EFFECTS OF SEMEN EXTENDER COMPOSITION AND COOLING METHODS ON
CANINE SPERM FUNCTION AND CRYO-SURVIVAL
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
bJ'
m L E N LOUISE BATEMAN
In partial fulfilment of requirements
for the degree of
Master of Science
April, 2001
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ABSTRACT
EFFECTS OF SEMEN EXTENDER COMPOSITION AND COOLING METHODS ON
CANINE SPERM FUNCTION AND CRYO-SURVIVAL
Helen Louise Bateman
University of Guelph, 2001
Superviosr:
Dr. K.L. Goodrowe
This study was undertaken to investigate energy source, glycerol addition, and pre-fieeze
cooling rates in canine sperm cryopreservation. Sperm were held at O°C, room temperature (RT),
and 37"C, in Trïs-egg yolk ( ' Y ) extender with 1 of 4 different sugars. No differences in sperm
motility between different sugar extenders was found- Room temperature best maintained sperm
motility, Motility of sperm held in modified Tyrode's solution (TALP) or extender, containing
glucose, £i-uctose, sucrose or pyruvatdlactate, was reduced in TALP containing sucrose and best
maintaïned in media containing glucose.
Fresh, cooled, and pst-thaw sperm were examined: 1) after fast or sIow cooling rates (ta 5
and 0°C);and 2) afier diEerent glycerolated extender addition methods. Cooling to 0°C more so
than to SOC, resulted in decreased intact acrosomes and morphologically normal sperm.
Regardless of pre-fkeeze treatment, decreased sperm motility, intact acrosomes, h~mologouszona
peneiration, morphology and increased absent acrosomes was observed pst-thaw.
ACKNOWLEDGEMENTS
The Moms Animal Foundation, Naturai and Sciences and Engineering Research Council
of Canada, and the Toronto Zoo Foundation supported this work.
1 would like to express my deepest gratitude to my supervisor, Dr. K. Goodrowe, not
only for ber teachings, guidance. and support, but also for the amazïng opportunities she
afforded me that went beyond the realm of my Master's degree and which taught me what
research in conservation and zoos is al1 about. 1 would also like to thank the members of my
advisory cornmittee, Dr. C. Gartley, Dr. M. Buhr, and Dr. A. King for al1 their guidance and
advice. AdditionalIy, I would like to thank Dr. C. Ganley for giving me her valuable time to
assist with dog semen collections, and also for giving me the perspective of clinicai, and
domestic dog applications of my research.
1 would like to thank Dr. C. Linde-Forsberg and the staff at the Obstetrics and
Gynecology department at the Swedish University of Agricultural Science (Uppsala
Sweden) whose assistance and guidance made conducting a small portion of my research in
their facilities such an educational and rewarding experience. 1 also would like to send my
appreciation to Dr. A. King and the European Union Exchange for making research
conducted in Sweden possible. 1 thank Dr. W. Farstad for not only sharing her expertise
while on this exchange but also for sharing her home in Oslo.
1 cannot begin to measure the ways in which the other mernbers of rny laboratoy,
Gabriella Mastromonaco, David Ryckrnan, Sue Walker, and Maria Franke, have supported
and heiped me with my research, but 1think the rnost important to me was their fnendship.
Likewise the support of my fellow ,pduate students and fnends helps me stay grounded. 1
thank the memben of the Animal Health unit at the Toronto Zoo, includinp those in our
Iaboratory, whose support, education, fun, and many cakes wilt remain with me for a long
time.
Finally. 1 wouid like to thank the staff at the local veterinary clinics: Markham, Progress,
and Whites Road vetennary clinics; the dogs: Teddy. Guinness, Hank, Taz, Beau, with their
owners Susan Baird. Cathy Park, Laura Colton, Carol Cunningham, and Neil Porter, and the
univenity housed dogs Moe. Joey,Brewster, and Kurt, whose participation, enthusiasm. and
interest made this research possible.
Last, but not ieast, 1 would like to thank my fvnily whose love, unwavenng support and
encouragement is what has got me this far.
DECLARATION OF \;VOIX PERFORMED
I declare that with the exception of the items indicated below, al1 work reported in this
thesis was performed by me,
Dog semen collection was done by myself, Dave Ryckman, Toronto Zoo, or Dr.
Gartley, Ontario Verterinary ColIege, University of Guelph. Canine ovaries collected
from the Toronto area uith the assistance of Gabreilla Mastromonaco and Dave
Ryckman, Toronto Zoo. Gabreilla Mastromonaco also assisted in sficing canine ovaries,
preparation of oocytes, and media preparation for the zona penetration assay. WhiIe at
the Swedish University of Agiculturai Sciences (SUAS) ovaries were collected from
SUAS veterinary ciinic, cleaned and stored frozen by Dr. StMm Holst, Department of
Obstetrics and Gynecology, SUAS.
LIST OF TABLES
Table 1. ................................................................................................................ -51
Canine spem charactenstics with or without
Equex addition to semen extender
Table II. ..............................................................................................................5 1
Morphologic canine spem characteristics
with or without Equex addition to sernen extender
Table III...............................................................................................................
Spem motility index of fiesh and cooled canine sperm
-52
Table N...............................................................................................................
Spem motility index of pst-thaw canine sperm
.57
Table V.. ..............................................................................................................
Characteristics of fresh, cooled, and pst-thaw canine spem
-62
Table VI. ..............................................................................................................
Morphologic characteristics fresh, cooled, and pst-thaw canine sperm
-62
.
.....................................................................................
Table VI1 ................... .
Fresh, cooied and pst-thaw sperm motility index for individual dogs
63
Table VIII ............................................................................................................
Percentage of intact acrosomes for fresh, cooled and post-thaw sperm
for individual dogs
.63
Table IX .............................................................................................................
Percentage of morphologically normal sperm for frcsh, cooled and
pst-thaw for individual dogs
..64
LIST OF FIGURES
Figure I .................................................................................................................. 7
Schematic of cooling and rewarming membrane alterations
Figure 2.. ...............................................................................................................
Schematic of temperame and ce11 water volume changes
during cryopreservation
-9
Figure 3................................................................................................................ - 1I
Schernatic of putative changes in sperm and extender during
cryopreservation
Figure 4.. ..............................................................................................................
Freezing rate of canine semen extender onto fiozen carbon dioxide
-35
Figure 5 ...............................................................................................................
Design for expenment 3
40
Figure 6................................................................................................................. 42
Design for expenment 4
Figure 7.................................................................................................................
Design for expenment 5
45
Figure 8.................................................................................................................
Sperm motility index of canine spem diluted in 4 different sugar
extenders and heId at RT, O, and 37°C.
45
Figure 9...............................................................................................................
Spenn motility index of canine sperm diluted in different
extenders and TALPs and held at RT
-.49
........................................................... 53
........
Figure 10..................... .
.
.
.
.
.
.
Mean fast and slow cooling rates
Figure 11.............................................................................................................
Acrosomal status of fresh, cooled (fast and slow to 5 or O°C)
canine sperm
3 4
Figure 1,7...............................................................................................................
Morphological charactenstics of fiesh, cooled (fast and slow to 5 or O°C),
and post-thaw canine s p e m
55
Figure 13..............................................................................................................
Zona pellucida penetration of fiesh, cooled (fast and slow to 5 or O°C),
and pst-thaw canine sperm
-56
Figure 14..............................................................................................................
Zona pellucida penetration of fiesh, combined cooled and cornbined
pst-thaw canine sperm
-58
Figure 15...............................................................................................................
Acrosomal status of pst-thaw canine sperm
59
Figure 16...............................................................................................................
Zona pellucida penetration of fresh, combined cooled and glycerol addition
treatments, and cornbined post thaw canine sperrn
61
LIST OF ABBREWATIONS
AI
ANOVA
BSA
Ca 2+
CCM
CTC
Equex
EY
h
HEPES
IVF
LN2
min
NaCI
PBS
PB 1
PI
PIPES
PSAEITC
PVP
RT
sec
SEM
SM1
sp TALP
TALP
Tes
Tris
v/v
ZBA
ZP
ZPA
Arti ficial Insemination
Analysis of Variance
Bovine S e m Albumin
calcium ion
Canine Capacitation Medium
Chlortetracycline
STM Equex paste
Egg Yoik
hou
N-[2-hydroxyethy l] piperazine-N'-[2ethanesul fonic acid]
In Vitro Fertilization
L iquid Nitrogen
minutes
sodium chloride
Phosphate B uEered Saline
PBS + 0.3% BSA
Propidium Iodide
Piperazine-N,N7-bis[2-ethanesulfonicacid]
Fluorescein isothiocyanate- labeled Pisum sar ivum
agglutinin
Polyvinlypyrrolidone
Room temperature
seconds
Standard Error of the Mean
Sperm MotiI ity Index
Sperm TALP
Tyrodes Albumin Lactate Pyruvate medium
N-Tris[hydro>iymethyl] rnethy l-2aminoetanesulfonic acid
Tris (hydroxymet hy 1) aminomethane
volume per volume
Zona Binding Assay
Zona Pellucida
Zona Penetration Assay
INTRODUCTION
Use of frozen sernen in domestic dog breeding over the past decade has been increasing
both internationally (Linde-Forsberg, 2000) and within North Arnenca (Greenbarik,
1997). With the establishment of a commercial interest in fiozen canine semen and easing
of Iitter registration procedures by the Canadian Kenne1 Club over the past 10 years,
there has been a surge of artificial insemination (AI) with frozen sperm within Canada
(Greenbank, 1997). Stud dogs located across the country or around the world can be
selected and their fiozen semen shipped and used without transport-associated stress to
the animals. For breeders with the foresight to bank sperm fron valuable dogs, frozen
sperm can be used afier the dog has passed peak reproductive age and even
posthumously. Furthemore, through international exchange of fiozen semen, inbreeding
within breeds can be reduced (Linde-Forsberg, 199 1,2000; Greenbank, 1997).
There are approximately 34 wild species in the family Canidae, 22 of which are
considered threatened or endangered under CITES (1998)or the United States Fish a d
WildIife Service (USFWS) Endangered Species Act. Of those 22 only 5: the Red (Canis
rufus), Mexican (Canislupus balieyi), and Maned wolves (Chrosycaon bral~yurus),
the
Ahcan WiId dog (Lycaonpictus),and the Bush dog (Speorhos venaticus) have captive
breeding and/or re-introduction programs (Canid TAG). The Canid k x o n Advisory
Group of the American Zoo and Aquarium Association has designated the Ethiopian
(Canis simensis), Red, Maned, and Mexican wolves, and the African Wild dog as priority
speciss for conservation efforts (Canid TAG, 1999).
Neither traditional in situ (habitat protection) nor ex situ (natural propagation in zoos)
approaches to conservation provide practical or adequate preservation of global wildlife
and resources. Many carnivore species susceptible to extinction ofien require
extraordinary home ranges which rnakes protection of habitat ofien expensive or socially
dismptive to human populations. Zoos do not have the financial or space resources to
effectively aid the hundreds of animals approaching extinction (Johnston and Lacy, 1995).
Furthemore, this type of ex situ conservation fails to offer a solution to preserving bioand genetic diversity (Wildt, 1992; Wildt et al., 1992). Therefore establisting a genome
resource bank (GRB) along with reproductive biotechnology techniques could benefit
both in situ and ex situ conservation. A GIiB is defined as the organized collection,
storage, and utilization of biological material including gametes.
The key to practical implementation of assisted reproductive technology for wildlife
conservation is the systernatic preservation and use of frozen gametes and ernbryos, the
primary constituents of a GRB (Wildt, 1992). However, in exotic canid species, the range
of possible reproductive matenal to be cryopreserved and stored at present is limited to
sperm, due to the difficulties associated with oocyte in-vitro maturation, superovulation?
and estrus synchronization in female canids (Farstad, 2000). Nevertheless, banking sperm
alone c m have extreme advantages. Banked sperm could be used simultaneously to
inseminate anirnals at geographically removed institutions without subjectinç animals to
transport-associated stress. Perhaps even more advantageous is the possible use of
banked spem posthumously or after re-introduction of the animal into the wild. This
type of reproductive techndogy then would also aiiow institutions to side step the
common problems associated with mate preference and sexual incompatibility that oflen
prevents successful copulation in captive breeding programs (Wildt, 1992). Furthemore,
a GRB would provide a reservoir of matenal from individuals genetically important to the
captive population and genetic material to protect against disease outbreak and n a t d
disasters (Wildt, 1992).
If a species cannot reproduce, its fate is exiinction; and understanding of the complex
mechanisms that control reproduction, and conservation techniques such as GRB are then
necessary if we are to manage the long-term survival of seiected endangered species.
Successhl banking of s p e m requires samples with high viability and fertilizing potential
when thawed to be effective for use in AI (Wildt, 1992; WiIdt et al., 1993; Johnston and
Lacy, 1995). To establish a successful GRB, a detailed understanding of gamete biology
is needed as well as the development of techniques which not only enable freezing of
these cells, but which yield viabie and functional samples when thawed.
Cryopreservation:
General Prinçiqals of Crvo~raervatipa;
The concept of freezing cells, gametes, organs, and even organisms which are then later
thawed, is not new. The theory behind cryopreservation is that if living cells can be
cooled and held at extremely low temperatures, then the rate of biological processes
required for ce11 life could be slowed to a point of suspended animation. At -196°C
(temperature of liquid nitrogen, commonly used for cold storage) water only exists in
crystalline or glassy States, which are so highly viscous that diffusion is insignificant, and
thus thermaliy dnven reactions cannot occur. In addition, at this low temperature there is
insufficient thermal energy for chernical reactions (Mazur, 1984). The only reactions that
c m take place are photophysiological, where formation of free radicals and breaks in
macromolecules result from background ionizing radiation. Over time this direct ionization
can produce enough breaks or other damage to DNA to become deleterious to the cell;
however, the dose of ionizating radiation required would take some 2000-1000 years to
transpire frorn terrestrial background radiation alone (Watson, 2000). It is not the survival
of cells at - 196°C ihat is challenging to the success of the cryopreservation process, but
the injurious intermediate temperature zone (-1 5 to -60°C) that the ceils must transverse
during freezing and again in thawinç (Mazur, 1984; Watson, 2000).
Hamrnerstedt et al. (1990) described 5 distinct processing steps in the
cryopreservation of sperm: 1) extension (dilution) and cooling; 2) penetrating
cryoprotectant addition and packaging; 3) freezing; 4) storage; and 5) thawing. Methods
for each of these steps must be developed for each species, as each step impacts uniquely
on sperm cryopreservation survivai.
Regardless of species, al1 extenders used for either fieezing or cold storage should: 1)
provide nutrients as an energy source; 2) buffer against harmfûl changes in pH; 3) be of
physiologic osmotic pressure and concentration of electrolytes; 4) prevent growth of
bacteria; 5) protect from cold shock during cooiing; and 6) have cryoprotectant(s) to
reduce the amount of fieezing darnage (review by Concannon and Battista, 1989). The
majority of e'xtender recipes result frorn modifications of the successful basic bovine
semen extender recipe. This standard recipe contains egg yolk (EY), a buffer, a
metabolizable sugar and glycerol.
Each ingredient is incorporated into the extender for its essential andor protective
function. Egg yolk, specifically the phospholipids in the low density lipoprotein fraction,
has been s h o w to protect ce11 membranes from cold shock. Consequently. EY is
regularly incorporated into semen extenders. The mechanism of EY protection is poody
understood; however, it appears to act at the plasma membrane surface possibly inducing
a non-permanent alteration of the membrane composition, yet also preventing gross
membrane disruption (Watson, l976,I99Oy 1995; Parks 2nd Graham, 1992; England,
1993). The metabolic activity of sperm resulu in the build-up of hydrogen ions;
therefore, a buffer in the extender is required for ion removal. Without this buffer, the
increase in hydrogen ions produced from sperm metabolism causes extender pH to drop
with a subsequent decrease in sperm longevil and fertility (Smith, 1984). Glycerol
addition to extenders increases the survival of sperm post-thaw and is discussed in more
detail in the cryoprotectant addition section below.
pre-fieeze C o u
The first temperature changes in cryopreservation (i.e. during cooling) are know-n to
alter the physical properties of al1 ce11 membranes (Hammerstedt et al., 1990). Although
not clear, there is probably a lipid/protein re-arrangement (Figure 1 ) resulting in the loss
of selective permeabili~that is characteristic of living biological membranes (HoIr and
North, 1983; Buhr et al., 1989; de Leeuw et al., 1990; Hammerstedt et ai., 1990; Watson,
1995). Integral membrane proteins are clustered by lipid phase separation, and thus a
lipid transition may alter the position of these proteins and their function, such as ion
channel proteins (Watson, 2000). For e.xample, cooling-induced membrane calcium flus
has serious comequences to ce11 function (Bailey and Buhr, 1994j. Because calcium
rellation is integral in the controi of capacitation (Visconti and Kopf, 1998; Dragileva et
al., 1999; Pamsh et al., 1999), reports of capacitation-like characteristics observed in
cooled and fiozen-thawed sperm of varying species (Cormier et al., 1997; Rota, 1998:
Bailey et al., 2000) may actually result fiom cold induced alterations in ce11 mediated
calcium regdation. Although poorly investigated prevention of this lipidiprotein rearrangement by slower cooling rates or dilution into esrenders rnay be unsuccessful (de
Leeuw et al., t 990). Moreover, there is a possibility of cytoskeletai elements beinç
COOL TO 4°C
AIID GLYCEROL
fj
-
I
AND HOLD
HOLD AND FREEZE
LIPID FAVOFUNG A BILAYER CONFlGUREATiON
Figure 1. Schrmatic representation of the possible effects of cooling and r e w m i n g on
the distribution of lipids around integral membrane proteins. A: represents the
sperm membrane at time of colIection. B: represents preferential clustering of
nonbilayer lipids as a result of cooling to ?OC. C: represents further clustering of
lipids during fieezing. D: represents possible alternative lipid patterns afier
r e w a r n i i n ~(Hammerstedt et al., 1990).
temperature sensitive, which could contribute to disorganized membrane fusion following
cooling or cryopreservation (Watson, 2000). Hence, alterations of sperm membranes
occur during early temperature changes, which may not be apparent by standard sperm
assessment and therefore require advanced investigation to observe membrane lipid reanangement.
-.
crvo~rotectantAdditlori;
The addition of glycerol to extenden substantia!ly reduces the amount of freezing
injuries; however, its addition and removal also have been shown to cause sperm damage
(Fahy, 1986; Parks and Graham, 1992; Gao et al., 1993; Watson, 1995; Rota, 1998).
GIycerol is osmostically active, and consequently its addition ternporarily causes ceIl
volume changes and cellular water ioss (Figure 2). The major benefits of glycerol are
thought to be extracellular through a direct effect on plasma membranes (see review: Parks
and Graham, 1992). Glycerol alters the colligative properties of bulk water to lower the
freezing point, thus providing a longer time for egress of water from the ce11 before it
fieezes and forms ice crystals that can damage intracellular organelles (Harnmerstedt et al.,
1990; Watson, 1995). Cryoprotectants, including glycerol, initially cause dehydration of
the ce11 by osrnotically inducing water egress. Glycerol was onginally thought slow to
pemeate membranes; thus, it was believed that addition of glycerolated extender to sperm
required suficient equilibration t h e for this compound to exert its effects before samples
could be frozen. However, it recently has emerged that glycerol permeates cells verquickly and therefore a long equilibration time may not be required (see revirws:
Hammerstedt et al., 1990; Watson, 1995).
Figure 2. A schematic of temperature and cell volume changes associated with
cryopreservation. Change in temperature is presented in the top panel and
changes in cell volume is presented in the bottom panel. Each phenornenon is
divided into 5 staees; extend and cool, glycero1 addition and package, fieeze,
storage, and thaw (Hammerstedt et al., 1990).
Once the c~yoprotectantis added and equilibrated, the extended semen is packaged
(e.g. straws, pellets, vials) and fieezing commences. Rates of freezing Vary depending on
species and packaging method As freezing begins and temperatures drop beween -5°C to
- 15"C, supercooling (defined as when ice crystals begin to form in the solution
surrounding the cells while cellular contents remain unfiozen) is observed. Presumably
because plasma membrane pores are too srnall for difision of ice crystals, growth of ice
crystals into the ce1 at this stage is blocked (Hammerstedt et al., 1990; Parks and
Graham, 1992). The supercooled water within the cell, due to a difference in chernical
potential with extracellular water, then flows out of the ce11 and freezes externally
(Mazur, 1983). As temperatures continue to decrease and the extracellular water freezes,
the ce11 is exposed to hypertonie conditions and high extracellular salt concentration
causing further eMux of water. Movement of water out of the ce11 lessens the probability
that darnaging ice crysbls will form within the ce11 (Figure 3). Therefore, the rate of
freezing becomes important to the arnount of intracellux ice crystal formation. Too rapid
a method provides insufficient time for water to leave the ce11 and large inmcelluar ice
crystals can form, increasing the probability of membrane damage by ice crystals or by
the resulting rapid volume changes which the ce11 membranes cannot accommodate
(Hammerstedt et al., 1990; Watson, 1995). Freezing too slowly could dehydrate the ce11
and reduce ice crystal formation; however, the cells are exposed to a prolonged excess of
solute concentration with ceIl damage only becoming apparent when rehydration occurs
csoling
20°C
* 5 or 0°C
+ -196°C
Freezing
wamiing
b 38°C
About -12°C
Suboptimal
(toorapid)
K
optimal
suboptimal
(too slow)
Figure 3. Schematic of putative changes in sperm extender during the cryopreservation
process. The effects of various cooling and fieezing rates on formation of ice
crystals (e ) within the cells and microcrystals flarge (JC) or small (x) stars]
outside the ceUs and the direction movement and q d t y [large
to
small ($ ) arrows] of cellular water are shown (Hamrnerstedt et al., 1990).
(t)
cryopreservation of dog sperm. After examining the effects of different b a e r ingredients
(citrate vs. phosphate vs. glycine), and the percentage of EY and pH on dog sperm stored
at 5"C, a 20% EY (v/v), 1.16% sodium citrate, 0.75% glycine, and 1% glucose extender at
pH 6.6 was found to be the best combination to preserve motility. Exarnination of postthaw motility ;however, demonstrated that the greatest survival resuited from 0.2M Tris
buffered-EY-glucose extender containing 1 1% glycerol (Foote, 1964a). Subsequently , the
use of Tris buffered extenders has repeatedly produced the best post-thaw motility
(Battista et al., 1989; Olar et al., 1989; Thomas et al., 1993). Smith and Graham (1983)
reported extender with 50% PIPES buffer to yield greater post-thaw motility to other
buffers including Tris; however, this benefit of PIPES on s p e m post-thaw survival has
not been repeated by others (Olar et al., 1989; England, 1992), except for Dobrinski et al.
(1993), who found no difference in canine sperm post-thaw progressive rnotility between
Tris and PIPES buffers in EY extender.
The benefits of buffer used in EY extender may be dependent on the type of
packaging. Tris buffer resulted in the greatest post-thaw motility and longevity when
samples were frozen in straws (Battista et al., 1989; Olar et al., 1989) or ampoules (Gill
et al., 1970), whereas lactose yielded the best results when used for freezing in pellets
(Battista et al., 1989; S e a g a and Platz, 1977) or alurninum tubes (Ivanova-Kicheva et al.,
1997). Thomas et al. (1993), found Tris buffered extender to be superior to lactose
regardless of packaging in pellets or straws, as did Anderson ( 1973, who found no
difference in post-thaw semen quality between either estender frozen in straws. For
storage at 4OC, Tris (Rota et al., 1995) or Tns/Tes (England, 1992) buffered extenders
have been observed to best preserve rnotility.
In fox (Vulpes vulpes), sperm fiozen in Tris-EY extender in straws has provided better
fertility than al1 other extenders tested thus far (see review: Fougner, 1989). Tris-EY
extender also has been used for fieezing Red wolf sernen (Goodrowe et al., 200 1).
However, Red wolf semen is currently banked using a commercial EY-based e~qender
{International Canine Semen Bank, ICSB; Sandy, OR, USA) and therefore the buffer is
(Goodrowe et al., 1998). Skim milk, ofien inciuded in semen freezing extender in
-own
other non-canid species, recently has been investigated in the dog with post-thaw sperm
motility and viability comparable to that obtained using Tris-based buffer (Rota et al.,
2000).
&g
Yak:
A large range of EY concentrations has been utilized in the preservation of canine
semen. Foote and Leonard (1964) reported 20% EY to be the most beneficiai and this
subsequently appears to be the standard percentage used by many others (Gill et al.,
1970; Farstad and Andersen Berg, 1989; Ferguson et al., 1989; Fontbonne and Badinand,
1993; Rodriques-Martinez et al., 1993; Nothling et al., 1995; Silva et al., 1996).
Unfortunately, the final EY concentration in extended semen is ofien unknown, owing to
variable and sometimes unreported dilution rates. By examining different percentages of
EY with final concentrations ranging from 0-20%, England (1992) found that the greatest
initial pst-thaw motility and percent of live sperm resulted from samples frozen in
extender with a final EY concentration of 10%. Still, the majority of reported successful
canine inseminations with fiozen-thawed semen used extender containing 20% EY (Gill
et al., 1970; Farstad and Andersen Berg, 1989; Ferguson et al., 1989; Fontbonne and
Badinand, 1993; Rodriques-Martinez et al., 1993; Nothling et al., 1995).
As previously mentioned both glucose and lactose are commonly found sugars in
canine semen extenders (see review Concannon and Battista, 2989). In addition, fnictose
is another commonly used sugar in both canine and fox semen extenders (Gill et al., 1970;
Farstad and Andersen Berg, 1989; Ferguson et al., 1989; Fontbonne and Badinand, 1993;
Rodriques-Martinez et al., 1993; Nothling et al., 1995). Initial investigation of energy
metabolism of fresh dog sperm incubated in 10 mM of glucose or fnictose indicates that
hctose is more efficient than glucose in obtaining higher energetic Ievels (ATP measurcd
enzymatically) from fresh sperm. Furthemore, there is some indication that fnictose may
possibly play a role as a sperrn activator after ejaculation (Rigau et al., 2000). The
majority of extenders reported for use in domestic dog sperm cryopreservation contain
either glucose or mictose (Gill et al., 1970; Fontbonne and Badinand, 1993; RodriquesMartinez et al., 1993; Linde-Forsberg and Forsberg, 1993; Linde-Forsberg, 1995; Ndthling
et al., 1995; Silva et al., 1996; Hay et al., 1997b; Rota, 1998).
phLL
The pH of the second fraction (spem rich) of domestic canine ejaculates is
approximately 6.2 (Rota, 1995; Whales and White, 1958) and the third fraction, or
prostatic fluid, is slightly more basic at a pH of 6.8 (Whales and White, 1958). In
agreement with measured ejaculate pH, Foote and Leonard ( 1964) found extender at pH
6.6 to be superior to both 5.9 and 7.3 for the maintenance of whole ejaculate canine sperm
motility stored at 5OC. While some investigators have titrated their extenders to a pH of
6.8 (Foote, l964a), and 6.7 mots et al., i 995, 1W8), others have maintained e'xtender at a
more neutral pH of 7.0 (Smith and Graham, 19841, 7.1 (Thomas et al., 1993), and 7.4
(Hay et al., 1997). In two studies comparing vanous extender pH, maximum canine
sperm motility was maintained in the range of pti 7.0-8.5 when held at room temperature
(Whales and White, 1958) or at a pH of 7.3 when held a 39°C (England, 1992).
Furthemore, England (1992) determined extender pH 7.3 to be the least delererious on
sperm morphology over time compared to pH 3.3, 5.3, and 9.3, and therefore deemed the
best overall extender pH.
GIy-1
* .
A-tEaub
ilrat'
- .
Ion;
In an early study, the addition of 8% glycerolated extender at 5"C, compared to 4 56
glycerd, \vas shown to be more h m f u l to dornestic dog sperm longevity when held at
5°C (Foote, 1964b). This decrease in sperm motility was reduced when glycerol was
initially incorporated in the exiender compared to addition after cooling. The addition of
8% viv glycerclated extender (final gIycero1 concentration 4%) compared to no gIycerol
addition, has s h o w to cause a decrease in the number of sperm penetrating homologous
oocytes afier pre-freeze coolinç (Hay et al., 199%). A study by Fontbonnt and Badinand
(1993) esamined the temperature at which glycerol was added (YC vs. RT), method of
addition (one or severai steps), and glycerol concentration, and found no difference in
post-thaw motility between different addition methods at any temperature, or between
IO
3.2 or 6.4% glycerol concentrations. However, post-thaw motility was drastically
reduced when 1.6% glycerolated extender was use4 suggesting that beneficial effects of
glycerol are not obtained at concentrations below a certain value (between 1.6 and 3.2%).
Y
Pefia et al. (2998) also found temperature of glycerol addition to have no effect on p s t thaw spem quality; however, they did find that both post-thaw motility and acrosornal
integrity was superior following use of 8% glycerolated exqender compared to 2,4, and
6% This benefit of higher glycerol concentrations also corresponded with results from
Foote (1964a), who found post-thaw motility increased by increasing the glycerol
concentration from 4 to 8%. Altematively, Olar et al. (1989), found post-thaw motility to
be best preserved by freezing with an extender containing 2-4% glyceroI. Interestingly,
Foote (1964a) also demonstrated a significant interaction between the percentage of buffer
in the semen extender and the required amount of glycerol; higher leveis of glycerol were
required as the amount of buffer increased.
Varying cooling and equilibration times, ranging from 45 min to 5 h, have been reported
for canids (Gill et ai., 1970; Farstad and Andersen Berg, 1989; Ferguson et al., 1989;
Fontbonne and Badinmd, 1993; Rodriques-Martinez et al., 1993; Nothling et al., 1995;
Silva et al., 1996). Olar et al. ( 1989) found that the best post-thaw motiliiy was achieved
with dog sperm cooled (from 37°C to 5°C) for 1 h and equilibrated at 5°C for 1 h or
cooled for 2 h and equilibrated at 5OC for 2 h using Tris-EY extender containing 3 - 4 6
dycerol. Post-thaw rnotili- was decreased when cooled for 2 h and equilibrated for only
b
1 h (OIar et al., 1989). England (1992) found that an equilibration time of 4 h at 5°C
resulted in the greatest post-thaw motility and percent live spenn when compared to 3
17
and 6 h equilibration times using Tris/Tes-EY extender with 6.5% glycerol. In contrast,
Hay (1996) observed that after fast cooling to O°C (- 40 min) with > 2 h equilibration
there was a decrease in cooled pre-fkeeze sperm moti!ity and in percentage pre-freeze
sperm with intact and undamaged acrosomes. It is important to note that these cooling
and glycerol studies never provided the actual pre-freeze cooiing rate, only the time spent
cooling and equilibrating. Therefore, the actud cooling rate may have more of an influence
on the post-thaw sperm survival than the equilibration time.
Overall, the optimal concentration of glycerol added tu e-xtenders is a compromise
between protective and adverse effects. In this respect, canids appear to be similar to
many other mammalian species where a range of 2-8% glycerol is standard with addition
at the beginning or end of pre-freeze cooling (Almid and Johnson, 1988; Olar et al., 1989;
Fiser and Fairfull, 1990; de Leeuw et al., 1990; Taggart et al., 1996; Hay et al., 1997b).
More recently, prolonged post-thaw maintenance of motility and plasma membrane
integrity was observed with the addition of Equex paste (a commercial paste containing
sodium and trielhanolamine lauryl sulfate) to canine Tris extender (Rota et al., 1997). The
addition of detergent to EY extenders is thought to act through alteration of the EY
possibly increasing its proteciive effects during the cryopresenration process (Pursel et
al., 1978). Post-thaw in vitro zona binding capability was shown to be significantly higher
in canine sperm frozen in extender with compared to without t q u e x paste (Str6m HoIst,
1999), but inclusio~iof Equex did not affect pregnancy rates after AI with frozen semen,
regardless of insemination method used (Rota et al., 1999). Addition of a sirnikir detergent
to Tris-citric acid-glucose estender did not result in significant differences in progressive
IS
motility and normal acrosomes of dog sperrn fiozen in pellets (Nizanski et al., 2000).
However, the percentage of progressive motile spem during post-thaw incubation at
38°C was better when the sperm were fiozen in the presence, compared to the absence,
of detergent (Srrom Holst, 1999; Nizanski et al., 2000). The si-giticance of this difference
increased with incubation time, therefore, warranting further investigation when fieezing
canine semen in pellets.
Chilled Extended Canine Semeri;
Very few studtes have systematically exarnined the effects of pre-freeze cooling on
semen quality both afier cooling and after freeze-thaw. Diluted semen in a Tris-citrateglycerol-EY extender (Rota et al., 1995; Mortan and Bruce, 1989) or dried milk-glucose
extender (England and Ponzio, 1996) preserved motility well at 4OC over 4 days. Those
who have investigated sperm quality after cooling and glycerolated extender addition have
shown varying degrees of sperm damage. Afier cooling to 5OC, addition of three different
glycerolated extenders (Tris-glucose, Tris-fmctose, and sucrose-lactose) and equilibration
for 180 min, Invanova-Kicheva et ai. (1995) found morphology had not significantly
changed compared to fresh sperm. In cornparison, afier cooling and glycerol addition, both
Oenlé ( 1986) and Hay et al. (199%) found a decrease in the percent of intact acrosomes
fiom fresh sperm values. Canine sperm stored at 5°C for 24 h had an increased percentage
of sperm that were acrosome reacted (Kumi-Diaka and Badtrarn, 1994) and capacitated
(Rota, 1998). Furthemore, Hay et al. (1997b) demonstrated a decrease in sperm zona
penrtrating capabili~afier cooling and glycerolated extender addition compared to
cooling without glycerol addition. Collectively these studies indicate that some damage,
19
particuIar1y acrosomal, occurs during cooling. It is not clear however, whether damage
during cooling affects the type of damage incurred during freezing or even if it c m be
prevented through different cooling methods.
Canine Smrm F r e e z i m Thawirie;
The freezing rates used for cryopreservation of canine semen \ ary widely and are
Iargely dependent on packaghg method With the rare exception, canine sperm usualljr are
frozen either in 0.5 ml straws (Andersen, 1972; Morton and Bruce, 1989; Olar et al.,
1989; Strom et ai., 1997; Rota, 1998) or pellets (Seager, 1969; Seager and Platz, 1977;
Ivanova-Kicheva et al., 1995). Direct cornparisons of pellet and straw freezing methods
have found no difference in post-thaw semen quality (Seager et al., 1975; review: England,
1993). However, M e systematic investigation on the effect of freezing rate on post-thaw
canine semen quality has been conducted. Foote (1964a) compared the rates of O.8"C min-
' and 3.C
min-' from 5 to -15OC, both followed by 5 T min-' from -15 to -40°C and 10°C
min" from 1 0 to -79°C and found the slower rate to yield greater post-thaw sperm
swival. Afier comparing 3 different freezing rates, a moderate rate of SOC min " from 5
to -1ST followed by 20°C min" from -15 to -100°C resufted in the best post-thaw
sperm quality (see review: England, 1993). Likewise, Hay et al. (1997a), found initial
pst-thaw canine sperm motility highest after freezing (from O to -70°C) at average rates
of 12 and 28°C min", compared to a slowver of 0.S0Cmin-', and faster rates of 99°C min"
and 2 14°C min-'.
In most studies which have cornpared fast vs. slow thawing rates, the fast thawing
rates comprised of extremely high thawing temperatures (>70°C) for mere seconds, which
was logistically possible when semen was fiozen in straws; however, it is difficult to
thaw for short tirne intervals at these higher temperatures when semen is frozen in pellets,
therefore pellets are usually thawed at 37°C (Seager and PMz, 1977). However, the
benefit of either fast or slow thawing rates on canine post-thaw sperm quailty is not clear.
Examination of 2 different thawing regimens for canine sperm fiozen in pellets; 55°C for 5
sec vs. 37°C for 8 sec, in 0.9% NaCl solution resulted in significantly increased pst-thaw
motility (29.3 vs. 26.1) and survival time (184.4 vs. 148.9 min) at 55 vs- 37OC,
respectively (Ivanova-Kicheva et al., 1995). Furthemore, samples thawed at 55°C
showed significantly lower acrosomal damage although the percentage of rnorphological
abnormalities \vas not found to be different between thawing regimens (Ivanova-Kicheva
et al., 1995). Greater post-thaw motility, after freezing in straws, also resulted from rapid
thaw rates of 70°C for 6 sec or 45 OC for 30 sec compared to slow rates of 37°C for 50
sec or 23°C for several minutes (Battista et al., 1989). Conversely, both Smith and
Graham (1984) and Yubi et al., (1987) found, after freezing semen in straws, slower thaw
rates (37°C for 2 min) resulted in the best post-thaw sernen quality, with a decline in
motility and percent live sperm after rapid thawing (75°C for 6 sec).
Artificial Insemination with Chilled or Frozen Canine Sperm
The first litter produced by AI with chilled canine semer; kvas in 1951 (Harrop,
1954) and the first pregnancy fiom AI of tiozen-thawed canine s p e m was not achieved
until 1969 (Seager, 1969). There appears to be a wide range of AI methods, and therefore
success rates, using frozen-thawed canine sprrm. Early attempts to perfonn AI with
21
fiozen s p e m (subsequent to Seager, 1969) were unsuccessfiil (Gill et al., 1970; Andersen,
1972). Even later studies, although successful, had ~i~gnificantiy
lower pregnancy rates
using frozen s p e m compared to fiesh (review: Linde-Forsberg, 1991, 1995). For e,uample,
Linde-Forsberg and Forsberg ( 1989, 1993) reported pregnancy rates of 623433.8% using
fresh semen as opposed to 5 1.1-69.3% using frozen sperm. Others have found similar,
but reduced, pregnancy rates after AI of frozen canine sperm (67%: Farstad and Andersen
Berg, 1989; 66%: Ferguson et al., 1989; 60%: Silva et al., 1996). In addition to differences
in pregnancy rates, litter size has reportedly been 23340.5% lower after AI with frozen
vs. fresh spem (Linde-Forsberg and Forsberg, 1989, 1993).
Differences in pregnancy rate and litter size cannot soleIy be attributed to the use of
frozen or fresh spem. More recent studies have found the marner of insemination to
have a major effect on pregnancy rate and litter size. Pregnmcy rates were 52.6-58.996 for
intravaginal vs. 73.6-84.4% for inmautenne insemination of frozen-thawed semen with
rnean litter sizes of 4.0-3.2 and 5.4-5.5, respectively (Fontbonne, 1993; Linde-Forsberg,
1999). Silva et al. ( 1996) reported a pregnancy rate of 60% regardless of insemination
technique, although different voiurnes were used for the inseminations. NotNing et al.
(1993), demonstrated that intravaginal insemination followed by addition of >3 mL of
prostatic fluid resulted in significantly higher pregnancy rates and litter size compared to
intrauterine insemination alone, providing a practical alternative to intrauterine
insemination. NevertheIess, intrauterine insemination is now the more commonly used
and preferred mrthod of insrmination, panicularly in Europe (see reviews: LindeForsberg, 199 1, 1995). FinalIy, upon investigation of the influence of insemination dosé,
and nurnber of inseminations on fertility rate and litter size, Fontbonne et al., (2000)
found that the nurnber of inseminations performed in the sarne bitch to be the only
influence which approached significance (P
= 0.07); with
100% conception for bitches
inserninated 3 to 4 times compared to 64% for bitches inseminated once or twice.
Likewise, Thornassen et al. (2000) found the whelping rate of bitches inseminated twice
(77%)to be significantlygreater than those inseminated once (60%).
RATIONALE
Due to the low number of individuals in endangered canid populations, assisted
reproduction and genome resource banking have the potential to be important tools for
genetically managing these populations. Banking frozen sperm from endangered canids is
particularly important, as banking of oocytes and embryos is cwrently not possible for
these species (Farstad, 2000; Goodrowe et al., 2000). Barked sperm used with other
assisted reproductive technologies could help ensure no m e r genetic Ioss from either
animal death or reintroduction. Before these technologies can be used for endangered
species the protocols are established üsing domestic species. Furthemore, growing
populanty of AI with fiozen semen in domestic dog breeding has increased the need for
research in do- sperm cryopreservation (Linde-Forsberg, 2000).
M i l e many investigations of dornestic dog semen preservation exist, there is stiil
room for improvement, and several areas within the cryopreservation process require
further investigation. For example, very Iittle information exists on the effects of prefreeze cooling and the damage this process incurs. Pre-freeze cooling has been shown to
cause acrosomal alterations to sperm in the domestic dog (Oettle, 1986; Hay et aI., 1997;
Rota, 1998) and the Red wolf (Goodrowe et al., 2000,200 1). However, most reports of
successful cryopreservation andor AI of frozen-thawed domestic dog sperm rarely
reported the actual pre-freeze cooling rate and at best onIy provide the tirne spent cooling
and equilibrating. Therefore, rate of pre-freeze cooling and its influence on both cooled
and post-thaw semen quality requires further investigation.
Cryoprotectant addition prior to freezing has been minirnally investigated in the
domestic dog. However, no reports exist on the possibIe reduction of osmotic shock
caused by glycerol through addition of glycerolated estender slowly throughout the entire
cooling process.
There are recent reports of improved longevity of post-thaw sperm motility with
detergent (Equex paste) addition to canine semen extenders and fieezing in straws (Rota
et al., 1997; Strom Holst, 1999), and onIy one report of detergent addition to canine
semen extender when pellet freezing was used (Nizanski et al., 2000). Therefore, further
investigation of Equex paste addition to extender composition in conjunction with pellet
freezing was conducted.
An important component of estender and other sperm holding media is the available
energy source. Several sugars have been reportedly used for short-term storage and
cryopreservation of canine sperm (see review: Concannon and Battista, l989), but these
sugars have not directly been compared The different energy sources in both canine
semen extender and sperrn TALP were investigated.
Determination of appropriate extender composition, glyceroi addition rnethods, and
cooling rates for canine sperm cryopreservation are important for the improvement of
domestic dog, and in turn exotic canid post-thaw spenn quality. Improving
cry-opreservation of canine sperm would increase the success of dornestic dog AI w-ith
frozen sperm for private breeding purposes as well as better enable the banking and
possible future use of exotic canid sperm in assisted reproductive technologies.
Based on deficiences in knowledge regarding the overall process of canine semen
freezing, the main objectives of this study were to: 1) to determine the influence of
cooling rate and fmal cooling temperature (5 or 0°C) on both cooled and post-thaw sperm
quality and fertilizing potential, 2) m e s s the influence of different glycerof addition
methods on cooled and post-thaw spem quaiity and fertilizing potential, 3) determine the
effect of glycerol addition on cooled sperm rnorphology, acrosornal and capacitation
statu, 4) detemine if detergent (Equex paste) addition prior to fieezing impacts postthaw sperm quality and fertilization potential, 5) determine the preferred energy source of
canine spem in extender and modifieci tyrode's solution (TALP) and 6 ) determine if
energy source preference is altered by the holding temperature-
It is hypothesised that a slow pre-freeze cooling rate would decrease the amount of
sperm damage due to cryopreservation, both afier cooling and pst-thaw. Furthemore,
with slow glycerol addition, throughout pre-freeze cooling, post-thaw s p e m quality will
be improved cornpared to glycerol addition over 3 min at 5°C. It is hypothesised that
addition of Equex paste to canine semen extender pnor to fieezing will reduce the
amount of sperm damage observed post-thaw and increase post-thaw spem longevity at
37°C. Extender and TALP containing glucose are hyporhesised to best maintain sperm
motility over time.
MATERIALS AND METHODS
Animals:
Dogs were either housed with their owners and fed varying commercial diets of sofi
and hard food, or were housed individually at the University of Guelph Veterinaly
Teaching Hospital (VTH), fed a commercial dry diet and exercised daily. Al1 dogs were
given water ad libidum. Dogs housed at the VTH also were used as veterinary teaching
dogs for extemal examination, blood donation, and semen collection; however, semen
collections were never performed on the same day if the dogs had already been used for
blood donation that day. Semen collection fiequency was no less than 3 days apart (to
rest the animal). Dogs ranged in age throughout the studies from 1.5 to 5 years. A total of
12 different dogs was used (see below), three were mongrels and 9 dogs were of proven
fertility (sired at least one litter).
Dog
Breed
pure bred
pure bred
mongrel
pure bred
mongrei
pure bred
pure bred
mongrel
pure bred
pure bred
pure bred
pure bred
(P -proven; NP -not proven)
Fertile Statu
Siberian Husky
Golden Retriever
Labrador .u
Grey Hound
Hound x
Smooth Collie
Sharpie
Hound x
Siberian Husky
Beagle
English Setter
Labrador
Sernen Collection:
Semen was cdlected by manual stimulation using a plastic artificial vagina (AV; AgTech, South Dakota). Ejaculates were collected in a 15 mL falcon tube (Fisher,
Unionville ON) secured at the base of the AV. KY jelly was applied to the inner top 1 cm
portion of the AV. n i e AV was used to push the prepuce back over the penis to expose
the urethral bulbs outside the prepuce and then the area immediately behind the bulbs was
vigorously massaged. Digital pressure behind the bulbs constricted the penile venous
return and erectiori commenced. The first, second (sperm rich), and a negligfile portion
of the third ejaculate fractions were collected. The AV with collection tube \vas rernoved
when the beginning of the third fraction was first observed as characterized by the
appearance of clear fluid (prostatic fluid). Dogs C, DyE, and H were collected using a
latex A V (latex bovine semen collecting cone), with ejaculate fractions collected
individually into a sterile glass tube (10 mL). The ejaculate was then transferred to a 15
mL falcon tube. Al1 dogs were monitored until the p a i s regressed to its ventral position
and was covered by the prepuce (Seager and Fletcher, 1972; Kibble, 1969).
After collection, semen was immediately diluted 1: l(v/v) in standard extender (Triscitric acid; Appendix 1), kept at -37°C and transported to the laboratory within 1 h after
collection. Unless othewise stated, when the extended sample amved at the laboratory it
was centrifuged for 10 min at 300 g and the pellet resuspended to original ejaculate
volume in warm (- 37°C) standard extender (Appendis 1). This centrifugation rernoved
seminal plasma previously found to be detrimental to ejaculate spenn over estended
penods of time (Rota et al., 1998). The resuspended estended sample was then aliquoted
for expenments. For assessrnenf samples were washed by being centrifugeci at 300 g and
resuspended in equal volume Sperm TALP (sp T a P ; Appendix 1).
Semen Evaluation:
Motilitv:
Motility and progressive status were assessed using subjective estimates, over 3-5
fields, at 37"C, under phase contrat light rnicroscopy at ZOOx rnagnification. Percentage
motility was estimated to the nearest 5%. For progressive status a range of 0-5 was used;
O = non-motile, 1 = slow non-progressive movement, 2 = rnildly progressive movernent,
3 = moderate fonvard movement kvith large head displacement, 4 = steady rapid fonvard
progression, and 5 = very rapid movernent usualiy reserved for hyperactivation (Howard
et al., 1990). Overali motility is reported as sperm motility index (SMI), which is a
calculation that combines the hvo rnotility readings (Howard et al., 1990):
SMI = [%rnotile + (progressive status x 20)] / 2
Approximately 10 pL of washed and centrifueed semen were smeared on a glass slide
(RT) and air dried. Slides were fixed in 2% paraforrnaldehyde in PBS for a minimum of
10 min, but could be lefi in fixative indefinitely. Fixed slides were then stained with
Spermac@ (Stain Enterprises, South Africa) and evaluated according to Orttld ( 1986).
Three series of 100 sperm were evaluated under light rnicroscopy at lOOOx magnification
for both morphology and acrosomal status and the average for each morphological
category calcdated-
Acroso-
To establish acrosomal status,
- 1O CILof washed and centrifüged semen was smeared
on a glass slide (RT) and air dried. Slides were then fvted in cold (- - t 8°C) methanol for
100 sec. Fluorescein isothiocyanate- labeled Pisum satlvum agglutinin (PSA/FITC;
Sigma, Oakville ON) was added (30 PL) to the slide and incubated at room temperature,
by placing a plastic strip over the stained siide for 20 minutes. Thirty pL of propidium
iodide (PI; Sigma, Oak~illeON) were then added and incubated in the sarne manner for a
M e r 10 min. The slide was then washed with distilled water, covered with FITC guard
(Fluroguard; Southern Biotechnology, USA), and examined under fluorescence at 1OOOx
magnification. The number of intact, partially intact, balloon, and absent acrosomes was
evaluated according to Goodrowe et al. (200 1); three series of 100 sperm were counted
and the average calculated for each category. Intact acrosomes were fully formed having
no apparent abnomalities with contents condsnsed and contained within the acrosomal
membrane, which was smooth and uniform in appearance. PartiaIly intact acrosomes
included any abnormality, such as disrupted, lipped or irregular shaped acrosomes, and
severely damaged membranes. Balloon acrosomes demonstrated intact acrosomal
membranes, but the outer acrosomal membrane was distended and therefore acrosomal
contents appeared diffuse and fluoresced less intensely.
Concentration;
Concentration was assessed using a Neubauer hemocytometer (Canadawide Scientific,
Toronto ON). Sperm suspension was diluted 1/20 with 2% paraformaldehyde in PBS, and
then mounted on the hemocytorneter. The sperm found in five 1 mm' squares in each of
the two counting chamben were counted and the total number of spenn then divided by 2
and multiplied by 107 to estimate the concentration of sperm/mL (Hay, 1996).
Zona Penetration Assav (ZPAI:
The ZPA followed the protocol established by Hay et al. (1996). Briefly, canine
avaries were collected after routine ovariohysterectomies, placed in a sterile container
filled with saline, and stored at 4°C for 24-48 h. Ovaries were warrned to RT and sliced
in 3 mL PB 1 with a #22 sterile scalpel blade to release oocytes. Selected oocytes had
uniformly dark ooplasm, an intact cumulus ce11 complex, and no apparent irregularities in
the ZP. Oocytes were washed in PB 1 then IM TALP (Appendix 1) and transferred to 95
PL droplets of IVF TALP and overlaid nlth PB 1-conditioned silicone oil (no more than
10 oocytes/drop). The oocytes were then incubated for 1-3 h in a humidified atmosphere
(5% CO*, 38°C).
Fresh, cooled, or frozen-thawed s p e m were first washed by being centrifugeci at 300 g
and resuspended in RT sp TALP. Motility and concentration were calculated before in
vitro insemination. Each spem sarnple was diluted to a concentration of 40 x 106 motile
s p e d r n L with sp TALP. Five pL of each s p e m ce11 suspension were added to the
droplets resulting in a final volume of 100 {LLand a concentration of 2 x 106 motile
sperm/ml. After 16-18 h incubation in a humidified atmosphere (5% C o , 3 8OC),
31
oocytes were washed in PBS and transferred into 1 rnL of 1% Na-citrate for 10 min.
Oocytes were then transferred to a microcentrifige tube (1.5 mL) wïth 200 pL Na-citrate
and stripped of cumulus cells and looseiy bound sperm by vortexing for 3 min.The
oocytes were then recovered and incubated for 20 min in 1 mL PBS containing IO0 PL of
Hoechst 33258 (Si-ma, Oakville ON). Following staining, oocytes were washed in PBS
and rnounted on glass slides with vaseline containing g l a s beads (-200 microns; Sigma,
Oah~illeON) at d l four coverslip corners supporting the cover slip. The oocytes were
examined under fluorescent microscopy at 400x magnification to identie the percentage
of oocytes with zona penetrated sperm, and the nurnber of peneîrated s p e d o v a . To
ensure poor zona penetration of frozen-îhawed spem was not due to poor egg andior
zona pellucida quality, a fiesh ejaculate from any previously used dog was utilized as a
control in the ZPA.
.
.
7 m d1i n g Assav GBM:
The ZBA foilowed the protocol of Strom-Holst (1999). Briefly, pairs of ovanes
collected from dogs afier ovariohysterectomies were seperated and frozen (-20°C) in
saline frlled containers and marked accordingly. When required, a single ovary from each
of 3-8 pairs were thawed and sliced with a #22 sterile scalpel blade to release oocytes
from ovarian tissue. The corresponding ovaries were thawed and the oocytes obtained
were used for pst-thaw sperm insemination. Ovaries were sliced in 3 mL PBS + 0.5%
BSA, and oocytes with uniformly dark ooplasm, a tight intact cumulus ceII cornplex, and
no apparent irregularities in the ZP were selected. Oocytes were then placed in 1 mL of
Na-citrate for 15 min followed by 15 min vortexing to remove cumulus c e k Zona intact
32
Hewin and England (1998). Approximately 400 yL of washed sperm suspension was
overlaid on 4 mL of PVP (polwinylpyrrolidone; Sigma, Oahcille ON) and centrifùged
for 5 min at 800g. The pellet was resuspended in 45 pL of PB 1 with 8 pL of CTC
fixative (Appendix 2). Forty-five pL of CTC working solution (Appendix 2) were then
added and the sample was kept in the dark. A smalI drop ( 4 0 PL) of fluorescence guard
was placed ont0 a g l a s slide and 10 pL of CTC stained sperm mixed into the
fluorescence guard, covered (20x40 mm slip), and carefully compressed for
approxirnately 3 min. The slide was then esarnined under fluorescence microscopy at
lOOOx magnification. Bright green fluorescence over the entire sperm head and postacrosomal region indicated acrosome intact non-capacitated (1j, green fluorescence over
the sperm head but not the post-acrosomal region indicated capacitated (C), and sperm
wïth only a du11 fluorescence dong the sperm equatorial region were acrosome reacted
(M).
Two senes of 100 sperm were evaluated and the average taken for each category:
I,C and AR.
Freezing and Thawing:
The pellet method as described by Seager and Platz (1977) was used for freezing.
Briefly, indentations in a block of solid carbon dioxide (dry ice) were made by pressing a
board of 2 mm diameter aturninum nails lined 2 cm apart, onto the dry ice for
approximately 10 sec. Fi@ PL drops of extended semen were placed into the dry ice
indentations, using a repeat pipettor wirh refrigerated sterile tips. Sperm pellets forrned in
less than 3 min. Freezing rate was approxirnately 1°C/sec (Figure 4) to -70°C determined
by placing a thermocouple inside a 50 yL drop of extender on dry ice and temperature
readings taken every IO sec. After 3 min, the pellets were dropped into LN2 (-196OC).
Five pellets were placed into a I .5 mL cryovial (with punctured hoies made with 22G
needle at the base of the cryovial) in LN2.Three cryovials for each treatment were placed
on a cane and stored in a tank of LN2 until thawed.
Five pellets (250 PL) were thawed directly into a 1.5 mL microcentrifuge tube (Fisher,
Unionville ON) containing 750 p.L of 37°C PB 1 in a 37°C water bath for 1 min. The
sarnple was then centrifuged at 300 g for 7 min. The resulting pellet was resuspended
with 250 PL RT sp TALP. This resuspended sample was used for al1 post-thaw spem
analyses.
Experimental Designs:
ent 1;
Extender with 4 different sugars: glucose, fnictose, sucrose, lactose.
Objectives: To investigate 1) the preferential sugar energy source of canine spenn in
extender using four standard extender sugars: glucose, fructose, sucrose, lactose and 2) if
storage temperature has an effect on the sperm sugar preference.
Ejaculates from dogs A, C, D, and 1 were coIlected once on separate days. After
semen collection, each sample kvas divided equally into 4, 15 mL Falcon tubes and then
diluted 1:1 (v/v)
with warrn standard extender containing 50 rnM of either glucose, hctose, sucrose, or
lactose (Appendix 3). The samples were then transported and centrifuged, as previously
stated, and resuspended to the originai volume with the corresponding extender (-37°C).
Three hundred and thirty pL of each extended sample were then placed into 3, 1.5 mL
(12 total) microcentrifuge tubes. One microcentrifuge tube fiom each of the 4 different
extended samples was held at 0°C (ice bath), 37"C,and RT (-2Z°C). The percentage of
sperm that were motile and progressive status of each sample in extender was assessed at
0,30,90,
150 min and then every 24 h. To avoid possible bias, different sugar extenders
were randomly numbered 2-4 with the corresponding extender unknown to the
investigator until ail experimental trials were cornpleted. AI1 4 samples at al1 3
temperatures were assessed in the same order at each time interval.
Statistics:
Differences between the SM1 of sperm were compared bebveen sugar treatments at
each time penod using a two way analysis of variance (two way ANOVA). Observations
fiom each time period were pooled and storage temperature compared by ANOVA.
Where significance was indicated (P<O.OS),
a Tukey HSD post hoc test was used to
compare SMI means for each holding temperature at each time period and between time
periods for each holding temperature (SPSS 10.0 for Windows).
&nerimu
Spem TALP and extender expenment
Objective: To evaluate the effects of energy source and concentration in both extender
and sp TALP on canine sperm motility in vitro.
Ejaculates from dogs A, C, D, and 1 were collected once on separate days. After
coIlection, each ejaculate was split into 10, 15 mL falcon tubes. Eight of the ejaculate
portions were immediately diluted 2: 1 with either w v m (-37°C) sp TALP prepared with
glucose, sucrose, or fructose at 50 and 22.6 mM concentrations, or warm extender
prepared with Na-pyruvate andNa-lactate (Sigma, Oakville ON) at 50 and 22.6 rnM
concentrations (Appendix 3). The rernaining 2 sarnples were diluted 2: 1 with warm
standard extender and sp TALP as controts. The sp TALP wiîh sugars had a pH of 7.4 2
0.1 and osmolarity adjusted to 300
0.02 and osmolarity 295
* 3.5 mOsm by adding NaCI. Extender pH was 7.45 *
5.0 mOsm (Appendix 3). Based on the optimum storage
temperature fiom experiment 1, sarnples were kept at RT and assessed for motility and
progressive status at O, 120, 180,240 min and then at 24 h and 48 h.
Statistics:
Differences in SMI between extending media at each time period were compared by a
two way ANOVA. Where significance was indicated (P<0.05),a Tukey HSD post hoc
test was used to compare S M means for each media at each tirne period (SPSS 10.0 for
Windowsj.
Es-riment 3:
Equex paste in extender (Figure 5)
Objective: To determine the effect of pre-freeze addition of extender containing 1%
Equex paste (Equex) on pst-thaw canine s p e m quality.
This experiment was conducted at the laboratory of Dr. C. Linde-Forsberg, Swedish
University of Agriculwal Sciences, Uppsala Sweden, and therefore their prorocols were
used.
On two separate occasions (6 days apart), ejaculates fiom three dogs J, K, and L were
collected within 30 min of each other and processed (Figure 5). Briefly, immediately
after collection of each ejaculate, the semen was diluted (1 :1 v/v) with standard estender
and held at RT until al1 dogs were collected. Each estended ejaculate was individually
centrifüged, resuspended to original ejaculate volume with standard extender, and then
ejauclates combined (pooled). One mL of the pooled sample was aliquoted into each of
two 10 rnL tubes, and then placed directly into a refngerator and cooled to 5°C (-45
min). The remainder of the pooled semen was used to assess fresh motility, progressive
status, morphology, acrosornal starus, and subjected to a ZBA. At 5"C, 8?6 glycerolated
extender, also at 5"C, was added 1:1 v/v over 3 min, or 8% glycerolated extender with
1% Equex (Nova Chemicals, Sictuate MA) was added 1: 1 v/v over 3 min. Once
glycerolated extender was added, the samples were frozen using the pellet method.
Samples were thawed (5 pellets) at 37OC for 1 min in thawing medium ( 750 PL; Str6mHolst, 1999). Once thawed, each sample was further divided into 2 portions and both
were centrifuged to remove extender. One of the 2 portions was resuspended in 250 pL
of CCM (Mahi and Yanagirnachi, 1978) and used for post-thaw assessrnent of motility,
progressive status, morphology, acrosomal status, and ZBA. The other portion was
resuspended in 250 ~ L of
L thzwing medium, incubated for 3 h in a humidified
atmosphere (506 CO2, 38°C) and then evaluated for al1 sperm characteristics including
ZBA.
Figure 5 . Design for experiment 3. Pooled canine semen fiozen with pre-freeze addition of
8% glycerolated extender containhg 1% Equex paste or not containing Equex
paste.
Dogs coilected individually
*
Extended (RT)
centrifuged Bi resuspended in extender
to original ejaculate volume
v
POOLED
COOLED to SOC
ADD (over 3 min)
8% glycerol +
1% Equex extender ( 1: 1 v/v)
8% glycerol
extender ( 1:i v/v)
50 pL pellets
STORED
in LN2
*
THAWED
3 7 T for f min
5 pellets in 750 pL thawing media
v
cenmfiiged & resuspended
CCM
Thawing
media-
v
4
Evaiuated
Ar"
motility
PS.4-acrosomal status
morpholoyy
ZBX
Statistics:
Due to the low number of replications no statistics were calculated. Oniy means with
SEM are reported.
Rate of cooling and fmal temperature (Figure 6).
Objective: To evaluate the effect of slow and fast cooling to 0°C and 5°C on cooled and
post-thaw canine spem characteristics.
Five ejaculates fiom each of 4 dogs, A, B,C, and D were collected, transported and
centrifuged as descn3ed previously. After centrifugation, the pellet was resuspended to
the onginal semen volume with warm standard extender and mixed. One mL of extended
semen was aliquoted into each of 4, 15 mL Falcon tubes. Two tubes were fast cooled by
placing the tubes directly into a refrigerator (- 4°C). The other 2 tubes were each placed
into a 50 mL, plastic tube (Falcon; VWR, Missauga ON) containing 30 mL warm water
(-37"C), which was then placed into a 250 mi, plastic beaker (Fisher Scientific,
Unionville ON) filled with 160 rnL of warm water (-37°C) and then the entire apparatus
placed into the refrigerator. One tube from each of the cooling rates, fast (FS) and slow
(S5), was cookd to 5°C and 1 rnL of 8% glycerolated extender added ( 1:1 v/v) over 3
min when the sample reached 5OC. The other tubes fiom each cooling rate, fast (FO) and
slow (SO) were transferred from the fridge at 5°C to an icehater bath and cooled to O°C,
with glycerolated extender added over 3 min when the sample reached 0°C. Cooling rates
were determined by placing a thermocoupie in the extended sample and recording the
temperature change every 5- 10 min. Approximately 1 mL of the cooled semen was
Figure 6. Design for expriment 4. Extended canine sperm cooled at Fast (average rates of
O.6"C/rnin
to 5°Cand or 0.25"C/minto 0°C) and Slow (average rates of
O. 14"C/min to 5OC and 0.09"Cto 0°C).
Semen Collection
Dogs A, B, C , D x 5 each (n = 20)
extended 1: l (v/v)
centrifuged & resirspended
in extender to orginal ejaculate volume
I
v
COOLED
v
Slow
Fast
5°C
Y
0°C
ADD
8% glycerolated extender
slowly over 3 min (1 :i v/v)
FROZEX
50 pL pellets
i
STORED
in LN2
v1
THAWED
37OC for 1 min
5 pellets in 750 pL spTALP
7
centrifùged an resuspended
in spTALP to onginai volume
Evaluated (n=5 ejacdateddog)
motility
PS A-acrosomal statu
morphology
ZPA (n = 2 ejaculates/doo for ZPA only)
pelleted in 50 Cu, drops onto dry ice using a repeat pipettor using refiigerated sterile tips.
The remainder of the cooled extended semen was centrifùged at 300 g for 10 min and the
pellet resuspended in an equal volume of sp TALP for assessrnent of cooled spem
characteristics and ZPA.
At least 5 pellets were thawed on a Iater date by previoudy described methods.
Samples were assessed fresh, after cooling to SOC, or to O°C, and post-thaw for motility,
progressive status, morphology, acrosomal status, and ZPA. Since the availability of
canine oocytes for ZPA for each ejaculate/dog could not be guaranteed, only 2
ejacuIates/dog (randomly selected) were used to assess for fresh, cooled, and post-thaw
ZPA, al1 other characteristics were assessed fresh, cooled, and post-thaw for each
ejaculate.
Statistics:
EEect of dog was accounted for in the statistical mode1 when comparing treatment
effect A two way ANOVA with replicates, grouped by dog, was used to determine
significant differences (Pc0.05)arnong treatments for each ~ h ~ c t e r i s t iWhen
c.
significant differences among treatments were found, a Tukey HSD post hoc test was
utilized to compare significance between means (SPSS 10.0 for W indows).
Cooling f a t and slow with different glycerol addition methods (Figure 7).
Objectives: To evaluate the effects of glycerol and method of glycerolated extender
addition on cooled and pst-thaw canine sperm quality.
Three ejaculates from each of 6 dogs (A, B, E, F, G, and H) were collected,
transported and centrifbged as described previously. After centrifugation, the pellet was
resuspended to original semen volume with warm (-37OC) standard extender and mixed.
The extended sarnple was then divided into 6 tubes with 0.5 mL in each tube. Two tubes
were assigned to one of 3 treatments: no glyceroi added to the extender (EY), 8%
glycerolated extender added (1 :1 viv) at SOC over 3 min (EGO), or 8% glycerolated
extender added throughout cooling (EGD). Al1 sarnples were cooled to 5OC, with one
tube in each treatment being exposed to the fast (F) and one tube to the slow (S) cooling
rates, as detailed in expenment 4. Tubes containing 0.5 mL of the 8% glycerolated
extender were cooled at the same rate as the semen, thus the glycerolated extender was
always at the same temperature as the extended semen sample it was being added to. For
fast and slow cooling in the EGD treatment, 75 pL of glycerolated extender were added
every 5 and 22 min, respectively. Samples in the EGO and EGD treatments were frozen
by pellet method once cooled to 5°C and glycerolated extender added. Samples without
glycerol addition (EY) were not fiozen, and were examined only afier cooling. At least
one cryovial(5 pellets) was thawed on a later date as described previously. Sarnples were
examined fiesh and cooled (with or without glycerol addition) and EGO-F,S and EGD-
F,S were examined post-thaw for motility, morphology, acrosomal status, capacitation
status, and ZPA. Since the availability of canine oocytes for ZPA for each ejaculate!dog
could not be guaranteed, and only 3 ejaculates/dog were being collected, only 1
ejaculate/dog (randomly selected) was used to assess fresh, cooled, and post-thaw ZPA,
al1 other characteristics were assessed fresh, cooled, and post-thaw for each ejaculate.
44
Figure 7. Design for experiment 5. Extended canine s p e m cooled (fast or slow) to 5OC,
with 8% glycerolated extender added slowly pre-freeze (over 3 min),
throughout cooling to 5°C (fast: 75 fi every 5 min; slow: 75
every 22 min),
or not at all.
Semen collection
Dogs A, D, E, F, G, H x 3 each (n = 18)
extended I :l ( d v )
centrifûged & resuspended
in extender to original semea volume
I
v
COOLED to 5OC
EY
EGO
Fast
Slow
4
no addition
EGD
Fast Slow
i
Fast Slow
h
'
ADD
8% glycerol extender
slowly over 3 min ( 1:L v/v)
/'
\
Evaluated
motility
FROZEN
morphology
50 pL pellets
PSX-acrosome status
CTC-capacitahon status
STORED
in LN2
Y
8% glyceroi extender
throughout cooling
(final 1:1 v/v)
I'
Evduated (n=3 ejaculates/dog)
S o t i l i t y
morphology
PSA-acrosome status
CTC-capacitation stams
ZPA (n = 1 ejacuiate/doç
1
for ZPA only)
THAWED
at 37°C for I min
5 pellets in 750 pL spTALP
v
resuspended in s TALP
4
Evaluated (n=3 ejaculrites/dog)
motility
morphology
PSA-acrosomal status
CTC-capacitation status
ZPX
(n = I ejaculatc/dog for ZPA only)
Statistics:
Effect of dog was accounted for in the statistical mode1 when comparing treatment
effect A two way ANOVA with replicates, grouped by dog, was used to detemine
significant differences (P<0.05) among treatrnents for each characteristic. When
significant differences among treatments were found, a Tukey HSD post hoc test was
utilized to compare significance between means (SPSS 10.0 for Windows).
anson of r
u from e x ~ ement 4 to exnement 5;
Cooling at the fast and slow rates to 5°C (F5, S 5 ) in e'rperiment 4 was the same
treatment as cooling at the fast and slow rates with giycerolated extender added m e r 3
min at 5°C (EGO-F, EGO-S) in experiment 5. For cornparison between experirnents 4 and
5; fresh, cooled, and pst-thaw sperm characteristics of dogs A and D (the only dogs used
in both experiments) from these equivalent cooling treatrnents were compared using an
one way ANOVA (SPSS 10.0 for Windows).
RESULTS
Eaperimcnt 1: - Extender with 4 different sugars: glucose. h c t o s e , sucrose, lactose.
There was no signïficant difference in SM1 of canine sperm diluted in any of the four
different sugar extenders (glucose, h c t o s e , lactose, sucrose) at any stonge temperature
(37°C. RT, 0°C). therefore the data for al1 sugars were pooled at each temperature. By 90
min. regardless of extender, RT maintained greater SMI compared to both O°C and 37°C
storage temperatures (Figure 8), and at every time interval thereafler SM1 at RT > 0°C >
37°C (P<0.01). For the 37°C treatrnent. there was a slight decline in SM1 at 30 min with
decreases in SM1 (P<0.001) at 90, 150 min and 24 h. For the 0°C trcatrncnt, SM1 decIined
slightly at 30 min with decreases (PcO.00 1) observed at 150 min, 24 h, and 48 h. For the
RT treatrnent, the first decrease in SM1 was observed at 24 h (P4.001). The next dccline
in S M at RT was observed at 48 h (P<0.01). with a further decline at 96 h (P<O.O5).
Motile sperm were obscrved at RT as late as 102 h in 2.14 dogs and one dog still had a low
percentage of motile sperm observed at 123 h (data not shown).
Expetimcnt 2 : - Sperm TALP and extendrr energy sources.
No significant diffcrence in SMi behveen samples diluted in any media was observed
until 24 h and 48 h (Figure 9). Samples diluted in standard extender consistently had the
greatest SMI. with sp TALP containing glucose consistently maintaining the second
highest S M 1 at every time interval. By 24 h and 48 h. the mean SM1 in sp TALP
containing sucrosc at 50 mM concentration was markedly lowcr than al1 other media
(P<O.OS). Appcndix 4 shows SM1 mcans and differenccs for al1 mcdia at 24 and 48 h.
Figure 8. Combincd spem motility index (SMI) of canine sprm diluted in extender with 4 different sugars (glucose, fructose, sucrose,
lactose) and held at room temperature (RT), O, 37OC.Values are mean + SEM of 1 ejaculate from each of 4 dogs.
indicates RT significantly different from both O and 37°C (P<O.OS).
*3 indicates significant differcnce between al1 holding temperatures RT>0>37 (P<0.05).
+ ind icites significant di fference between all holding temperatures RD0>37 (W0.00 1 )
Erperiment 3: - Equex paste in extender.
Data fiom experiment 3 were not statistically analyzed due to the low number of
replicates. Motility, acrosomal status? and zona binding results of pooied semen with or
vithout pre-heeze addition of Equex paste were similar both post-thaw and afier 3 h
incubation (Table 1). The SM1 o f the pooled semen decreased between Fresh and postthaw sarnpies, and further decreased by 3 h incubation post-thaw. The perccntage of
spenn with intact acrosomes also decreased between Fresh and post-thaw evaluation, with
essentially no intact acrosomes and a large majority of sperm rvith absent acrosomes aker
3 h incubation post-thaw. The percentage of rnorphologically normal sperm was
moderately greater in post-thaw sarnples and in incubated sarnples with pre-freeze
addition of Equex than samples with no Equex addition (Table II). The percent of
rnorphologically normal sperrn slightly decreased between fresh, post-thaw, and afier 3 h
incubation s m p l e s with an increase in the percentage of bent tails between fresh. postthaw. and after incubation sarnples. There also was a sIight increase in sperm head and
midpiece abnormalities d e r 3 h of incubation compared to immediately post-thaw.
Sperrn zona binding capability decreased between fresh. post-thaw, and after 3 h
incubation (Table 1). This decreze in zona binding capability corresponded with the
decreases in S M I and percentage of intact acrosomes.
TabIe 1. Characteristics of each pooled (1,2) canine sperm fiesh, pst-thaw (PT)in
extender containing 1% Equex (w/ EQ) or not containing Equex (no EQ), and afier
3h incubation post-thaw (PT) at 37°C (+3h).
Acrosornal Status
ZBA~
Pool S M " Intact Partial Absent Penetration sp/ova
Fresh
1
2
2
b
30.0
4.6
28-0
67.33
64.0
1.7
Spem Motility Index
Zona Binding Assay
Table II. Morphologie characteristics of each pooled (1,2) canine sperm fiesh, after
frozen (PT) in extender containing 1% Equex (w/EQ) or not containing Equex
(no EQ), and after 3h incubation post-thaw (PT) at 37°C (+3h).
Pool Normal coiled tail bent tail bent tip
othe?
12.3
6.0
2.8
7 1.6
6.3
Fresh
1
11.3
2.0
1.7
2
74.0
11.0
4.6
1.8
5.3
36.0
52.3
PT no EQ
1
27.3
2.6
1.2
63 -3
5 -6
2
26.3
4.3
2.5
4.6
PT w/ EQ
1
62.3
PT no EQMh
PTw/ E Q G h
2
1
72.0
56.6
2
1
2
57.0
60.0
65.6
' includes head and midpiece abnormalities
3.3
6.6
7.3
3.6
2.6
20.0
28.3
27.0
26.3
25.3
3.0
4.6
4.0
3 -3
2.3
1.7
3.9
3.7
5.8
4.2
Experiment 4: - Rate of cooling and fuial temperature.
The fast cooling rate, measured by thermocouple, was as follows (Figure 10):
1S°C/min for the first 15 min, from 32 to 1 1OC; O. 13OC1'min fkom 15-45 min (to reach
SOC); and O.O6OC/min from 45-130 min (to reach OT). The slow cooling rate, measured
by thermocouple, \vas as follows (Figure 10): 0.47"C/min for the fint 15 min, from 32.9
?O
283°C; 0.19°C/min from 15-45 min, to
- 19S°C; 0.09°C/min fiom 45-200 min (to
reach 5OC); and O.O3"C/min from 200-365 min (to reach O°C). The mean SM1 was not
different between fresh sperm and sperm cooled slow or fast to YC, whereas SMI
showed no significant decrease when cooled at the fast rate to 0°C and a ~i~gnificant
decrease (P<0.05) when cooled at the slow rate to 0°C (Table III).
Table III. Sperm motility index of fresh canine sperrn and sperm cooied at the fast rate to
5OC (F5) and 0°C (FO), and cooled at the slow rate to 5OC (S5) and O°C (SO).
Mean
SEM
Sipnificance
Fresh
81.3
1-1
A
F5
79.9
1.O
A
FO
78.2
1.2
AB
S5
78.8
0.8
A
SO
75.4
1.O
B
A,B ; Different letters indicate significant dserence (Pc0-05)berween treatrnents.
The percent of intact acrosomes appeared to decrease progressively from fast to slow and
5°C to O°C, respectively (Figure 1l), and was mirrored with an increase in partially intact
acrosomes. Differences in percentage of sperm with balloon or absent acrosomes were not
observed between treatments. S p e m slow cooled to 0°C had fewer normal sperm than
Figure 12) which appeared to bs largely due to a significant
did fiesh semen (P~0.05,
increase in the percent of spem with bent tips. Although there appeared a trend for the
'CI
F.
C
Cr.
'n
C1
C
Cl
W.
f..
(3,)aml=admaJ,
--
C
n
G
'n3
cc
e-
uf
percent of oocytes with sperm penetrated and the number of sperrdova to increase with
cooling, no difference was found among treatrnents (Figure 13).
Table N. Post-thaw sperm motility index (SM) of canine sperm cooled pre-fieeze at a
fast rate to 5OC 0 5 ) and O°C (FO), or at a slow rate to 5°C ( S 5 ) and 0°C (SO),
and combined post-thaw SMI.
F5
Mean
SEM
58.9
3.5
FO
59.3
2.9
S5
58.3
3 -6
SO
55.9
3.3
combined
58.0
1.7
There were no differences in pst-thaw motility (Table IV), acrosomal status,
morphology, or ZPA (data for treatment not shown) between cooling methods (Figures
12, 14, 15). Sperm motility index decreased markedly (P<O.OO 1) post thaw from fresh
and cooled values (Tables III, IV). The percentage of sperm with intact acrosomes (PSA)
decreased post-thaw, with the percentage of partially intact acrosomes increasing, from
both fresh and cooled values (Figures 11). For post-thaw samples, the percent of sperm
with absent acrosomes also increased, whereas the percentage of sperm with balloon
acrosomes only slightly decreased compared to fiesh and cooled samples (Figure 15).
Post-thaw, the percentage of morphologically normal sperm remained approximately
equivalent to cooled values; however, the percentage of spem with bent tails increased
coinciding with a decrease in the percentage of sperm with bent tips cornpared to cooled
samples (Figure 12). The percent of oocytes with spem penenated and the number of
s p e d o v a for post-thaw sperm were both drastically lower than fresh and cooled values
(Figure 14).
0
= C -=
El
0
25,
>crr CI-
O
.E
F
3
L
c e
C
=
O
* O
Q
Ec -c">
&=
~ -e:
Egsà?
c
.
L
c;
U
L S C
5 - f f i U
.->
" '3,cLj
-
-
eE*=
% p = ü
5
Experiment 5: - Cooling fast and slow with different glycerol addition methods
There were no significant differences in SMI, acrosomal status, rnorphology, or
capacitation status between either cooling rate (fast or slow) or method of glycerolated
extender addition arnong cooled or among pst-thaw values. Therefore, within cooled or
post-thaw data sets sperm characteristics resulting fiom fast or slow cooling, with
glycerolated extender addition at 5°C or throughout coding, were pooled. The post-thaw
SM1 value was substantially (P<O.Ol) lower than fkesh and cooled values (Table V). The
percentage of acrosome intact sperrn decreased afler cooling, and decreased M e r postthaw, compared to fiesh values, with an increase in partially intact acrosomes after
cooling and a M e r increase in both partial and absent acrosomes pst-thaw (Table V).
The percentage of non-capacitated sperm was smaller, and the percentage of acrosome
reacted spem greater, in pst-thaw compared to fiesh and cooled samples, as indicated
by the CTC assay. The percentage of morphologically normal sperm was lower in postthaw samples compared to fiesh and cooled values, with an ïncrease in the percentage of
sperm with coiled tails, bent tails and tips (Table VI). Percentage of zona penetration
drastically decreased pst-thaw compared to both fiesh and cooled values (Figure 16).
Table V. Cornparison of canine sperm characteristics fresh, cooled, and post-thaw.
SMI
Acrosome status
CTC*
'
Fresh
SEM
Cooled
SEM
Post-thaw
SEM
1
C
AR
3.2"
33-2"
60.7=
6 2
1.2
3.7
3.2
1.2
10.4"
3.7"
28-2a
6 1
10.Sa
O-9
1.2
0.3
1 -6
44.4'
7.1"
Intact
Partial
Bailoon
82.6"
67.9"
24.2"
5.2"
1.0
2.6
1.6
1.5
81.5"
57.4b
29.7b
0.5
1.4
63.9'
32.8"
15.6~
0.9
1.2
14.0~ 61.5"
~ 4 . 2 ~
0.8
1 .O
1.1
0.6
0.8
0.9
0.9
1.3
l Sperm motility index
Chlorotetracycline assay 1: acrosome intact, not capacitated; C: capacitated; AR: acrosome reacted
ayb*cSuperscript letters indicate significant difference (PcO.0 1) within coiurnns.
'
Table VI.Morphological characteristics of fresh, cooled, and post-thaw canine sperrn.
Noxmal
Coiled
Bent tail Bent tip
0ther'
--
- -
p
p
--
--
--
Fresh
73.8"
1.5"
7.4"
16.5"
0.8"
SEM
1.2
0.2
1.1
1.2
O. 1
7 1-9"
1.6"
7.6a
17.7"
1.0"
0.5
O.1
0.3
0.5
O.1
2.4b
24.7b
8.gb
0.8"
0.8
0.4
0.2
Coolec!
SEM
Post-thaw
SEM
63.2b
0.8
0.4
' Includes head and midpiece abnormalities.
a*bSuperscript
letters indicate significant difference (P<O.O 1 ) within colunns.
Dog Variation
Statistical analysis in expenments 4 and 5 demonstrated between dogs differences
for al1 fresh, cooled, and post-thaw sperm characteristics [except ZPA). Fresb- cooled,
and post-thaw S M , percentage of intact acrosomes, and percentage of morphologically
normal sperm combined fiom each dog in ex~eriments4 and 5 are shown in Tables
ViX,VIfI, IX. Dog C consistently had lower Sm, percent of intact acrosomes and percent
of morphologically normal sperm compared to the other dogs for fresh, cooled and postthaw treatments. Dog E, while not having the highest fresh sperm characteristic values
compared to the other dogs, did have the highest sperm characteristic values post-thaw.
Table VIL Sperm Motility Index (mean I: SEM) for fiesh, cooled, and pst-thaw canine
spem fiom each dog used in experiments 4 and 5.
(5)
D
(3
E
(3)
F
(3)
G
(3 )
H
(3
83.5"
76.0"
83.0"
82Sa
78.3"
81.6"
83.3a
2.5
3.6
2.0
1 -4
4 -4
3 -3
1.6
80.0"
77.8"
72.gb
81.1"
79.6"
79.0"
81.1"
81.1"
0.8
0.7
1.2
0.7
0.7
1.7
1.1
0.8
58Xb
49. l b
1.8
2.4
(8)
B
(8)
83.0"
1.5
A
ejacdated
C
(4
Fresh
SEM
Cooled
SEM
Post- thaw
SEM
57.6ab
2.6
6 0 . 4 ~ ~ 68.5"
3.5
1.7
56Sb
1.2
61.0"~ 62.gab
1.O
1.4
Different letters within each row indicate significant difference @'<O.OS).
Table VIII. Percent of intact acrosomes (mean + SEMj for fresh, cooled, and post-thaw
canine sperm from each dog used in experiments 4 and 5 .
dog (n)
Fresh
SEM
Cooled
SEM
Post-thaw
SEM
73.6"
80.2"
67.3"
71.5"
66.1"
70.3"
66.6"
74.2"
4.6
3.2
4.4
3.2
4 .O
1.8
5.5
2.8
6 1. gab
1.7
70.6"~ 57.2"b
2.1
2.4
56.gab
2.7
32.0"~ 3 ~ . 6 " ~ 18.6~ '21.9~
1.8
3.6
2.4
2.3
46.2b
3.3
35.4"b
2.1
Different letters within each row indicate significant difference (W0.05).
59.0"~ ~ 6 . 4 " ~68.4"
1.8
1.9
1.2
~ 5 . 2 ~3 0 . 3 ~ ~ 36.7"
1.23
3.85
1.2
Table IX.Percent of morphologicdly normal spexm (mean ISEM) for fiesh, cooled, and
pst-thaw canine sarnpies fiom each dog used in experiments 4 and 5.
A
B
C
D
E
F
G
H
ejacuiated
(8)
(8)
(5)
(5)
(3)
(3
(3)
(3
dog (n)
Fresh 81.0" 7 ~ . 7 " ~~ 9 . 7 ~75.6"
72.2"b 73.3"b 74.8ab 73.8"
SEM
Cooled
SEM
Post-thaw
SEM
qb
2.0
1.5
74.3ab 66.73b
4.3
2-6
50Sb
69Sab
2.9
3 -5
70.6"~ 71Sab
4.1
0.9
75.3"
69.6ab
1.9
1.5
3 -8
1.7
I .O
1.2
0.8
0.8
67.0"
62.2"
52.6"
61.9"
69.3"
57.9"
61.8"
63.6"
1.7
0.7
3.9
1.8
1.2
1.2
1.7
1.9
Different letters within each row indicate si-nificant dserence (P<0.05).
DISCUSSION
Short-term storage of canine sperm is often required when AI cannot occur irnmediately
afier semen collection. In domestic dogs, a selected stud dog rnay be geographically
distant to the bitch requiring that semen be shipped ovemight rather than undertake the
expense and stress of transporting animals. UsualIy for prolonged semen transport the
semen is extended and cooled to 5 or 0°C and then warmed upon arrivai for AI, or directly
inseminated into the bitch Extended and cooled canine spem up to 2 days has been
shown to have supenor motility, morphology, acrosomal status, and longevity (after
rewarming and being held at 39OC) compared to the same samples afier freeze-thaw
(England and Ponzio, 1996). Not only does cooled canine semen appear to be less
darnaged compared to s p e m subjected to the cryopreservation process, but it a h
generally yields higher pregnancy rates afier Ai compared to frozen-thawed spem when
the same method of insemination is used (Linde-Forsberg and Forsberg, 1993; Linde-
I is usually surgical
Forsberg, 1995; Linde-Forsberg, 2000). In exotic canid species, A
(compared to intravagiml or transcervical insemination in dornestic dogs) and requires
anesthetizing the animal and surgical preparation. This ofien can result in a delay of a few
hours between semen collection and surgical AI. To enable the same transport for nonfrozen exotic canid sperm over long distances the appropriate storage and transport
conditions must be determined.
In experirnent 1, the type of sugar contained within the estender had no effect on
domestic dog sperm longevity; however, a holding temperature of -22°C (RT) resulted in
the best maintenance of motility compared to O and 37°C. Room temperature also has
65
been found to best maintain red wolf sperm motiIity, morphology, and acrosomal status
compared to O and 37"C,both fresh and afier holding for l h at these temperatures
(Goodrowe et al., 2000). Convenely, Bouchard et al. (1990) found using a differem
extender, non-fat dried milk-glucose, that storage at 4OC best preserved motility over 22
and 3j°C, respectively. Furthermore, sperm motility preservation at RT negates the
convention that by reducing the temperature to 5"C, or lower, sperm metabolisrn is
reduced and therefore sperm longevity prolonged (Wishart, 1984). In dog sperm stored at
5°C (Oeitlé, 1986; Kurni-Diaka and Badtram, 1994; Rota, 1W8), and in sperm cooled to
5°C in this study, plasma membrane and acrosomal darnage was observed. No study has
cornparatively investigated sperm characteristics at.different holding temperatures.
Therefore, a more detailed investigation is neecied to determine the canine sperm storage
temperature which best maintains spem qualitv, as well as motility. While cooling has
been s h o w to decrease such sperm parameters as rnorpho!ogy, acrosomal and
capacitation status (Oettlé, 1986; Hay et al., 1997b; Rota, 1998)- it is not h o w n if these
sperm parameters are altered when canine sperm are held at RT.
Despite no detected differences in SMI benveen sugars added to canine semen extender,
glucose appeared to be the preferred energy source in TALP solutions cornpared to other
sugars, includins standard TALP energy sources of pyruvate and lactate. Moreover,
standard (Tris-glucose) extender consistently maintained the highest motility over al1
other extender and TALP solutions, suggesting that a combination of glucose and EY best
supports maintenance of sperm motility. Benefits of EY and glucose on canine spenn
motility are supported by a recent prelirninary report (Verstegen et ai., 2000) which
66
observed that addition of egg yolk to commercial extenders significantly improved
motility parameters of canine sperm held at 4"C, with sperm motility best maintained in a
laboratory prepared Tris-glucose extender. Verstegen et al., (2000) also demonstrated that
medium exchange and energy substrate supplementation re-stimulated sperm motility
fiom 15 to 60% and progressive rnotility from O to 22% with medium change after 19
days. This medium change returned what was a continually decreasing glucose content
and pH to the original levels. The medium, Tris-citric acid-glucose extender (Iguer-ouada
and Verstegen, 200 l), is similar to the standard extender used in expenments 1 and 2
except the pH (6.8) and osmolarity (364 mOsm) are quite different, which may suggest
that the ideal extender osmolarity and/or pH for canine short-terni sperm storage requires
further investigation. Nevertheless, these data demonstrate the importance of the energy
source in storage of canine sperm and suggest, along with the preliminary findings here,
that both glucose and EY are beneficial to the storage of canine sperm. Holt et al. (1988),
demonstrated that EY prevented the looping of ram s p e m tails, by the EY lipids
interacting with the plasma membrane surface, therefore protecting rnotility. A sirnilar
interaction of EY and plasma membranes resulting in maintenance of dog sperm motility
rnay also have occured in the present study, accounting for the greater motility observed
in Tris-glucose extender.
As in previous experiments (Oettlé, 1986; Hay et al., 1997b; Rota 1998), the most
extensive damage to canine sperrn during cryopreservation \vas acrosornal. Electron
microscopy of fiozen-thawed canine sperm fias shown rarefiaction of the acrosome and
loss of acrosomal electron-dense material (Rodriguez-Martinez et al., 1993). While it
67
seems some acrosomal darnage occurs as a result of cooluig to 5°C- the majority of
acrosomal damage occurred afier fieezïng and thawing. What is not ciear however, is
whether the acrosomat darnage observed after freezing and thawing is a result of
undetected injury during cooling which only becomes apparent pst-thaw or is a direct
result of fieezing and thawing.
There is increasing evidence that during sperm cooling, a lipid phase transition in the
plasma membrane bilayer occurs, possibly causing the acrosomal and capacitation-like
damage observed after cooling and post-thaw (Hammersdt, 1990; Parks and Graham,
1992). At B ° C , both bu11 and boar sperm head and tail plasma membranes exhibited a
random distribution of intramembranous particles observed through freeze-fracture
e~ectronmicroscopy(de Leeuw et al., 1990). Subsequent exposure to 0°C resulted in a
redistribution of these intramembranous particles in the acrosomal, post-acrosomal, and
tail regions of the plasma membrane which was aîtributed to lateral phase separation of
membrane lipids. Neither dilution of sperm in extender nor cooling more slowly prevented
the cold induced redistribution of the intramembranous particles. Rewanning the buIl and
boar sperm to 3 8°C did reverse the membrane redistribution (de Leeuw et al., 1990).
However, isolated head plasma membranes from cryopreserved boar sperm do not
undergo the fluidity changes observed in fresh or even cooled sperm (Buhr et al., 1989),
suggesting these ultrastructure changes are not fully reversible. In fact, cryopreservation
does affect lipid composition and organization of spenn plasma membranes in rams
(Hinkovska-Galcheva et al., 1989; Bailey et al., 2000). Therefore, it is entirely IikeIy that
reorganization and ultrastruchual damage of plasma membranes due to cooling
predisposes sperm to gross rnorphologic defects, such as abnomal acrosomes.
Moreover, integral membrane proteins are clustered by lipid phase separation and if
these protein-lipid associations are altered due to cooling- and cryopreservation-induced
membrane reorganization, protein function could be altered; especially proteins which
undergo structural modification to function, such as ion charmel proteins (Watson, 2000).
Increased intracellular calcium has been observed after sperm cooling and may be a result
of cooling-induced lipid phase alteration of calcium ion charnel proteins. Since an increase
in intracellular calcium is associated with initiation of capacitation, this calcium increase
after cooling could be contributing to the capacitation-Iike characteristics observed in
cooled spenn (Bailey and Buhr, 1994; Bailey et al., 2000; Watson, 2000). Similady, the
obsewed increase in sperm membrane permeability afier cooling rnay be due to effects of
cooling on specific protein charnels (Bailey et al., 2000; Watson, 2000). Therefore,
alteration to sperm membranes as a resuIt of cooiing could be causing sperrn damage either
directly or through predisposition of the sperm to damage, culrninating in the observed
increase in cryopreserved canine sperm that have undergone capacitation or exhibit
acrosomal and morphological damage.
Cooling to O°C appeared to have a detrimental effect on sperm motility and acrosornal
status compared to cooling to 5°C regardless of cooling rate. While motility of sperm
cooled slowly to 0°C was found to be statistically lower than fresh and other cooled
spem samples, motility was subjectively estimated and there fore such a small observed
diffirence may seem too minimal to be considered biologica!ly significant. However, there
69
was a clear trend for sperm motility of each dog to be lower after slow cooling to O°C
than to YC. The percent of intact acrosomes significantly decreased after cooling, dong
with a corresponding increase in the percent of partialfy intact acrosomes, as was
previously observed (Oettlé, 1986; Hay, 1996). Interestingly, the decrease in intact
acrosomes coincided wïth the amount of time spent cooling; the longer the time to cool,
the fürîher decrease in the percent of intact acrosomes observed, with slow cooling to 0°C
(-5 h) having significantly smaller numbers of intact acrosomes than al1 other cooled
sarnples. Hay ( 1996) observed a sirnilar time-related phenornenon, with canine sperm
cooled at the same rate for 3 h vs. 30 min demonstrating significantly lower numbers of
intact acrosomes. Similarly, a time related trend also was observed in the percent of
rnorphologically normal s p e m after cooling. This decrease in morphologically normal
sperm afier cooling was mirrored by an increase in the percent of bent tail tips, with
sperm slow cooled to O°C having significantly greater proportions of bent tips compared
to fresh sperm. Yubi et al., (1987) also found the number of abnormal spenn to increase
after pre-freeze cooling but did not report the specific abnormalities which increased.
Therefore, this is the first study to report that cooling increases the percent of bent tail
tips in canine sperm. This increase in acrosomal damage and percentage of sperm with
bent tails is likely a result of cooling induced membrane transitions in the plasma and
acrosomal membranes (c'cold shock"; Parks and Graham, 1992). Perhaps the prolonged
cooling times provided an environment in which damaging membrane redistribution could
occur, resulting in the observed increase in the s p e m population with partial acrosomes
and brnr tails tips. Consequently, short-term storaçe of canins sperm below 5°C could be
70
more detrimental to the sperrn than the observed rnotility would indicate, M e r
supporting storage at RT. However, characterisation of canine spenn over tirne at RT is
still required,
Freezing and thawing caused the majority of sperm with bent tips to transfom to
fully bent tails, as these were the only morphologie categories to alter post-thaw. While
the percentage of morphologically normal sperm in experiment 5 did decrease slightly
post-thaw, this was coincident with an increase in the percentage of bent tails. This
increase in bent tails could be more likely amiburable to cooling-induced sperm tail
plasma membrane changes only becoming apparent afier fieezing and thawing, given the
observed results in experiment 4. Furthemore, it appears that these cooling-induced
sperm tail plasma membrane changes are irreversible afier fieezing and thawing.
In experiment 4, both the percentage of canine oocytes penetrated and the number of
s p e d o v a appeared to increase after cooling, but were not significantly different from
fresh values. This trend for sperm zona penetration to increase afier cooling may indicate
a cooling induced spenn alteration, such as sperm capacitation, that promoted the in vitro
sperm/oocyte interactions. To detennine if capacitation \vas a possible reason for the
increased zona penetration obsewed in experiment 4, a CTC assay was incorporated into
the sperm evaluations in experiment 5 . Yet, the same increase in zona penetration by
cooled sperm was not obsewed in experiment 5 and the percentage of sperm capacitated
did not increase afier pre-freeze cooling (discussed later). Post-thaw zona penetration was
drastically reduced from fresh and cooled values, which is consistent with previous canine
zona penetration (Hay, 1996) and zona bindins (Strom Holst, 1999) assays. Since the
71
acrosome generally is vital for sperm binding, penetration, and fertilization of the oocyte,
it rnay be that the poor post-thaw acrosomal statu contnbuted to the reduced post-thaw
zona penetration. The reduced longevity of fiozen-thawed canine sperm at 37°C (Olar et
al., 1989; Morton and Bruce, 1989; England, 1992) rnay also have conmbuted to the
reduced zona penetration. Frozen-thawed canine sperm incubated at 37°C take 2 h to
reacn peak percent capacitation and to show an increase in the percentage of acrosome
reacted sperm at 2 h cornpared to 4 h for fiesh sperm (Rota, 1998). Frozen-thawed ram
sperm also capacitate more quickiy than fiesh (Perez et al., 1996). Earlier capacitation
should lead to earlier oocyte penetration; however, in the b a r , the nurnber of capacitated
and acrosome reacted sperm increased after the Erst hour of incubation post-thaw, but
actual oocyte penetration by the frozen-thawed sperm required 4-6 h (Wang et al., 1995).
Whether canine oocyte penetration occurs earlier with fiozen-thawed vs. fresh sperm, in
addition to the possible influence of accelerated capacitation on oocyte penetrating
capability in vitro remains to be investigated and would require induction of fiesh and
frozen-thawed sperm capacitation prior to spedoocyte incubation, compared to noncapacitation induced sperm, and assessrnent of sperm binding and penetration at r e g d a
time intervals (e-g.every 2 h).
hterestingly, the percent of capacitated sperm did not differ among fresh, cooled or
post-thaw values. The lack of change in the percent of capacitated spem seems
inconsistent with reports of capacitation-like changes in cooled dog and other domestic
species sperm observed by CTC staining (Cornier et al., 2 997; Fuller and Whittingham,
1997; Gillian et al., 1907; Maxwell and Johnson, 1997; Rota, 1998; Bailey et al., 2000).
72
Kowever, ultrastructure studies of both hurnan (BartheIemy et al., 1990) and bu11 sperm
(Jones and Stewart, 1979) demonstrated that membrane alterations occur during
dilution/extension of s p e m comparable to those during cryopresemation. Recently,
Schembri et al. (2000) observed in equine sperin assessed by the CTC assay, that the
largest increase in capacitated sperm compared to fiesh spenn was afier seminal plasma
removal and dilution with an EY-extender, followed by only a slight increase after cooling,
and a moderately larger increase post-thaw. A higher percentage of human s p e m held at
5°C in TES/Tris-EY were acrosome-reacted compared to sperm in standard non-EY or in
the same TESiTris medium without EY (Bielfeld et al., 1990). For experirnent 5, seminal
plasma initially was removed fiom al1 samples and fresh s p e m were held at RT in €Y
extender until al1 samples were cooled and then assessed together. Therefore, perhaps like
in equine and hurnan, seminal plasma removal and dilution with EY extender stimulated
capacitation of sperrn, which would also account for the higher percentage of capacitated
sperm observed in fresh sperm in experirnent 5 and a previous report (Rota, 1998) of
fiesh and cooled canine sperm, where sperm were held in Tris-fructose medium (no EY).
Nevertheless, Rota (1998) did demonstrate an increase in the percent of capacitated
sperm afier cooling to 5°C for 24 h compared to fresh sperrn in non-EY media.
The mechanism of EY protection against cold shock is still poorly understood
(Hammerstedt et al., 1990). Conceivably EY lipid interaction wïth the sperm tail plasma
membrane is beneficial in preventing tail bending, while its interaction with diEerent
sperm head plasma membrane domains has yet unknown detrimental effects leading to
capacitation-Iike staining observed after extender dilution. Such an alteration in the sperm
73
head plasma membrane could then favour in-vitro fertilization conditions resulting in the
non-significant increase seen in the number of sperrn penetratinç homologous oocytes in
esperiment 4. Fwther investigation on the role of EY andior coolinç is necessaxy to
understand the capacitation-like effects obsewed in extended and cooled sperm.
Freezing and banking of sperm enables semen fiom both domestic and exotic canids to
be stored and shipped to different institutions and/or internationally, and also allows the
use of semen long after animal death. These advantages are particdarly important for
genetic management of endangered captive breeding populations as mentioned in the
introduction. Sperm must overcome many barriers within the female reproductive tract
afier insemination before reaching and finally fertilizing the oocyte. One such bamer is
maintenance of sperm motility at body (37OC) temperature (Doak et al., 1967). Longevity
of frozen-thawed canine sperm rnotility at -37OC is known to be reduced compared to
fresh sperm (Olar et al., 1989; Morton and Bruce, 1989; England, 1992). Recently, the
addition of a commercial detergent, STM Equex paste, significantly improved the
longevity of post-thaw caaine sperm motility at 37OC compared to samples fiozen in the
sarne extender without Equex paste (Nothling et al., 1995; Rota et al., 1997). These
studies demonstrated a benefit of Equex addition to canine sperm extenders when freezing
in straws. Therefore a prelirninary study was conducted to investigate the effect of Equex
addition on post-thaw canine sperm quality when frozen in pellets. While there was no
obvious benefit in canine sperm quality immediately post-thaw or after 3 h incubation at
37"C, addition of 1% Equex paste to standard extender did not have any detrimental
effects on sperm quality. Perhaps M e r replications of pelleted sperm frozen with
74
Equex is needed in order to detect any possible benefit of pre-fieeze Eques addition on
post-thaw dog sperm quality. Nevenheless, fuither investigation of Eques paste addition
to canine sperm extender when fieezing in pellets is warranted and couid include,
exarnining different concen~ationsof Equex, earlier Equex addition in the cooling process,
or a longer holding/equiiibration time with Equex present before Freezing.
At firsi glance, the cooled and post-thaw sperm quality in experiment 5 appeared
-greater than in experirnrnt 4. However, direct cornparison of the same doçs (A and D),
cooling rates and glycerol addition method (experiment 4: F5 and S5; expenment 5 fast
and slow cooied with glycerol addition over 3 min) between experiments 4 and 5
demonstrated no difference in fresh, cooled or post-thaw semen quality between the two
experiments. The apparent differences may be a reflection of more animafs with less
replicates in eïpenment 5 than experiment 4, and therefore the averaçes would be less
likely to be affected by an individual animal. Between dog differences were detected in
fresh, cooled and post thaw semen quality for both experiments. It would appear that
semen sampIes £tom particular dogs were more susceptible to fieezing damage than
others. Semen sarnples are heterogeneous populations of sperrn each having varying
resistance to cryopreservation stresses (Bailey et al., 2000; Watson, 2000). Perhaps
certain characteristics of sperm membrane structure are genetically determined,
predisposing these spem sub-populations toward survival under cryopreservation stress,
thus accounting for the classification of individuals in other species as 'good freezers' or
'bad freezers' (Watson, 2000) ,including the fox (Fougner, 1989). Subsequent
investigations with more dogs and replicates would be required to corroborate these
75
observed variations between dogs. Moreover, increased number of doçs and replicates
would allow for greater detection of significance. That is, large apparent differences,
especially among doçs, found not significant in these experiments rnay actually be found
sig-iificant with increased sarnpling, assuming low withîn do- variation. For example, a
greater than 20% difference among cooled spem with intact acrosomes was found not to
be significant; however, with increased sample size and replicates this may show sorne
signifiant differences.
While sperm cryopreservation and AI with frozen sperm has relative success in
domestic dogs (Fontbonne, 1993; Nothling et al., 1993; Silva et al. 1996; Linde-Forsberg,
1999) this is not the case with exotic canid species (Goodrowe et al., 3000; Fartstad et al.,
2000). Linde-Fonberg, (2000) and Thornassen et al. (2000) reported thar much of the AI
success using frozen domestic doç sperm cornes fkom the ability to inseminate the bitch
several times during esmis. This generally is not possible for wildlife species, and
therefore post-thaw semen quality must be maximized when the bitches are inseminated
only once. Through methodical investigation of domestic dog sperm cxyopreservation it is
hoped the ctyopreservation success of wild canjds can be improved. Based on the results
here it is suggested that canine sperm be held at RT in a Tris-glucose extender or TALP
containing glucose. However, further investigation into the interaction of E Y with sperm
plasma membranes is required. For cryopreservation of dog sperm cooling below 5°C is
not recornmended and a cooling rate of approximately 0.6OC/min (fast) wlth glycerol
addition once 5°C is reached would be adequate. Pre-freeze cooling of dog spem was
shown to cause both acrosomal and morphological damage. It is M e r suggested that
76
prevention of this cooling-induced sperm darnage requires firrther understanding and in
depth investigation of cooling-induced spem membrane alterations. Electron microscopy
investigation of cooling-induced changes and membrane interaction of egg yolk would be
an important first step.
1. Short tenn storage of domestic dog spexm was best achieved at RT in standard extender
or TALP solution containing glucose.
2.Addition of 1% Equex paste prior to freezing had no detrimental effect on post-thaw
sperm quality .
3. Pre-freeze cooling of semen to 5°C compared to 0°C resulted in better cooled sperrn
quality. However, final coo!ing temperature had no effect on pst-thaw sperm qualit)..
4. Neither cooling rate (fast vs. slow) or method of glycerol addition (over 3 min at 5°C
vs. throughout cooling) had any influence on pst-thaw sperm quality.
5. Both acrosomal and rnorphologic damage occurred to sperm d e r cooling. Frezzing and
thawing dramatically decreased motility, homologous zona penetration and 1 ncreased
acrosornal damage, with either no or slight decrease in the percentage of
morphologically normal spem.
6. Cooling resulted in an increase in sperm with bent tail tips, which shified to sperm
with fuily bent tails after fieezing and thawing.
7. Capacitation of sperm \vas not significantly increased by cooling or freezing and
thawing, but may have been increased after extender addition.
8. Male to male differences were detected in the quality of fresh, cooted, and post-thaw
spenn.
Almid T. Johnson LA. Effects of glycerol concentration. equiiibration tirne and
temperature of glycerol addition on post-thaw viability of boar spermatozoa fiozen in
straws. J Anim Sci 2 988; 66: 2899-2905.
Andersen K. Fertility of frozcn dog semen. Acta Vet Scand 1972: 13: 128-1 30.
Bailey K, Bilodeau JF, Cormier N. Semen cryopreservation in dornestic animals: A
damaging and capacitating phenomenon. J Androl2000: 21 : 1-7.
Bailey JL and Buhr MM. Ciyoprescnration aiters the Ca" flux of bovine spermatozoaC a .J A n h Sci 1993; 71: 45-5 1.
Battista M. Parks J, Concannon P. Canine sperm post-thaw survival following freezing in
straws or pellets using PIPES. lactose, Tris or Test estenders. Anim Reprod Sci 1989: 3:
229-23 1.
Barthelemy C, Royere D, Hammahah S, Lebos C, Tharanne MJ? Lansac J. LrItrastructural
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Bielfeld P, Jeyendran RS, Holmgren WJ, Zaneveld LJD.Effect of egg yolk medium on the
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CITES. The Convention on International Trade of Endangered Species of Wild Fauna and
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Concannon PW, Battis- M. Canine semen freezing and artificial insemination. In: Kirk
RW (eds.) Current Veterinary Therapy 10th edn. 1989: 1247-1258.
Cormier N. Sirard MA. Bailey JL. Premature capacitation of bovine spermatozoa is
initizited by cryopreservation. J ,hdroI 1997; 18: 461-468.
de Leeuw FE, Chen HC, Colenbrander B. Verkleij .45. Cold-induced ultrastructural
changes in bu11 and boar s p e m plasma membranes. CryobioIogy 1990; 27: 171-1 83.
Do& RL. HaIl A. Dale HE. Longevity of spermatozoa in the reproductive tract of the
bitch. J Reprod Fertil 1967: 13: 51-58.
Dobrinski 1, Lulai C, Barth AD, Post K- Effect of four different extenders and three
different freezing rates on post-thaw viability of dog semen. J Reprod Fertil 1993:47
(Suppl): 29 1-296.
Dragileva E, Rubinstein S1 Breitbart H. Intmcellular Ca2+-Mg2+-ATPase replates
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APPEh?)IX 1
Media Recipes
Glucose
Tris
Citric Acid (anhydrous)
Egg yolkb (EY)
Penicillin
Streptomycin
50
200
60
20% (v/v)
1000 ICT/mL
1000 u&lL
I A U
Componentsa
Spenn TALP
IVF TALP
(mM)
NaC1
KCI
NaH2P04
KH2P04
CaClz
MgCl2
NaHC03
Na-lactate (60% syrup)
Na pyruvate
HEPES
Glucose
114.0
3.16
0.35
-----
2.0
0.5
25.0
10.0
1.O
-----
-----
Canine Capaciation
CCM (mM)
83 -49
4.78
-----
1-19
1.71
-----
37.6 1
2 1.55
0.25
-----
2.78
for 100 mL
Gentamicin (50 mg/mL)
K-Penicillin G
BSA FrV (3 5% solution)
BSA FrV
Heparin (2mghn.L)
PH
Osmolality (mOsm)
" Al1 components were obtained fiom Sigma Chernicals (Oakville ON), except Gentarnicin
(Canadian Life Teçhnology; Mississauga ON).
and Penicillin~Streptomycin
Egg yolk was obtained from fieshly laid white leg hom chickens (held in the Toronto
Zoo Health unit). Cleaned eggs were cracked in haif and egç white discarded. The
remainder of egg white, which was still adhered to the yolk, was removed by siowly
moving the yolk around a 12cm diameter filter paper, (Whittman ff3;Fisher Scientific,
Unionville ON) careful to keep the yolk intact. The yolk was then punctured with a
sterile needle and the intemal egg yolk extracted.
Mahi and Yanagimachi, 1978
APPENDIX 2
ChIorotetracycline(CTC) Stain Solutions
. .
Fixative B u E r
Tris-HC1
12.11 g
ddH20
100 nL
(double de-ionized water)
Paraformaldehyde
Fixative buffer
Tris
NaCI
d m 0
CTC
Cysteine
CTC Buffer
Standard Extender with:
glucose
fi-uctose
sucrose
lactose
50 m M pyruvateAactate
22.6 m M pyruvate/lactate
TALP with:
pyruvate/lactate
glucose 50 mM
23.6 mM
fructose 50 mM
22.6 mM
sucrose 50 mM
22.6 mM
Osmolarity only read for media used in experiment 2
APPENDIX 4
Table A. Sperm rnotility index (SM) means for canine sperm after dilution in 10 different
media for 24 h at room temperature.
Media
Meansa
SEM
simificanceb
Standard Extender
73.7
3.1
A
Glucose TALP 50 uM
2.1
AB
70.6
Glucose TALP 22.5 uM
65.6
3.4
AB
Sp TALP
65.0
3-9
AB
Fructose TALP 22.5 uM
64.4
2.6
AB
P L Extender 22.5 uM
61.9
4.5
ABC
Fructose TALf 50 uM
60.6
4.1
ABC
P L Extender 50 uM
58.1
1.8
BC
Sucrose TALP 22.5 uM
48.7
2.6
CD
Sucrose TALP 50 uM
2.1
D
40.6
" S M means are listed from highest to lowest.
bDifferent leîters indicate significant difference between treatrnents (P~0.05).
Table B. Sperm motility index (SM) means for canine spem after diluted in 10 different
media for 48 h at room temperature.
SEM
si,gificanceb
Media
Meansa
Standard Extender
A
68.0
4.1
Glucose TALP 50 uM
57.5
2.5
AB
Glucose TALP 22.5 uM
57.5
4.3
AB
Fructose TALP 22.5 uM
50.0
6.1
AB
Fructose TALP 50 uM
47.5
4.7
AB
Sp TALP
4 1.2
6.5
ABC
P L Extender 22.5 uM
6.9
BCD
39.3
P L Extender 50 u M
34.4
5.6
BCD
Sucrose TALP 22.5 u M
17.5
8.1
CD
Sucrose TALP 50 uM
12.5
4.9
D
"SM1means are listed fiom highest to lowest.
bifferent letters indicate significant difference between treatments (Pc0-05).