
60.0±3.8
A
0.127±0.005
0±0
0.5±0.2
B
0.129±0.007
40.6±3.3
47.3±3.7
B
0.136±0.005
30.0±3.7
44.1±4.4
24 hours
48 hours
72 hours
96 hours*
B
0.127±0.004
16.1±3.5
24.6±4.6
*Due to tissue degeneration, Treatment A could not be continued after 48 hours of sampling. The testicles of
the remaining group of mice were separated into a Treatment B, 72-hour group and a Treatment B, 96-hour
group.
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Table 2. Effects of storage at 22°C (Treatment A) and 4°C (Treatment B) on epididymal dog sperm
(mean values ± standard error of the mean)
Time
Treatment
(n)
Sperm motility (%)
Membrane-intact
sperm (%)
Concentration
(million/ml)
0 hours
(Control)
A
22
86.1±1.8
90.6±1.5
394±32.5
B
22
86.6±1.8
90.3±1.2
390±32.5
A
22
55.7±2.4
67.5±2.5
421±42.2
B
22
79.3±1.7
86.5±1.5
315±37.9
A
22
1.0±0.7
7.4±2.7
270±47.5
B
22
69.5±2.3
80.8±1.9
227±26.8
24 hours
48 hours
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Table 3. Protocols for Eosin-B/Fast Green Stain Preparation
Prepare Lakes A Extender:
Sodium glutamate: 1.92 g
Potassium citrate monohydrate: 0.128 g
Sodium acetate: 0.5132 g
Magnesium chloride: 0.0670 g
Fructose: 1.0 g
q.s. to 100 ml with deionized water and pH to 7.2-7.4
Prepare EosinB/Fast Green:
2.5 ml Lakes Extender
0.5 g Fast Green
0.2 g Eosin B
Filter sterilize, store in amber tubes at refrigeration for up to 3 months.
136
VITA
Karla Sioux Stilley was born to J. Warren and Nancy Stilley in Hammond,
Louisiana on September 20, 1972. She was raised in a small town in south
Louisiana and graduated from the Louisiana School for Math, Science and the
Arts in May of 1990.
She then attended Centenary College of Louisiana and received her
bachelor of science degree in applied science in May of 1994. She went on to
work as a zoo keeper at the Greater Baton Rouge Zoo for 3 years. She worked
as an embryologist at a fertility clinic in Tulsa, Oklahoma and is currently
working as a research embryologist in Englewood, Colorado. She is a
candidate for the degree of Master of Science in reproductive physiology in the
Department of Animal Science at Louisiana State University, Baton Rouge,
Louisiana, under the supervision of Dr. Robert A. Godke, Boyd Professor.
137