1. health and fitness
2. disease
3. infertility

Mestrado Integrado em Medicina Veterinária
Ciências Veterinárias
Comparison of canine sperm quality under
different temperature storage
Paulo Alexandre Paulos Borges
Orientador:
Professora Doutora Rita Maria Payan Martins Pinto Carreira
Co-orientador:
Professor Alain Fontbonne
UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
VILA REAL, 2011
Not everything that can be counted counts and not
everything that counts can be counted.
Albert Einstein
ii
ABSTRACT
Successful gamete cryopreservation is essential to preserve the genetic pool of several
species. The canine semen is known to possess a certain resilience to chilling procedures, which
has a limited interest for long term gamete preservation; however, when frozen, canine sperm
suffers an important decrease in quality. This feature determined the development of different
extenders aiming the protection of the cell and to increase the success of canine sperm
freezing/thawed procedures. In parallel, development of new techniques allowing a more
objective evaluation of putative sperm fertility were also developed. However, those methods do
not explain why some stud males are “good freezers” while other are “bad” freezers, when both
were selected at start as adequate for freezing. Further, no clear definition exists on the cut-off
values for specific seminal parameters to distinguish between a fertile and infertile stud, although
the analysis of semen is regularly performed to assess the male fertility.
In this study we plan to evaluate cold treatment associated changes on sperm cells by using
two different approaches. For that, semen was collected from 8 dogs frequently used as semen
donors. Freshly ejaculated samples were assess (Control group) before its use for chilling
(Treatment A) or freezing (Treatment B), using routine procedures. For treatment A, three
different times were considered: at 1.5h, at 4h and at 24h of chilling (respectively times 1, 2 and
3). Sperm characteristics were assessed by the convention methods, CASA and hypoosmotic test
(HOST) for motility and movement parameters, velocity fractions, cell morphology and
membrane integrity. Additionally, a molecular approach was tried by the study of heat sock
protein 70 (HSP70) immunoexpression in the spermatozoa. HSP70 has protective effects over
cells under conditions of thermal or proteotoxic stress.
In the study presented here, the more conservative tests confirmed that chilling is not an
aggressive procedure for dog semen in comparison to freezing, provided that the samples have a
minimum acceptable quality at start. In frozen/thawed samples it was found a decrease in sperm
motility and an increase in the percentage of static cells, along with a loss of the sperm
membrane integrity. In parallel, a decrease in HSP intensity of immunostaining and a dislocation
of the immunoreaction towards the flagellum were observed in the frozen samples (treatment B),
while for treatment A no significant changes were found in the pattern of HSP
immunoexpression. The use of molecular marker may reprosent an increase value in the study of
the mechanisms underlaying the cenine sperm sensitivity to cryopreservation.
iii
RESUMO
A criopreservação de gâmetas é essencial à conservação do pool genético das espécies. O
sémen canino é conhecido por ter uma capacidade de resistência à refrigeração aceitável, mas
quando congelado, perde potencial fecundante. Esta sensibilidade do sémen canino à congelação
incentivou o desenvolvimento de vários diluidores que potencializassem o sucesso da técnica,
tendo sido neste processo uma parte integrante o desenvolvimento de métodos relativamente
objectivos para avaliação do potencial fecundante do sémen ou dose seminal. No entanto, estes
métodos não conseguem explicar os motivos subjacentes à perda de fertilidade observada nem
porque alguns machos se apresentam como “bons congeladores” e outros como “maus
congeladores” quando todos eles foram seleccionados como adequados para congelar. E apesar
de a análise de sémen ser rotineiramente usada para avaliar a fertilidade do macho, não está
claramente definido que valores dentro dos parâmetros de sémen distinguem um macho fértil de
um infértil.
Neste trabalho propusemo-nos a avaliar as alterações associadas ao tratamento térmico
pelo frio por dois tipos de metodologia. Assim foram obtidas amostras de sémen de 8 animais
dadores regulares de sémen. As amostras foram analisadas a fresco (grupo controlo) e depois
processadas de forma rotineira para refrigeração (Tratamento A) e congelação (tratamento B).
No grupo de tratamento A foram ainda considerados 3 tempos (tempos 1, 2 e 3, respectivamente
às 1.5h, 4h e 24h de refrigeração). O estudo comparativo do potencial fecundante foi realizado
por intermédio de métodos convencionais, por CASA e pelo teste hipoosmótico(HOST), para a
motilidade e parâmetros de movimento, características de velocidade e integridade funcional da
membrana. Esta avaliação foi complementada pelo estudo de imunoexpressão de HSP70
(proteína de shock térmico 70), uma molécula com efeitos protectores sobre a célula em
situações de stress térmico e proteotóxico,
No trabalho agora apresentado, os testes mais conservadores permitiram confirmar que a
refrigeração não é uma técnica muito agressiva para o espermatozóide canino, comparativamente
à congelação. Associada a esta última foi observado uma perda na motilidade e na velocidade
além de um aumento do nº de células com alteração na função de membrana. Em simultâneo, foi
observado um decréscimo na intensidade de marcação para a HSP70, associada a uma
deslocação da marcação para a cauda do espermatozóide. A utilização de marcadores
moleculares pode revelar-se útil na identificação de mecanismos biológicos subjacentes à
sobrevivência do espermatozóide à congelação.
iv
CONTENTS
ABSTRACT .................................................................................................................................. iii
RESUMO.................................................................................................................................... IV
PART I. ASSESSMENT OF CANINE SEMEN AFTER CHILLING AND FREEZING: A REVIEW .................... 1
1. SEMEN COLLECTION .............................................................................................................. 4
2.1 METHODS OF SEMEN COLLECTION ..................................................................................... 7
2. THE SEMEN ........................................................................................................................... 8
3. THE SEMEN QUALITY ............................................................................................................. 9
3.1 SEMEN ASSESSMENT ....................................................................................................... 10
3.1.1 CLASSICAL METHODOLOGY ....................................................................................... 10
3.1.2 ADVANCED METHODOLOGY ....................................................................................... 15
3.2 SPERM MOTILITY ASSESSMENT ........................................................................................ 16
3.3 HYPO-OSMOTIC SWELLING TEST ...................................................................................... 17
4. HEAT SHOCK ....................................................................................................................... 19
4.1 HEAT SHOCK PROTEINS ................................................................................................... 20
PART II. COMPARISON OF CANINE SPERM QUALITY UNDER DIFFERENT TEMPERATURE
STORAGE: EXPERIMENTS .................................................................................................... 21
1. INTRODUCTION AND AIMS ................................................................................................... 22
2. COMMON MATERIAL AND METHODS .................................................................................... 23
2.1 ANIMALS......................................................................................................................... 23
2.2 SAMPLE PREPARATION .................................................................................................... 23
2.3 EXTENDERS..................................................................................................................... 24
2.4 CHILLING PROCEDURE..................................................................................................... 25
2.5 FREEZING/THAWING PROCEDURES................................................................................... 26
3. EXPERIMENTS...................................................................................................................... 29
3.1 EXPERIMENT 1 - EVALUATION OF DOG SEMEN QUALITY AFTER CHILLING AND FREEZING . 29
3.1.1 GOALS ....................................................................................................................... 29
3.1.2 SPECIFIC METHODS .................................................................................................... 29
3.1.3 STATISTICAL ANALYSIS ............................................................................................. 30
3.1.4 RESULTS .................................................................................................................... 30
3.2 EXPERIMENT 2- IDENTIFICATION OF HSP70 CHANGES IN CHILLED AND FROZEN
SPERM SAMPLES ................................................................................................................... 39
3.2.1 GOALS ....................................................................................................................... 39
3.2.2 MATERIALS ............................................................................................................... 39
3.2.3 METHODS .................................................................................................................. 39
3.2.4 STATISTICAL ANALYSIS ............................................................................................. 41
3.2.5 RESULTS .................................................................................................................... 41
4.DISCUSSION ......................................................................................................................... 45
5.FINAL CONSIDERATIONS ....................................................................................................... 51
REFERENCES ........................................................................................................................... 52
ANNEXES ................................................................................................................................ 59
ANNEX 1 ................................................................................................................................. 60
ANNEX 2 ................................................................................................................................. 61
v
LIST OF FIGURES
Figure 1 – Morphological structure of the canine spermatozoon .................................................. 9
Figure 2 – Sperm cell abnormalities (vetmed.lsu.edu) ............................................................... 14
Figure 3 - Semen collection material ......................................................................................... 23
Figure 4 – For preservation, either for chilling or freezing, the collected semen
is centrifuged to obtain a pellet with spermatozoa. .................................................... 24
Figure 5 – Sequential procedure of egg yolk addition ................................................................ 25
Figure 6 – From left to right, removal of the supernatant, addition of half of the
extender; addition of the second half of the extender ................................................ 25
Figure 7 – Process of storage in the refrigerator ......................................................................... 26
Figure 8 – From left to right, sequential process of supernatant removal,
extender addition and storage in the refrigerator ....................................................... 26
Figure 9 - Addition of the second extender ................................................................................ 26
Figure 10 - On the left image, equipment for casing the straws; on the right,
procedure of filling up the straws. ............................................................................ 27
Figure 11 - From left to right, procedure of casing the straws..................................................... 27
Figure 12 - From left to right, procedure with the liquid nitrogen ............................................... 28
Figure 13 - Storage of the straws ............................................................................................... 28
Figure 14 - Concentration determination with a spectrophotometer and pH analysis .................. 30
Figure 15 – Immunocytochemical evidence of HSP70 in the spermatozoa head
from the control group spermatozoa . ....................................................................... 42
Figure 16 – Immunocytochemical evidence of HSP70 in chilled spermatozoa .......................... 43
Figure 17 - Immunocytochemical staining for HSP70 in frozen/thawed samples ....................... 43
LIST OF GRAPHS
Graph 1 – Individual variation of the total motility with treatment ............................................. 32
Graph 2 – Individual variation of the progressive motility with treatment . ................................ 33
Graph 3 - Representation of the sperm total motility (in the left) and progressive
motility (on the right) for the group of samples according to the treatment................ 33
Graph 4 – Individual variation of the velocity of the spermatozoa with treatment . ..................... 36
Graph 5 - Individual variation of the hypo-osmotic swelling test (HOST) of the
spermatozoa with treatment. ..................................................................................... 38
Graph 6 – Effect of the cryopreservation process on the coiling of spermatozoa......................... 38
Graph 7 – Distribution of HSP 70 intensity in the spermatozoa’s head ....................................... 44
Graph 8 – Distribution of HSP 70 in the spermatozoa’s tail ....................................................... 44
vi
LIST OF TABLES
Table 1 - The most common causes leading to semen collection in dogs ...................................... 2
Table 2- CASA parameters....................................................................................................... 17
Table 3 – Composition of the extenders used for chilling and freezing/thawing procedures ........ 24
Table 4 – General characterization of the individual semen samples used in this study. .............. 31
Table 5 – Spermatozoa morphology for the freshly ejaculated samples ..................................... 31
Table 6 - Total and progressive motility .................................................................................... 32
Table 7 - Velocity of spermatozoa movement ............................................................................ 35
Table 8 – Hypo-osmotic swelling test ........................................................................................ 37
Table 9 – Immunocytochemical classification of the HSP70 in the spermatozoa ........................ 45
Table on Annex 1 – Total and progressive motility and consequent treatment losses .................. 60
Table on Annex 2 - CASA parameters for sperm cells under cold treatments . ........................... 61
vii
List of abbreviations, symbols and units
% - percent
® - registered brand
AI – artificial insemination
ALH - amplitude of lateral head displacement
ARTs - artificial reproductive techniques
AV – artificial vagina
BCF - beat cross frequency
CA – California
CASA – computerized assisted semen analysis
CERCA - Centre d'Étude en Reproduction des Carnivores
C-FDA - 6-carboxyfluorescein diacetate
DAB - 3,3’-diaminobenzidine tetrahidrocloret
DHS – dihydrostreptomycin
ENVA - École Nationale Vétérinaire d’Alfort
EthD – 1-ethidium homodimer
h - hour
HOST – hypo osmotic swelling test
HSP - heat shock proteins
Hz – hertz
IL - Illianois
IVF – in vitro fertilization
KG – kilogram
L – liter
LIN - linearity of sperm movement
ML – mililiter
ng – nanogram
p – significance level
PBS – phosphate-buffered saline
PGF2α – prostaglandin F2α
PM – progressive motility
rpm – rotations per minute
s - second
STR – straightness
SP – seminal plasma
TM – total motility
UK – United Kingdom
USA – United States of America
UTAD - University of Trás-os-Montes and Alto Douro
VAP – velocity average pathway
VCL – curvilinear velocity
VSL – velocity straight line
WHO – World Health Organization
WI – Wisconsin
x - average
ZBA – zona pellucida binding assay
ZP - zona pellucida
μm – micrometer
μl – microliter
viii
ACKNOWLEDGEMENTS
To both, the University of Trás-os-Montes and Alto Douro (UTAD) and the École
Nationale Vétérinaire d’Alfort (ENVA) for all the support given, which allowed the development
of this study.
To Professor Rita Payan Carreira, my coordinator, I recognize all the dedication applied,
the long hours of work, the patience, the friendship, all the wise advices given and the
opportunities provided, along with the priceless knowledge she transmitted me throughout my,
still short, career.
To Doctor Alain Fontbonne, who provided me everything required for the elaboration of
my work, along with very important knowledge and opportunities to evolve in the area of small
animal reproduction. Within the ENVA and specially the CERCA (Centre d'Étude en
Reproduction des Carnivores), I thank Fernando Mir, Emeline Leblond, Cindy Maenhoudt,
Natalia Santos and Emmanuel Fontaine for all the attention, knowledge, advices and tender that
they offered me during my internship. Also I would like to thank Karine Reynaud and all the
laboratory staff that received me very well and helped with everything I needed.
To Professor Maria dos Anjos Pires I leave a special acknowledgment, for the special
attention and care that she always showed towards me, being available every instant needed and
offering all the help and means required.
To the Histology and Pathological Anatomy Laboratory of UTAD, Professor Anabela
Alves and Professor Maria dos Anjos Pires as its directors along with its technician and staff,
Mrs. Lígia Lourenço, Ms. Ana and Mrs. Glória, I thank all the help provided.
To my laboratory partners, who were of extreme importance, Inês Santana and Inês
Carvalho, I leave a very special thanks for making me embrace the work every day with a smile,
for all the teachings, attention, reprimands and specially the friendship provided.
ix
To all my great friends in Vila Real, Badano, Luis Moreira, Rapper, Verguinhas, Diana,
Tatiana, Afilhada, Francisco, Rui, Marta, Ana Margarida, Xami, Zero, João Pires, Magda and
Ana Andrade for sharing and giving me so much throughout this so long, though short years.
To Professor Wojtek Nizanski who induced and enhanced in me this reproduction
thematic and to Natalia Mikolajewska who has been a great support and a very good friend in all
the occasions, I thank all the care and preoccupation shown and most importantly the friendship.
To Nuno Escudeiro, Vitor Lopes and Ana Lúcia a special thanks for telling me what I
need to hear, no matter what, making them true very best friends.
The last, but definitely not the least, I would like to thank my family, specially my
mother, my father and my little sister, with whom I shared all the tears and joys along my path
and who made me the person who I am today most importantly, because without them this would
mean nothing.
Muito Obrigado!
x
PART I. ASSESSMENT OF CANINE SEMEN AFTER CHILLING AND
FREEZING: A REVIEW
1
FUNDAMENTALS
The collection of semen from a male dog may represent an extended procedure during
reproductive examination technique. Most frequently, it is used for three major purposes: artificial
insemination (AI), semen cryopreservation or diagnostic purposes (Kustritz, 2007). Artificial
insemination may be needed in the eventuality of vaginal anomalies of the bitch (such as vaginal septum,
vaginal hyperplasia or ptosis, narrowed vagina or vaginal-vestibular stricture), whenever a stud dog is
required to produce a seminal dose for breeding at distance or due to other sort of problems, such as
behavioral issues between the male and the female (Kutzler, 2005). Canine semen may also be collected
in a regular basis for cryopreservation, allowing dog owners to preserve the genetics of their male
specimens, thus enabling breeding, even, when the male is no longer able to copulate, if it becomes
infertile or if it is physically absent due to the distance or to its death (Johnston et al., 2001; Kutzler,
2005). The most common factors that usually lead to semen collection are described in Table 1.
Table 1 - The most common causes leading to semen collection in dogs
MOST COMMON CAUSES FOR SEMEN COLLECTION
FOR MEDICAL OR OTHER REASONS
ASSOCIATED WITH THE REPRODUCTIVE ACTIVITY
- Breeding soundness examination
- Evaluation of abnormal clinical signs (i.e.
prepuce discharge)
- Males kept from copulation for several years
- History of infertile breedings
- Following the treatment of different
reproductive tract diseases
- AI procedures with fresh or preserved semen
- For research
- Behavioral issues
- Female opposition to the mounting
Nowadays, semen cryopreservation and artificial insemination are two of the major
biotechnological techniques currently applied in animal breeding (Foote, 2002). Its large-scale
introduction in cattle breeding around the 1940’s had the purpose of preventing genital infections
transmissible through natural mating (Wilmot, 2007). Semen cryopreservation has also been seen
for a long time as a way to improve the breeding of animals of greater farm importance, as a
contributor to the conservation of endangered species and also as a way to overcome some
aspects of the male infertility (Watson, 2000).
Successful preservation of semen is also important to improve the results of the main
artificial reproductive techniques (ARTs), such as in vitro fertilization and, most commonly, AI
(Luvoni, 2006). Semen cryopreservation eases the transport and exchangeability of genetic
2
material across short or long distances, contributing effectively to widen the restricted gene pool
of some species (Johnston et al., 2001; Kutzler, 2005; Kustritz, 2007). Chilling and freezing can
also turn into reality the creation of gene banks for particular wild species, which are for the
moment maintained under controlled hunting, but may eventually become endangered in the
future, as in the case of the Eurasian lynx (Lynx lynx) in Northeastern Europe, or the wild felids
in Africa or South America (Swanson, 2006). Cryopreservation of spermatozoa has been well
studied in different species, including the dog, having the first pregnancies been successfully
achieved
with
frozen/thawed
spermatozoa,
using
lactose
and
Tris-(hydroxymethyl)-
aminomethane (Tris)-based extenders (Seager, 1969; Andersen, 1972).
Canine AI with cryopreserved semen has received increased interest from dog breeders in
the past years due to its versatility and to the possibilities that it brings to genetic exchange
between distant geographical areas (Thomassen et al., 2009). Furthermore, interest for in vitro
technologies for canine species is increasing worldwide and to accomplish the aimed outcomes it
is demanded good quality standards for preserved semen (Larsson et al., 2000; Johnston et al.,
2001; Kutzler, 2005).
The chilling process of dog spermatozoa has been reported to induce less damage in the
spermatozoa than freezing (Oettlé, 1986; England et al., 1996). When compared to the observed
for the frozen spermatozoa, chilled sperm quality measured by motility, sperm morphology,
acrosome status, hypo-osmotic swelling test and longevity, at a temperature of 39ºC, has been
reported to be superior, for up to 4.9 days of chilling, despite some deterioration may occur
during cold storage (England et al., 1996). Along with motility, the acrosome reaction in dogs
seems to be more affected by freezing and thawing than by chilling (Oettle, 1986; Burgess et al.,
2001). Recently, few studies have been developed in dogs to determine if the spermatozoa can be
chilled and then successfully frozen, having in the end similar post-thawing viability to the
semen that follows the normal freezing/thawing procedure (Hermansson, 2006).
Cryopreservation of semen, taking into account the process from cooling to thawing, has
been proposed to present several negative effects on sperm viability, which could be related to
injury of the plasma membrane, associated to changes in lipid phase transition, mechanical
stress, efflux of water and high salt solutions and, possibly, by interfering with intracellular ice
crystals formation (Woelders, 1997).
The type of semen extender and the freezing rates are determining factors on sperm
survival during cryopreservation procedures and might explain the variability in the response of
canine semen to freezing and thawing, between studies (Peña et al., 2000; Yildiz et al., 2000;
3
Nöthling et al., 2005). The major side effect of the freezing/thawing is a loss of sperm fertilizing
ability, mainly associated with reduced motility, promotion of the capacitation and loss of
viability (Yildiz et al., 2000). Some of these parameters are associated with sperm membrane
damages, which are considered to be the primary cause of freezing induced injuries to canine
sperm (Ström Holst, 1998). Furthermore, the same authors also report the existence of significant
changes in the elemental composition of post-acrosomal region of the canine spermatozoa after
freezing/thawing. In addition, important individual variations were found between stud dogs on
what concerns sperm quality after freezing procedures, leading to the common designation of
“good” and “bad” freezers (Silva et al., 2003).
1. Semen collection
Prior to semen collection a thorough and complete historical review of the dog’s previous
health and breeding experiences should be obtained; in addition, information regarding the
medication or supplements administered over the previous 6 months (at the minimum) and on
the genetic or familiar background could be important (Johnson, 2006). There should also be
collection of information about the status of vaccinations, dewormings and heartworm protection
history, as well as on the duration of the ownership and accuracy of the history of the animal
even before the establishment of the ownership (Freshman, 2002; Ettinger et al., 2010).
The anamnesis should be completed with a physical examination, which must consist of a
visual, an auscultatory and a palpable examination of the entire animal, with special focus on the
parameters or characteristics known to be heritable (CBRA, 1998; Johnson, 2006). This is the
moment to talk with the owners and to advise them whether the animal should or should not be
bred (Johnson, 2006). This evaluation should be followed by the examination of the reproductive
system and it should include the palpation of all the reproductive structures, such as, the scrotum
and its contents, the penis and the prepuce and finally the prostate (CBRA, 1998; Simpson et al.,
1998). The order of the examination, like the palpation, can and should be postponed till after the
collection of the semen, depending on the male’s behavior (Ettinger et al., 2010). The
examination of the scrotum and its contents should start with the location of both testes within
the scrotum and if that is not the case, the history should confirm a reliable information about the
missing testis in order to consider the animal proper for breeding or not (Ettinger et al., 2010).
The size and consistency of the testis should be evaluated and registered, along with the position
of the tail of the epididymis (to check for example, for a possible torsion of the testicle), as well
as the evaluation of the vas deferens, the spermatic cord, the thickness of the scrotal skin and the
4
state of the skin tissues, correlating them with a possible cause of temperature imbalance
(CBRA, 1998). The palpation of the prostate should also be included in the physical examination
of every male dog, considering its size and shape (Ettinger et al., 2010). Brucella screening tests
should also be done in all breeding males (Wanke, 2004).
Following the physical examination, it is important to assess the libido, using or not a
bitch in heat to facilitate the procedure, but always taking into account that the libido is
influenced by several factors, such as the physical conditions of the collection site, the existence
of olfactory cues or other specific and unknown reasons related to the bitch used for the effect
(Simpson et al., 1998; Johnson, 2006).
Many factors influence semen quality, including the animal’s age, the size of the testicles,
the degree of sexual arousal, the frequency of ejaculation, the collection procedure and the
amount of seminal fluid collected (Johnson, 2006). It is expectable that males may react
differently to the surroundings during semen collection and the procedures that precede it
(Kutzler, 2005). The collection area should be calm, isolated and interruptions during the
procedures should be prevented; the soil should be of secure footing for the animal, using for
example a rubber-backed mat that besides providing good footing can also provide olfactory
cues to the stud dog (Simpson et al., 1998; Freshman, 2002). Furthermore, in case of a toy dog it
could be of benefit to position him on a grooming table (Freshman, 2001; Nelson et al., 2009).
The presence of the owner is contradictory, as some dogs can be more comfortable with the
owner standing next to them, whilst others can show reluctance to their master’s presence in the
room (Purswell et al., 1992; Freshman, 2001). If there are any, the owner should bring the toys
or other accessories that the dog associates with the breeding. The collector should avoid any
“doctor” paraphernalia, like the white coat or the stethoscope (Freshman, 2002). It is important
to remember that the collection of semen should be performed prior to the physical examination,
injections, venipunctures or other stressful procedures. As last resort the physical or
complementary exams can be even postponed to a different session (Feldman et al., 1996;
Johnston et al., 2001). The equipment needed for the procedure should be promptly available and
warmed approximately to body temperature (Seager, 1986). Some concerns have been raised on
the effect of the usage of latex materials, on the canine spermatozoa motility, for example in the
use of artificial vaginas (AV) (Althouse et al., 1991). There are some studies that correlate a
decrease in the motility of spermatozoa and the prolonged time of contact with latex, not only
with AV but also with gloves; however, this is not an issue when the different techniques are
properly chosen and performed (England et al., 1992; Johnston et al., 2001). Another
5
inconvenience of the use of latex collection cones, is the need for a careful cleaning between
collections, including the removal of all disinfectant residues, soap and water prior to the next
use, taking into account that these are spermicidal (Kutzler, 2005). A sterile non-spermicidal
lubricant can be used in minimal quantities in the AV, as so it decreases the friction with the
penile mucosa, reducing the risk of injury of the superficial vessels and the penile mucosa
(Froman et al., 1983).
Previously to the collection procedure, the stud dog should be walked, allowing him to
urinate; afterwards, ideally, he should be trotted, to help the cleaning of the urethra from residual
urine (Freshman, 2002). The presence of a teaser bitch is usually of benefit, especially if she is in
proestrus or estrus and has the same proportions as the stud dog, although a calm bitch can also
be used (Freshman, 2002). The typical scent from estrus can either be obtained from frozen
swabs or sponges in plastic bags, taken from vaginal secretions (Purswell et al., 1992; Kutzler,
2005) or through the use of a chemical pheromone, methyl p-hydroxybenzoate (Aldrich
Chemical, Milwaukee, WI), having them used in the vulvar area and in the tail head of a teaser
bitch (Johnston et al., 2001; Kutzler, 2005). It has also been described that administration of
prostaglandin F2α (PGF2α; Lutalyse®, Pfizer) in a dosage of 0.1mg/kg, given subcutaneously 15
minutes before the collection, enhances the concentration of the ejaculate, having also a positive
effect on the libido. However secondary effects such as salivation, defecation and vomiting can
be present due to the use of this drug (Nelson et al., 2009), and the sensitivity of individual males
should be tested prior to collection. Allying the additive effects of both a teaser bitch and PGF2α
the number of spermatozoa may increase in 300% (Kustritz, 2007). The bitch should be
restrained and a muzzle may be used if she seems to be aggressive. The stud dog should be
walked behind her, allowing him to sniff and make himself at ease before the collection
procedure begins (Schubert et al., 1991).
Regarding the collection itself, dog’s prepuce should be vigorously massaged at the
region of the bulbus glandis until a partial erection develops, time at which the bulb should be
extruded from the prepuce to avoid possible pain for the animal (Freshman, 2001; Kutzler, 2005;
Ettinger et al., 2010). If the bulbus glandis becomes too enlarged to be extruded from the
preputial opening, removal of the bitch is indicated so that the erection can subside. Once the
erect penis is completely extruded, a gentle, firm, circular pressure should be maintained, so that
the male dog starts to ejaculate (Ettinger et al., 2010). After the collection, the bitch should be
withdrawn from the room and the dog should be monitored till the penis returns to its normal
position, inside the prepuce (Freshman, 2002).
6
2.1 Methods of semen collection
Semen collection in animals became easier due not only to the development of the AV in
1914 (Gordon, 2004), but also due to the use of the electroejaculator in 1936 (Gunn, 1936).
However, nowadays, those methods have been discarded in dogs, in favor of the digital
manipulation technique. Besides the digital manipulation of the penis, there are other methods,
such as pharmacological ones, that can be used. Nevertheless, the studies that have been done are
not very complete, regarding the parameters needed to privilege one method to the others; as an
example, an essay has been done consisting on collecting male dogs using pilocarpine
hydrochloride dissolved in saline solution, that showed better results than digital manipulation,
according to the concentration of spermatozoa, however, no data has been registered relatively to
the quality of the collected semen (Juniewicz et al., 1989).
Depending on the intended use for the semen, there are some particularities on the way
the semen sample should be collected from a dog, taking into account the different fractions that
compose the semen. Nevertheless, when there is the need to perform a breeding soundness
examination on a dog or if there is suspicion of a reproductive disease, all the three fractions
should be collected and evaluated (Kutzler, 2005; Lagishetty et al., 2011). Briefly, if there is the
likelihood of risk regarding the fertility, it is important that a complete ejaculation occurs, which
can be verified by the analysis of the seminal plasma alkaline phosphatase that should be
superior to 10.000 U/L in the combined fractions (Kutzler et al., 2003).
For preservation reasons, either semen freezing or chilling, or for fresh semen AI, it may
be of benefit to perform two collections, with an interval of 45 to 75 minutes among them.
Although the number of spermatozoa is rather low in the second collection comparing with the
first, the amount of both collections is in average, 70% more than if the collection is performed
only once (England, 1999).
The total volume of a dog’s ejaculate may vary from 1.0 mL up to 30.0 mL (England et
al., 2010; Ettinger et al., 2010). The canine ejaculate is composed of 3 distinct fractions
(Johnston et al., 2001; Kustritz, 2007). The first or pre-spermatic fraction is composed of clear
seminal plasma, devoided of sperm cells, originates from the prostatic gland and its main
function is to flush the urethra (England et al., 2006; Nelson et al., 2009; Ettinger et al., 2010).
The volume of the pre-spermatic fraction usually varies between 0.5 and 2.0 mL (Feldman et al.,
1996; Freshman, 2001). The second fraction, also denominated sperm-rich fraction, has a cloudy
7
and opalescent appearance with an opaque consistency, varying in volume between 0.5 and 5.0
mL, depending on the testicular size and on the individual variation; moreover, in its
composition there should be no cellular components besides sperm cells (Boucher et al., 1958;
England, 1999; Nelson et al., 2009). It originates from the storage in the tail of the epididymis as
from the daily sperm output (Johnston, 1989). The dog can take up to 2 minutes to achieve the
emission of the sperm-rich fraction (Kutzler, 2005). Ideally there should be a separation between
the first and the second fractions, especially for cryopreservation, as it is described that the
sperm’s contact with both the first and third fractions may diminish the motility after 2 hours
(England et al., 1992). The last portion, the third or prostatic fraction, is usually collected in
order to increase the volume of the ejaculate for fresh semen AI and to perform a cytology or a
culture (Ettinger et al., 2010). This last fraction can reach up to a total volume of 30 mL,
depending on the duration of the pressure maintained around the bulbus glandis (Johnston,
1989).
The penile erection can remain for up to 10 minutes and it should be supervised till it
wears off, in order to avoid complications (Freshman, 2002; Ettinger et al., 2010).
2. The semen
Whenever a semen sample is collected from a dog it should be assessed in order to
determine its quality (Kutzler, 2005).
At the time of semen collection, information regarding the sexual abstinence should be
checked and ideally the stud dog should have 4 to 5 days of sexual rest prior to collection
(Freshman, 2002). Nevertheless, it has been reported that more than 10 days of sexual rest can
result in an increase of morphologic abnormalities and a decrease in the motility related not only
with the ageing of the spermatozoa but also with the increase of debris (Purswell et al., 1992;
Johnston et al., 2001).
The dog’s ejaculate is composed of cells - spermatozoa (Spz)-, suspended on a fluid - the
seminal plasma (SP). Among its constituents, diverse glandular products, proteins and electrolyte
were identified (Kustritz, 2007). Several analyses have been performed in order to create a
pattern within the normal values of SP mineral contents, specifically zinc, iron, calcium,
magnesium and copper (Johnston et al., 2001). Their amounts usually drop till minimum values
after the semen collection, due to the metabolism of the spermatozoa and its enzymatic
8
degradation. These parameters can also be influenced by the time interval of the last ejaculation,
the sexual arousal of the male and the physiological status of the sexual accessory glands
(Kustritz, 2007).
Structurally, a spermatozoon can be divided in 3 parts (Figure 1): the head, the midpiece
and the tail (England et al., 2010). The sperm head contains a nucleus, which is covered
proximally by the acrosome (Cunningham et al., 2007). The midpiece has approximately 1.5
times the length of the spermatozoon’s head (Feldman et al., 2004). A normal dog spermatozoon
has approximately 7.0 µm, a midpiece with 1.1 µm and a tail with the length of 5.0 µm divided
in its main piece and an end piece (Johnston et al., 2001).
Figure 1 – Morphological structure of the canine spermatozoon
3. The semen quality
Considering that semen contains live cells, the collector should handle it with special care
while executing the procedures, since the interval from the collection till the evaluation should
be as brief as possible, in order to prevent cellular modifications or even death of the cells
(Feldman et al., 2004). The semen should be maintained between 20ºC to 30ºC (room
temperature or water bath) and kept away from direct sunlight (England et al., 2010). Some
authors defend that for the analysis of chilled semen, the tubes containing the samples should be
kept in a glassed warm water recipient in order to prevent thermal shock during the chilling
process, along with temperature variations during the period when the samples stand outside the
fridge, for analysis (Iguer-Ouada et al., 2001). Immediately after the end of the collection
procedure, the ejaculate should be assessed, since some parameters start to change soon after the
collection (Kutzler, 2005).
9
3.1 Semen assessment
Each time that a dog is collected with the purpose of insemination, the sample should be
assessed in order to determine its quality and to evaluate the reproductive and fertility potential
for both the male and the seminal dose (Kutzler, 2005). Usually, available assessment methods
vary mainly according to the use of an in-house or a research laboratory on what concerns its
profundity or subjectivity (Kutzler, 2005).
Although several methods have been proposed for semen evaluation in dogs, the analysis
of conception rates should remain as the ultimate test to assess male fertility (Oettle, 1993).
The assessment techniques used to determine semen quality may be divided into:
- a classical methodology, which include the evaluation of the physical parameters of the
collected sample and the assessment of motility, concentration and morphology
(Simpson et al., 1998);
- a variety of sophisticated assessment techniques, less subjective than the classical, that
can be used to assess the motility (like the CASA system) and quality of sperm cells,
including the evaluation of the viability, the integrity of the plasma membrane, the
capacitating status and the acrosome reaction, among others (Nelson et al., 2009).
3.1.1 Classical methodology
The macroscopic parameters in the evaluation of semen are an important component of
the classical method and include the volume, the color and the pH (Johnston et al., 2001).
The volume, by itself, is not a parameter that defines the quality of the semen, once it
depends on the amount of prostatic secretion collected, the age and size of the animal and also
the frequency of ejaculations of the dog (Feldman et al., 2004). Nevertheless, it is an essential
parameter in order to calculate the total number of spermatozoa in the ejaculate, being of
outmost relevance for the semen quality (Simpson et al., 1998). From a laboratorial point of
view, the small volume obtained in each ejaculation consists of a great challenge in the semen
processing, limiting the number of experiments possible to be done per dose (Farstad, 2009).
Usually the appearance of dog’s semen is white, cloudy or opaque and the intensity of its
opacity depends on the concentration of spermatozoa (Feldman et al., 2004). The samples that
possess a slightly cloudy coloration should be examined under the microscope, to check for the
presence of spermatozoa, once that the cloudy color can characterize a sample with no
spermatozoa, but can also be associated with a great amount of adipose tissue, bacteria or
10
inflammatory cells (Johnston et al., 2001). The yellow color can indicate the presence of urine,
pus or some inflammatory exudates, while the green color normally indicates the presence of
pus. The red or brown color can traduce the presence of fresh or haemolyzed blood, respectively
(Johnston et al., 2001).
The pH should be expected to be within the range of 5.5 to 8.0. Specifically, the normal
pH values of the third fraction should vary between 6.0 and 7.4 (Freshman, 2002). As an
example, pH measurement may be important regarding the choice of an antimicrobial drug in the
eventuality of a prostatitis (Johnston et al., 2001; Freshman, 2002).
The microscopic characteristics that should be taken into consideration during the
assessment of dog’s semen are included in the protocol used by many andrologic centers and
laboratories where light microscopic techniques are routinely used to evaluate the three
conventional sperm parameters: concentration, motility and morphology (Johnston, 1991).
Semen concentration, along with the volume, is not per se, an indicator of the quality of
the semen, as it also depends on the amount of the collected seminal secretion (Johnston et al.,
2001; Freshman, 2002). Despite all the technological investment, the method still considered to
be the gold standard is also the most traditional one, which uses a counting chamber, such as the
Neubauer or the Bürker chambers (Kustritz, 2007). The total number of spermatozoa is achieved
multiplying the concentration (millions per mL) by the ejaculate’s volume (mL); the value can
vary between 300 million and 2 billion spermatozoa (Ettinger et al., 2010). This wide range of
normal values enhances the theory that semen production is directly influenced by the quantity
of testicular mass (Johnston et al., 2001; Freshman, 2002; Feldman et al., 2004; Kustritz, 2007).
The total and progressive motility can be assessed subjectively on a pre-warmed glass
slide and analyzed under a light microscope (Johnston, 1991; Strom et al., 1997). The method
should include five different fields of the microscope, in a total of 200 spermatozoa (Kustritz,
2007). The evaluation of the motility can become extremely difficult because of the high
concentration of the sample, making it necessary to dilute the semen with a solution that can be
the prostatic secretion (natural extender) or a synthetic extender (Feldman et al., 2004; Martinez,
2004). Some saline solutions used as extenders can lead to a decrease on the progressive motility
of spermatozoa, due to its pH (Johnston et al., 2001). Other extenders, either due to the presence
of viscous substances in their composition (such as egg yolk), the dilution factor or even the
temperature at which the analysis is made, may induce a change in the velocity of the
spermatozoa, along with the linearity of its movements (Schafer-Somi et al., 2007). The
reference value for the progressive motility of dog semen is ≥ 70% (Johnston et al., 2001). The
11
progressive motility of a dog’s semen sample is not affected by the frequency of collections
performed (Kustritz, 2007). Nonetheless, the first ejaculate after a long period of abstinence may
present a great number of older spermatozoa that have been stored in the epididymis, resulting in
a significant decrease in the total spermatozoa with progressive motility in the sample (Feldman
et al., 2004). The percentage of morphologically normal spermatozoa is positively correlated
with the percentage of spermatozoa with progressive motility (Johnston et al., 2001; Kustritz,
2007), once the motility is considered to be a manifestation of the structural and functional
competences of the spermatozoa (Martinez, 2004; Volpe et al., 2009). Although it might not be
the best parameter to define the capacity of fertilization of the spermatozoa, motility is an
evaluation parameter fairly easy to assess and it can be used as a guideline for the evaluation of
fresh and cryopreserved semen (LeFrapper, 2010). Even with all the development around this
area and the improvement of more objective techniques for semen evaluation, the progressive
motility is still the most used indicator for the spermatic function, once that it is unquestionable
that the motility is essential for the progression of the spermatozoa throughout the oviducts
(Martinez, 2004).
The abnormal motility can be associated with morphologically abnormal spermatozoa
and with decreased fertility; however, there can be morphologically abnormal spermatozoa with
a normal motility, making it important to evaluate the morphology (Ettinger et al., 2010).
The percentage of morphologically normal spermatozoa in a semen sample is considered
acceptable over a value of 70% (Feldman et al., 2004). The sperm morphology can be evaluated
through the use of different staining techniques, such as eosin/negrosin, Diff Quick or trypan
blue (Bangham et al., 1955; Dott et al., 1972; Johnston et al., 2001; Freshman, 2002; Risopatron
et al., 2002; WHO, 2010) and it is regarded as an important parameter in the conventional semen
analysis (Johnston, 1991; Oettle, 1993). The two most used staining techniques are eosinnegrosin and modified Giemsa (Johnston et al., 2001; Freshman, 2002). The eosin-negrosin as a
vital staining has the purpose of emphasizing the borders of the spermatozoa than the cell itself.
It also allows, through the use of a contrast phase microscope, the evaluation of the acrosome of
live and dead spermatozoa, having the live and acrosome intact spermatozoa a white color with
round, regular and defined borders, whilst the live Spz with a reacted acrosome show the
acrosomic region in a dark coloration with an unidentifiable apical extremity (Martinez, 2004).
This method of coloration allows the differentiation between live and dead spermatozoa,
considered as dead ones, the spz that become stained due to the absorption of eosin (staining),
12
once that they are considered to have lesions at the level of the cellular membrane (Kustritz,
2007).
It has been described the use of Spermac®. This staining allows a quick staining with
unique characteristics, marking the spermatozoon’s nucleus in red, the acrosome, midpiece and
tail in green and the equatorial region of the acrosome in pale green (Feldman et al., 2004); this
makes this technique a very interesting staining to evaluate the morphology of the acrosome
(Martinez, 2004; Monteiro et al., 2009).
Apart from the staining technique used, there are always some changes in spermatozoa,
resulting from the cytology execution, called artifacts, such as detached heads, coiled or folded
tails and folded intermediate pieces, so it can be concluded that the consistency used in the
technique is essential for the accuracy and precision of the semen evaluation (Johnston et al.,
2001). The use of stainings that are osmotically not similar to the spermatic samples, result in the
detachment of the acrosome of some spermatozoa. This osmotic effect is more pronounced in the
frozen/thawed than in the fresh semen (Martinez, 2004). Besides the Spz alterations caused by
the processing of the semen samples, there are other factors such as testicular traumas, fever or
infection of the reproductive tract, that can origin seminal changes, due to the increase of
temperature in the testes (Martinez, 2004). However, these changes should only be noted some
time after the initial problem, as the spermatogenesis on the dog has a length of 62 days to which
the time for epididymal transit should be added (Johnston et al., 2001; Freshman, 2002). Other
possible causes for the morphological changes of spermatozoa can be associated with the
decrease of LH, a disturbed testosterone secretion or even iatrogenic causes (Martinez, 2004).
Healthy dogs that change either of owner or environment can show an increase in the number of
abnormal spermatozoa, possibly due to the increase of endogenous corticosteroids (Martinez,
2004).
The morphological changes of spermatozoa can be classified according to the cellular
location where they occur: head, acrosome, intermediate piece and tail (Figure 2). Furthermore,
they can be divided in primary, occurring during spermatogenesis, and secondary if occurring
during maturation and for some authors also as tertiary, if during the preparation of the sample
(Johnston et al., 2001). Another classification that tries to associate the sperm morphology to the
sperm capacity for fertilization, divides the abnormalities of the spermatozoa in minor defects, as
those which do not influence the fertility, and major defects, as those which are negatively
correlated with the ability to fecundate the oocytes (Johnston et al., 2001).
13
Figure 2– Sperm cell abnormalities (vetmed.lsu.edu)
The morphological abnormalities that have been linked to infertility include defects in the
connection with the intermediate piece, microcephalic spermatozoa and spermatozoa that possess
proximal cytoplasmatic droplets (Feldman et al., 2004).
Two main problems that subsist with the light microscopic methods are the subjectivity
and the variability (Oettle, 1993; Hewitt et al., 1998). Visual sperm motility assessment is
difficult and may be influenced by both the temperature and the evaluator’s skills, leading to
high variability among laboratories and observers (Verstegen et al., 2002). On the other hand, the
evaluation of the morphology is also problematic due to the fact that it not only depends on the
fixation, on the staining technique and on the quality of the microscope, but most importantly it
further depends on the observer’s experience and skills (Pena et al., 1999; Pena et al., 1999).
The low number of spermatozoa assessed with the conventional techniques is also a
factor for inter-laboratory variation (Pena et al., 1998).
14
3.1.2 Advanced methodology
Aiming to overcome the principal disadvantages of the current methods of canine semen
assessment, other techniques have been proposed, such as fluorescence microscopy, flow
cytometry, computerized sperm analysis systems and zona pellucida binding and penetration
assays (Gunzel-Apel et al., 1993; Hewitt et al., 1998; Pena et al., 1999; Strom Holst et al.,
2000b). One advantage of some of these techniques, such as the use of fluorophore combinations
is to evaluate live and dead cells at the same time, either by fluorescent microscopy or by flow
cytometry, the last one allowing the evaluation of large numbers of spermatozoa in a short period
of time (Rijsselaere et al., 2005). The use of these methods allows also the simultaneous
evaluation of several sperm characteristics, relatively to different organelles or sperm domains
(Rijsselaere et al., 2005).
Wide variations have been described on what concerns the analysis of sperm motility
(Chong et al., 1983; Jequier et al., 1983; Mortimer et al., 1986) along with the morphological
parameters (Baker et al., 1987; Kruger et al., 1995) of the same ejaculate. The need for higher
objectiveness in the methods used, recurring to its standardization for both practical and research
purposes, was one of the major concerns in the past two decades (Smith et al., 2001; IguerOuada et al., 2001a). The gathering of these factors opened a window for the development of
several semi-computerized (England et al., 1990; Iguer-Ouada et al., 2001b) and computerized
measuring devices (Gunzel-Apel et al., 1993; Smith et al., 2001; Iguer-Ouada et al., 2001a;
Iguer-Ouada et al., 2001b) for the evaluation of canine sperm motility, morphology and
concentration.
In order to fertilize an oocyte, a sperm cell should possess the ability to play several
functions, including the cascade of reactions that allow the sperm/oocyte interaction at
fertilization (Rijsselaere et al., 2005). A great deal of evolution has been gathered around the
domains of the assessment of several sperm functions, allowing a more detailed evaluation of
dog semen quality (Hewitt et al., 2001). There are some functional assays that are able to
examine the interaction between the spermatozoa and the oocyte, thus giving a good estimation
of sperm fertilizing capacity, by testing for example the ability of the spermatozoon to bind and
penetrate the ZP and fertilize in vitro (i.e. achieved in vitro fertilization, IVF). The ZP binding
assay (ZBA) has been used in several species, including the dog (Hay et al., 1997; Strom Holst et
al., 2000a; Strom Holst et al., 2000b). In general, the techniques available to assess the semen
can be resumed in fluorescent staining, computer-assisted sperm analysis, zona pellucida-binding
assay, oocyte penetration assay/in vitro fertilization, and sperm-oviduct interaction (Rijsselaere
et al., 2005).
15
3.2 Sperm motility assessment
Several commercial computer assisted sperm analysis systems, generally named CASA,
such as Strömberg-Mika Cell motion analyzer, CellSoft computer videomicrography; Hobson
Sperm Tracker and Hamilton-Thorne, base predominantly on individual spermatozoon assessment
and have been validated for use in dogs (Gunzel-Apel et al., 1993; Smith et al., 2001; Iguer-Ouada
et al., 2001a; Iguer-Ouada et al., 2001b). These kind of devices allow an accurate and quick
calculation of various semen parameters such as the total and progressive motility, the static, slow,
medium and rapid moving spermatozoa, the linearity of sperm movement (LIN), the beat cross
frequency (BCF), the amplitude of the lateral head displacement (ALH) and quite a few other
velocity parameters (Table 2) (Gunzel-Apel et al., 1993; Smith et al., 2001; Iguer-Ouada et al.,
2001a; Iguer-Ouada et al., 2001b). There are several studies reporting the existence of high
correlations among the computer-calculated motility, progressive motility and concentration, and
the conventional light microscopic evaluation (Gunzel-Apel et al., 1993; Iguer-Ouada et al.,
2001a). In addition, these systems have proved to be useful when assessing various semen
characteristics simultaneously and objectively and are endowed to detect subtle changes in the
sperm movement, unnoted with the conventional semen analysis (Gunzel-Apel et al., 1993; Rota
et al., 1999; Verstegen et al., 2002). Also, a very important characteristic is that a high number of
spermatozoa can be analyzed in a short period of time (Iguer-Ouada et al., 2001a).
The main disadvantages pointed out for those devices are the need for high investments
and the notorious need for standardization and validation of the software and the system
previously to its practical use (Smith et al., 2001; Iguer-Ouada et al., 2001b; Verstegen et al.,
2002). Moreover, to avoid a new source of subjectivity among laboratories, associated to the
definition of the computer parameters to be applied, the selection of the software and the
microscope conditions, the standardization of the technical settings became in focus (Smith et al.,
2001; Iguer-Ouada et al., 2001b). In addition, it still needs to be determined which sperm
movement characteristics are of best clinical value for the prediction of in vivo fertility in dogs,
along with the standardization of the parameters, which differ between users or laboratories and
the different commercial systems (Verstegen et al., 2002).
16
Table 2 - CASA parameters (modified from Iguer-ouada et al., 2001a; Verstegen et al., 2002; Rijsselaere et al., 2005)
Parameter
Definition
TM
Total motility
PM
Progressive motility
VAP
Velocity average pathway
VSL
Description
Unit
Percentage of spermatozoa with movement
%
Percentage of spermatozoa that progress; STR > x
(straightness threshold cut-off to determine the
progressive spermatozoa)
%
Average velocity of the smoothed cell’s pathway
μm/s
Velocity straight line
Average velocity measured in a straight line from the
beginning to the end of one track
μm/s
VCL
Curvilinear velocity
Average velocity measured over the actual point-to-point
track followed by the cell
μm/s
ALH
Amplitude of lateral head
BCF
Lateral amplitude of the head displacement
μm
Beat cross frequency
Frequency at which the sperm cell’s head crosses the sperm
cell’s average pathway
Hz
STR
Straightness
Average value of the ratio VSL/VAP in percentage
(estimates the proximity of the cell’s pathway to a straight
line with 100% corresponding to the optimal straightness
%
LIN
Linearity
Average value of the ratio VSL/VCL in percentage
(estimates the proximity of the cell’s track to a straight line)
%
The CASA is basically a computer analyzer for sperm motility and morphometry. Its use
has become more popular, but due to its high cost it is still not available to the majority of the
veterinarians (Verstegen et al., 2002; Kustritz, 2007). With a contrast microscope integrated, this
equipment allows a quick and objective evaluation of many parameters, some of them described
in Table 2.
As this equipment allows to identify even very small variations in the spermatozoa
motility, that would, otherwise be impossible to detect (Rijsselaere et al., 2005), it allows the
classification of small populations of spermatozoa in a sample that may react differently when
submitted to processes, such as cryopreservation (Kustritz, 2007).
3.3 Hypo-osmotic swelling test
The morphological and functional integrity of the sperm membrane has been intensely
studied, given its importance as a cellular barrier and in the cell to cell interaction (RodríguezMartínez, 2000). The integrity of the plasma membrane is crucial for the fertilizing capacity of
spermatozoa. As described to the morphology, in the classical methods, the membrane of dog’s
17
spermatozoa was routinely assessed by means of light microscopic stainings, such as
eosin/negrosin (Bangham et al., 1955; Dott et al., 1972) or trypan blue (Risopatron et al., 2002).
An indirect method to evaluate the membrane integrity is by exposing spermatozoa to hypoosmotic conditions, for example through the hypo-osmotic swelling test (Kumi-Diaka, 1993),
once that the number of swollen spermatozoa was shown to be inversely proportional to the
number of membrane damaged spermatozoa (England et al., 1993).
This test is considered, by many laboratories, as an appropriate method to evaluate the
integrity of the membrane and due to its simplicity, it can be introduced into the routine semen
analysis (Pinto et al., 2008). This technique consists of mixing the semen sample in a hypoosmotic solution and incubating it at 37ºC, for 45 to 60 minutes (Rodríguez-Gil et al., 1994).
When submitted to a hypo-osmotic solution, a viable spermatozoa possessing an intact, active
and functional plasmatic membrane, will react, allowing the fluid to enter the cell, causing the
swelling and coiling of the tales of spermatozoa (Kustritz, 2007). It has been proved that no
significant differences exist between incubation times of 1 or 60 minutes, and also that this test is
easily performed by any veterinarian (Pinto et al., 2008). The solution used in this test should be
enough hypo-osmotic to cause the swelling, without causing lysis to the cell (Bencharif et al.,
2008). The hypo-osmotic solutions described for this test include sodium citrate, fructose in
distilled water and a solution of sacarose (Kustritz, 2007).
The swelling effect is more easily identifiable on the tails of the spermatozoa for two
main reasons: the membrane of the tail is more flexible and is less adherent to the internal
structures comparing to the membrane of the head, and at the spermatozoon’s head the
intracellular fluid compartment is rather small making it difficult to observe the variation in the
volume when the water enters the cell (Jeyendran et al., 1984). Previously to the realization of
the hypo-osmotic swelling test, it is of high relevance that the percentage of spermatozoa with
tail abnormalities is measured, in order to subtract this value to the counting made after
incubation, obtaining the true value of spermatozoa with an intact plasmatic membrane (Kustritz,
2007).
This test shows a good correlation among the spermatozoa that suffer swelling and the
percentage that were able to fertilize the oocytes in vitro. In the same study, the correlations
obtained between this parameter, the percentage of progressive motility, the percentage of
morphologically normal spermatozoa and the percentage of live spermatozoa (negative with vital
staining) was not so relevant (Jeyendran et al., 1984). There is no relation described between the
18
hypo-osmotic swelling test results and the fertilization capacity of dog spermatozoa (Martinez,
2004), however there is a correlation between these results and the motility and viability for both
fresh and frozen dog semen (Pinto et al., 2008).
4. Heat shock
A reaction to an induced heat shock is inherent to the procedure of semen
cryopreservation, which is accompanied by the irreversible loss of viability when the
spermatozoa rapidly reach neutral temperatures. This co-exists with a decrease in sperm motility
(Quinn et al., 1966), a reduction in the energetic ability and an imbalance of the membrane’s
permeability (Watson, 1981). Thus, the refrigeration of the semen during the freezing process
should be done with extreme precaution, in a slow procedure, even if it does not prevent all the
damage to the membrane (Watson, 2000).
It has been reported that the spermatozoa become susceptible to the heat shock in the
proximal region of the body of the epididymis, during the epididymal maturation process; more
precisely it occurs when the cytoplasmatic droplet moves towards the distal portion of the
midpiece. It has been concluded that acquisition of motility and heat shock susceptibility may
occur at the same time (White, 1993). The freezing-associated heat shock and the irreversible
lesions that it causes, seem to result from the changes in the lipid organization in the sperm cell
membrane (Drobnis et al., 1993).
The heat shock has different impact according to the species, being the sperm cells of
both swine and bull the most susceptible. There are relevant changes in the ion exchanges, with
Ca2+ increase and loss of K+ and Mg2+. On the other hand, the sperm of humans, dog and
rabbit seems not to show so intense changes (Quinn et al., 1966). Nevertheless, freezing/thawing
processes in canine semen are usually referred as being detrimental to the sperm fertility, mainly
due to reduced longevity (England et al., 1993; Bessa, 2005) and to an increase in the proportion
of capacitated cells after thawing (Rota et al., 1999; Bessa, 2005). These issues were at the origin
of intense scientific work aiming to identify the most adequate extender for dog semen.
19
4.1 Heat Shock Proteins
Heat shock as other stressing factors stimulate every cell to synthesize a small group of
protective proteins, which are named heat shock proteins (HSPs) (Schlesinger et al., 1982; Craig,
1985; Schlesinger, 1986). HSPs are classified into several families, named according to their
approximate molecular weight (Sonna et al., 2002).
Among several stress stimuli, elevated temperature or heat stress have been described as
inducers of the HSPs production in certain tissues (Nollen et al., 1999). The concentration of
stress proteins increase, responding to stress factors, therefore acting as cell protectors and
facilitating cell survival, proportioning renaturation of partially denaturated proteins (Parcellier
et al., 2003).
There have been important and relevant studies about the heat shock proteins throughout
the last years. These studies have been more incisive in a special set of evolutionary conserved
heat inducible and related proteins of molecular weight of around 70 kDa (Lawson et al., 1984;
Craig, 1985; Schlesinger, 1986).
HSPs, and specially HSP70, under physiological conditions act as molecular chaperones
as well as assisting protein folding, transport and degradation; on the other hand, subjected to
stress conditions, they prevent aggregation and promote refolding after denaturation, protecting
cells of stress or other hazardous conditions (Georgopoulos et al., 1993). Induction of heat shock
response is related to the physiological temperature range (Liu et al., 1994) and can occur either
when the temperature increases upwards a threshold or when it decreases below a specific value.
Responding to heat stress, heat shock genes are stimulated and new molecules of HSP are
produced within the cells, in an attempt to prevent or to correct denaturing of cell proteins. Thus,
induction of the heat shock response should be read as a manifestation of temperature-induced
cell damage (Liu et al., 1994). One important implication of this finding in sperm post-thawing
fertility is that events involved in the expression of the heat shock response require active cell
metabolism, such as ATP production or macromolecule synthesis, which are of limited capacity
in sperm cells due to their highly specialization.
20
PART II. COMPARISON OF CANINE SPERM QUALITY UNDER
DIFFERENT TEMPERATURE STORAGE: EXPERIMENTS
21
1. Introduction and aims
The specific thematic for this work was chosen accordingly to the work developed
throughout the 3.5 months of practical training in the École Nationale Vétérinaire d’Alfort, in the
area of small animal reproduction, CERCA (Centre d'Études en Reproduction des Carnivores),
which is a highly regarded centre for reproduction and specifically cryopreservation.
In the process of cryopreservation, the sperm cells, in particular dog sperm cells are highly
susceptible and suffer substantial alterations, especially on what concerns sperm motility. As
proposed aims for this study, we ought to:
1 – Evaluate the sperm quality upon chilling and freezing/thawing procedures and to
compare it to the freshly ejaculated sperm.
2 – Characterize the pattern of immunolocalization of HSP70 in the freshly ejaculated
canine spermatozoa and to study putative changes after being chilled and frozen/thawed.
We further will attempt to study eventual association between data gathered in those two
studies, and to ascertain possible influences of the temperature stress induced by the two cold
treatments on sperm quality.
To achieve the proposed objectives we performed two different studies where the same
samples were used. For the first experiment, freshly ejaculate samples were obtained from 8
dogs. Those samples were processed in straws as for routine seminal doses and submitted to
chilling for 24h and to freezing in liquid nitrogen with posterior thawing. The quality of each
sample was assessed by the use of both classical and advanced methods, for each temperaturechallenge tests, compared to controls (fresh ejaculates) and differences among treatments and
individual dogs were analyzed. In this study, the following parameters were evaluated:
concentration, pH, volume, color, morphology and HOST, as well as CASA characteristics (total
and progressive motility, VAP, VSL, VCL, ALH, BCF, STR, LIN).
For the second experiment, cytological specimens were prepared from each sample, fixed
at 95% ethanol and processed for immunocytochemistry. The characterization of the normal
expression pattern of HSP70 was studied in the control samples (fresh ejaculates). The intensity
of immunostaining as well as the sub-cellular location of the protein was recorded. Comparison
between controls and the cold-treated groups (chilling and freezing/thawing) was evaluated on
what concerns the intensity of immunoreactions and any possible changes in the sub-cellular
location of HSP70. With this approach we ought to test the possible involvement of HSP70 in
some treatment-associated changes found in more conventional sperm assessment.
22
2. Common material and methods
2.1 Animals
In this work, fresh ejaculates were obtained by digital manipulation, according to the
method described by Linde-Forsberg (Linde-Forsberg, 1991), from 8 dogs, with ages ranging
between 3 to 4 years, that either belonged to the École Nationale Vétérinaire d’Alfort, Paris or
were received in CERCA (Centre d'Études en Reproduction des Carnivores) for consultation.
There was no specificity neither according to the breed chosen, nor the age of the dogs, besides
that all of them were considered to be healthy and reproductively within the accepted fertility
parameters, since a reproductive exam and a prostatic ultrasound were previously performed.
The collected sample included the sperm-rich (second) fraction and a small amount of the third
fraction Figure 3.
Figure 3 - Semen collection material
2.2 Sample preparation
For chilling procedures, after dilution the samples were stored in test tubes in the
refrigerator for up to 24h. For frozen samples, 0.50 mL straws were used and stored in liquid
nitrogen for a period of 2 days.
From fresh ejaculates, and after semen evaluation, the straws were prepared in order to
obtain a concentration of 150 x 106 spermatozoa in each straw. The following formula was
applied:
23
Fresh samples were centrifuged for 10 minutes at 1.500 rpm, as displayed in figure 4. The
exceeding fluid was withdrawn and only the sperm pellet was kept. The pellet was then resuspended in the adequate extender, according to the temperature treatment to be followed:
chilling or freezing.
Figure 4 – For preservation, either for chilling or freezing, the collected semen is centrifuged to obtain a
pellet with spermatozoa.
2.3 Extenders
The extenders used in the study, for both chilling and freezing/thawing of the semen,
correspond to the method of Uppsala, (Linde-Forsberg et al., 1999). The extenders were prepared
with Tris, citrate, glucose, antibiotics, glycerol, Equex and distilled water (Table 3) and were
frozen.
Table 3 – Composition of the extenders used for chilling and freezing/thawing procedures
Components
Chilling extender
Freezing extender 1
Freezing extender 2
Thawing
extender
TRIS
6.025 g
3.025 g
3.025 g
3.025 g
Citrate
3.4 g
1.7 g
1.7 g
1.7 g
Glucose
-
1.25 g
1.25 g
1.25 g
Fructose
2.5 g
-
-
-
Peniciline
200 000 UI
0.06 g
0.06 g
0.06 g
DHS
0.2 g
0.1 g
0.1 g
0.1 g
Distilled water
Till 200 mL
77 mL
72 mL
Till 100 mL
Glicerol
12 mL
3 mL
7 mL
-
Equex
-
-
1 mL
-
Egg yolk
20 mL
20 mL
20 mL
-
24
The preparation was made in advance, by mixing the powder components in water and,
for the extender 2, the final adding (without mixing) of the glycerol or the Equex. It was filled up
to 100 mL or 200mL of water, according to the extender, and it was strongly mixed. It was
stored in plastic tubes in the amount of 4 mL each and frozen to future use. At the time of use 1
mL of egg yolk was added to both extenders (Figure 5), having a total of 5 mL of each extender
to use for the chilling or the freezing procedure.
Figure 1 – Sequential procedure of egg yolk addition
2.4 Chilling procedure
After the withdrawn of the supernatant with a Pasteur pipette, one half of the calculated
chilling extender was added to the sperm pellet. After waiting one minute, the second half of the
extender was added, with the proper care of not jetting it harshly, as shown on figure 6. It was
stored in the refrigerator or chilling chamber, as displayed in figure 7, for 24 hours at 4ºC, having
been collected small samples to analyze at the time of 1.5, 4 and 24 hours.
Figure 6 – From left to right, removal of the supernatant, addition of half of the extender; addition of the
second half of the extender
25
Figure 7 – Process of storage in the refrigerator
2.5 Freezing/thawing procedures
After the supernatant removal, with a Pasteur pipette, the calculated freezing extender 1
was added to the tube with the sperm pellet, and then stored in the refrigerator or the chilling
chamber for 1.5 hours at 4ºC (Figure 8). In a separate container, the second extender was also
placed in the refrigerator, to chill and equilibrate the temperature with the pre-diluted sperm
sample. Then, the second extender was carefully added, avoiding jetting it harshly (Figure 9).
Figure 2 – From left to right, sequential process of supernatant removal, extender addition and storage in
the refrigerator
Figure 3 - Addition of the second extender
Immediately after mixing, the semen was shed in a leaned metallic container, in order to
case the semen in straws. The end of each straw with no cotton was submerged and the semen
26
was aspirated, with a pipette or another aspiration method, as shown in Figure 10. A small air
chamber was left inside each straw, to prevent its explosion during freezing. To seal the straw,
the end devoided of cotton was inserted in a specific powder for several times and then
submerged into water, to become solid, as displayed in Figure 11.
Figure 4 - On the left image, equipment for casing the straws; on the right, procedure of filling up the
straws.
Figure 5 - From left to right, procedure of casing the straws
The straw was then placed under nitrogen vapors for 20 minutes, and therefore completely
submerged in the liquid nitrogen, as seen in Figure 12. The straws for this study were finally stored in a
specific nitrogen container for 2 days, as shown in Figure 13.
27
Figure 6 - From left to right, procedure with the liquid nitrogen
Figure 7 - Storage of the straws
For thawing, the straws were submerged directly in a water bath at 37ºC, for one minute,
and dried up afterwards. Its powder end was cut off, while the remaining of the straw was kept
inside a plastic tube already containing 0.5 mL of pre-warmed PBS (phosphate-buffered saline
solution). The cotton end was then also cut off to allow the seminal doses to flow.
28
3. Experiments
3.1 Experiment 1 - Evaluation of dog semen quality after chilling and freezing
3.1.1 Goals
In this study, we ought to evaluate the differences in sperm quality following two
different temperature challenges (chilling vs. freezing/thawing), using fresh ejaculated semen as
controls (Time zero). For this experiment, we considered 3 different times during chilling
treatment (at 1.5h, 4h and 24h, respectively treatment A, times 1, 2 and 3) and the freezing
treatment (treatment B).
3.1.2 Specific methods
Fresh ejaculates were assessed by classical methodology, the CASA technology and the
hypo-osmotic swelling test. Temperature-treated samples were subjected to both CASA device
analysis and hypo-osmotic swelling test.
The macroscopic parameters evaluated included the semen volume, color and pH. Also,
the morphology of the spermatozoa was evaluated in a cytology stained with eosin-nigrosin. The
abnormal forms found were classified according to Johnston (Johnston et al., 2001).
Within the microscopic parameters the concentration of the seminal sample was
determined with a spectrophotometer, whilst the morphology was assessed in eosin-negrosin
stained slides (Figure 14). In order to maintain the coherence, massal and individual motility
parameters were assessed by CASA, as for the treatment samples.
All the semen samples were left for 2-3 minutes to warm up, in a warm bath at 37ºC. A
volume of 3μl was placed, with the help of a micropipette, in a pre-heated Leja® standard count
4 chambers slide of 20 micron and the assessment with the CASA device was initiated 1 minute
after placing the Leja® slide in the warmed plate of the microscope.
The parameters taken into consideration from the CASA analysis were the total and
progressive motility, the velocity of the Spz movement (rapid, medium, slow, static) and the
different characteristics of Spz agility, VAP, VSL, VCL, ALH, STR and LIN.
29
Figure 8 - Concentration determination with a spectrophotometer and pH analysis
3.1.3 Statistical analysis
Data gathered in the two experiments were organized in Microsoft Excel . Statistical
comparisons were performed by using the IBM SPSS Statistics Base 19.0 statistical software for
Windows®. Statistical analysis of the effects of treatment on motility, velocity and HOST were
performed using the ANOVA, with Bonferroni adjustment. A p value ≤ 0.05 was regarded as
statistically significant.
3.1.4 Results
Semen samples collected from our 8 dogs showed variable individual semen
concentrations (Table 4). DPAR’s semen was the less concentrated, with a 70x106 Spz/mL, in
contrast to the one from ELWOOD, which was the most concentrated, with a value of 657x106
Spz/mL (Table 4). The collected seminal volume, by gathering both the 2 nd and part of the 3rd
fraction, varied between 2.5 and 5 mL (Table 4). The samples showed a pH within the normal
range for the canine semen (between 6 and 6.5, as shown in Table 4) and remained unchanged
throughout the cryopreservation process. The color of all the semen samples collected was
opaque white (Table 4), aiding in this way to prove the good reproductive status of the animal
and not evidencing a bad method of semen collection.
Considering the morphology of the spermatozoa, all the samples showed acceptable and
similar percentages of normal spermatozoa, with DPAR´sample being the one with the lowest
percentage of abnormal sperm forms. DINGO´s sample was the exception as it presented a
higher amount of abnormal sperm forms when compared to our other samples and even to
reference values. In this individual, it was particularly notorious the high percentage of tail
defects.
30
Table 1 – General characterization of the individual semen samples used in this study.
Animals
Concentration
(x106 Spz/mL)
Volume
(mL)
pH
Color
DPAR
70
4
6
Opac white
DLIT
261
2,5
6
Opac white
ELTON
421
3
6.5
Opac white
ELIOS
200
5
6.5
Opac white
TECH
186
4
6.5
Opac white
DINGO
114
4
6.5
Opac white
UROCH
309
3
6
Opac white
ELWOOD
657
3
6
Opac white
Table 2 – Spermatozoa morphology for the freshly ejaculated samples (control group)
Animal
DPAR
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
%
Normal
%
Abnormal
Abnormal
heads (n)
Intermediate
pieces (n)
Abnormal
tails (n)
Droplets
(n)
Detached
heads (n)
98,5
89,5
87
90,5
91
77,5
93
93
1,5
10,5
13
9,5
9
22,5
7
7
0,5
1,5
3
5
0,5
3
2
3
0
5
4,5
2
1,5
2
2
1
0
3
4
2
4
16
2
3
0,5
0
0,5
0,5
1
0
0
0
0,5
1
1
0
2
1,5
1
0
The results of total and progressive motility of all the 8 dogs can be observed in Table 6
and more clearly in Graphs 1 to 3.
Regarding the evaluation of spermatozoa motility in the CASA system, in the control
group ELTON and DINGO´s samples showed the lowest total motility values and consequently
also the lowest results for the progressive motility. For Treatment A (chilling), no overall
significant variations were observed, having the majority of the animals better total motility for
time 3 (24h chilled) than time 2 (4h). The one exception was DLIT, which showed a slight
decrease in the total motility and lower values for progressive motility at time 1 (1,5h chilled)
(Graphs 1 to 3; Table 6).In treatment B group a significant decrease in the motility parameters
were observed in all the samples of the group. Again, the most deleterious effects of
freezing/thawing over sperm motility were found for DINGO´s (Graphs 1 to 3), with a total and
progressive motility of the frozen samples reduced to 16 % and 12% respectively (Table 6).
31
Table 3 - Total and progressive motility
Total motility (%)
Samples
Controls
DPAR
Treatment A
Treatment B
Time 1
Time 2
Time 3
89
86
85
84
71
DLIT
85
66
67
69
53
ELTON
68
86
72
81
53
ELIOS
80
87
80
88
55
TECH
89
76
81
85
50
DINGO
61
82
71
81
16
UROCH
88
81
87
87
60
ELWOOD
73
81
77
89
61
Progressive motility (%)
DPAR
61
49
52
59
39
DLIT
61
39
40
44
34
ELTON
49
49
53
53
43
ELIOS
54
49
52
50
33
TECH
55
53
52
47
31
DINGO
32
58
50
53
12
UROCH
52
52
54
47
32
ELWOOD
50
51
53
54
43
30
20
10
0
DPAR
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
-10
-20
-30
-40
-50
Chilling T1
Chilling T2
Chilling T3
Freezing
Graph 1 – Individual variation of the total motility with treatment, compared to control samples (Time 0).
The units in Y-axis correspond to the unitary differences between the total motility (%) between the
temperature treated samples (Chilling time 1, 2 and 3, and freezing/thawed) and the controls.
32
30
20
10
0
DPAR
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
-10
-20
-30
Chilling T1
Chilling T2
Chilling T3
Freezing
Graph 2– Individual variation of the progressive motility with treatment, compared to control samples
(Time 0). The units in Y-axis correspond to the unitary differences between the progressive motility (%)
between the temperature treated samples (Chilling time 1, 2 and 3, and freezing/thawed) and the controls.
Graph 3- Representation of the sperm total motility (in the left) and progressive motility (on the right) for
the group of samples according to the treatment. Motility values were obtained from the CASA analysis.
The central bar means the median value, the lower line stands for the lowest value, while the upper one,
its opposite. Outside values correspond to samples considered to be “outsiders” to the core population.
33
Cold treatment was found to significantly influence the total motility as well as the
progressive motility (p<0,0001). However, those differences were found only for
freezing/thawing group in comparison to chilling (treatment A) and to controls. No differences
were found between the controls and treatment A group concerning the total motility or the
progressive motility.
The velocity of movement of spermatozoa was also evaluated and scored as rapid,
moderate, slow and static, by using the CASA system (Table 7). The results from this evaluation
reinforce the information provided by the motility assessment. On what concerns this parameter,
both ELTON and DINGO samples showed lower percentage of rapid spermatozoa, 61 and 50%
respectively, regarding the control group. Regarding the treatment A group, at time 1 DLIT
shows the most important decrease in the rapid spermatozoa, from 81% to 55%, while both
ELTON and DINGO show very acceptable values, an increase in these values being noted (78
and 76%). According to the treatment B, the animal that shows a higher decrease is TECH with a
decrease of 42% of rapid spermatozoa considering the control group (Table 7).
For the analyzed CASA parameters (VAP, VSL, VCL, ALH, STR and LIN), only
averages were taken into consideration (Table 8). On what concerns the velocity of average
pathway (VAP), small changes were observed in treatment A group, with half of the animals
presenting a decrease (DPAR, DLIT, ELTON and UROCH), which was more pronounced at
Times 2 and 3, whilst the other half showed an increase (ELIOS, TECH, DINGO and
ELWOOD). Regarding the treatment B, all the samples presented a decrease in VAP value, with
the exception of TECH and DINGO´ samples. A similar situation was found for the velocity of
straight line (VSL), where for both treatment A and B groups the samples from the
abovementioned animals showed similar fluctuations. Attending the curvilinear velocity (VCL)
for treatment A, a decrease in values was found for Time 1 in DLIT, TECH and
UROCH´ samples, which were also registered towards Time 2 for DLIT, ELTON and UROCH.
For Time 3, all the samples showed increased values in comparison to controls. In contrast, for
treatment B all the samples used showed a decrease in VCL values (Table 8).
34
Table 4 - Velocity of spermatozoa movement
Rapid (%)
Samples
Controls
DPAR
Treatment A
Treatment B
Time 1
Time 2
Time 3
84
78
79
79
60
DLIT
81
55
57
60
44
ELTON
61
78
66
75
49
ELIOS
75
82
76
82
47
TECH
83
71
76
79
41
DINGO
50
76
65
76
13
UROCH
83
74
81
82
47
ELWOOD
67
75
71
84
53
Medium (%)
DPAR
4
8
6
5
11
DLIT
4
10
10
8
9
ELTON
7
8
6
6
3
ELIOS
5
6
4
6
8
TECH
5
5
5
6
9
DINGO
11
5
6
5
3
UROCH
5
7
6
5
13
ELWOOD
6
7
7
5
8
Slow (%)
DPAR
12
13
14
14
28
DLIT
14
32
31
24
41
ELTON
31
14
25
18
36
ELIOS
20
12
19
12
40
TECH
11
23
18
15
46
DINGO
30
17
26
16
26
UROCH
12
18
12
12
38
ELWOOD
27
17
20
11
26
Static (%)
DPAR
0
1
1
2
1
DLIT
1
3
2
8
6
ELTON
1
0
3
1
12
ELIOS
0
0
1
0
5
TECH
1
1
1
0
4
DINGO
9
2
3
3
58
UROCH
0
1
1
1
2
ELWOOD
0
1
2
0
13
35
30
Medium velocity fraction
Rapid fraction
20
10
0
-10
-20
-30
-40
-50
Chilling T1
60
Chilling T2
Chilling T3
Freezing
Static fraction
Slow fraction
50
40
30
20
10
0
-10
-20
-30
Chilling T1
Chilling T2
Chilling T3
Freezing
Graph 4– Individual variation of the velocity of the spermatozoa with treatment, compared to control
samples (Time 0). Upper: for rapid and medium velocity fractions; bottom: for the slow and static
fractions. The units in Y-axis correspond to the unitary differences between the total motility (%) between
the temperature treated samples (Chilling time 1, 2 and 3, and freezing/thawed) and the controls.
When evaluating the lateral amplitude of the head (ALH), in treatment A group, there was found
a decrease in TECH, DINGO and UROCH’s values at Time 1, with only TECH and DINGO
remaining with lower values at Time 2. At Time 3 an increase of ALH values was found for
most samples used, with exception of ELWOOD´s which registered a pronounced decrease for
36
this parameter. In the treatment B group, DLIT and ELIOS´ samples failed to evidence a
decrease in ALH values. Concerning the straightness (STR) movements of the spermatozoa, at
the initial Time for treatment A group samples TECH, DINGO and UROCH showed an increase
toward the controls, with only Dingo´s sample maintaining such increase by Time 3. For
treatment B group, both DPAR and ELIOS showed lower STR-values in comparison with the
controls. For the spermatozoa’s linearity (LIN), in the treatment A, an increase in the values was
found for samples from TECH, DINGO and UROCH at Time 1, and for TECH and DINGO in
Time 2, which were maintained in Time 3. For treatment B, only samples from ELTON, TECH
and DINGO showed an increase in LIN values (Table 8).
Regarding the hypo-osmotic swelling test (HOST) the largest variation in terms of coiled
spermatozoa were noted in DINGO comparing the frozen and fresh semen, while for the chilling
process, a decrease was also noted though in an equilibrate way among all the animals (Table 9).
Throughout the chilling process a slight decrease was noted in the animals, apart from DINGO,
which presented a small increase in coiled spermatozoa at 24h (Graph 5). No statistical
differences were found between the controls and the group for treatment A (p>0.05), no matter
the time considered, however both the controls and treatment A group statistically differed from
the treatment B group (p<0.0001).
Table 5 – Hypo-osmotic swelling test
DPAR
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
Fresh
90.9
94
95
97
92
88
95
ELWOOD
D
96
Chilled 1.5h
96
84
89.1
86
93
93
94
93
Chilled 4h
96
85
89
98
92
91
93
96
Chilled 24h
96
79
89
86
89.1
97
91
93
Frozen
74
50
57
49
45
31
56
49
Fresh
9.1
6
5
3
8
12
5
4
Chilled 1.5h
4
16
10.9
14
7
7
6
7
Chilled 4h
4
15
11
2
8
9
7
4
Chilled 24h
4
21
11
14
11
3
9
7
Frozen
26
50
43
51
55
69
44
51
Hiposmotic Test
HOST
(coiled)
HOST
(not
coiled)
37
Graph 5- Individual variation of the hypo-osmotic swelling test (HOST) of the spermatozoa with
treatment, compared to control samples (Time 0). The units in Y-axis correspond to the unitary
differences between the total motility (%) between the temperature treated samples (Chilling time 1, 2
and 3, and freezing/thawed) and the controls.
Hyposmotic swelling test
100
75
Not
coiled
50
Coiled
DPAR C
DLIT C
ELTON C
ELIOS C
TECH C
DINGO C
UROCH C
ELWOOD C
DPAR R 24
DLIT R 24
ELTON R 24
ELIOS R 24
TECH R 24
DINGO R 24
UROCH R 24
ELWOOD R 24
4
4
4
4
4
4
4
4
DPAR R
DLIT R
ELTON R
ELIOS R
TECH R
DINGO R
UROCH R
ELWOOD R
DPAR F
DLIT F
ELTON F
ELIOS F
TECH F
DINGO F
UROCH F
ELWOOD F
0
DPAR R 1,5
DLIT R 1,5
ELTON R 1,5
ELIOS R 1,5
TECH R 1,5
DINGO R 1,5
UROCH R 1,5
ELWOOD R…
25
Graph 6 – Effect of the cryopreservation process on the coiling of spermatozoa.
38
3.2 Experiment 2- Identification of HSP70 changes in chilled and frozen sperm samples
3.2.1 Goals
In this study, we proposed ourselves to evaluate the presence of the HSP70 in the canine
spermatozoa. Another purpose of this project was to observe the distribution and relocation of
the HSP70 on the canine sperm, under the influence of two different cryopreservation processes,
such as chilling and freezing, treatment A and B, respectively.
The final aim was to compare the distribution of the HSP70 and the quality of the semen,
after the two cryopreservation procedures.
3.2.2 Materials
For the purpose of immunocytochemical analysis, 8 semen samples were collected and
processed,
submitting
them
to
a
smear
procedure
in
silane
coated
slides
(3-
Aminopropyltriethoxysilane, Sigma ®), according to the conventional methodology and fixated
in alcohol at 95%. For each cryopreservation procedure 10 slides of the different samples were
prepared. In this study, HPS70 immunoreaction was study in samples from treatment A, at 24h
of chilling (time 3) and from treatment B, using the fresh semen samples as controls for the
normal intensity and pattern of immunoreaction.
The expression of the proteins in the study was evaluated by the indirect method of the
Streptavidin biotin-peroxidase, using the solution 3,3’-diaminobenzidine tetrahidrocloret (DAB)
as the cromogenic agent.
The immunocytochemical expression of the antibodies was obtained through the indirect
method with the use of the complex streptavidin-biotin peroxidase, using the kit Ultravision
Detection System, Labvision Corporation®, Fremont, CA, USA.
3.2.3 Methods
3.2.3.1 Immunocytochemical study
In order to make the technique as accurate as possible, preliminary studies were
performed, so that the dilution of the antibodies could be determined, the method of the antigenic
recuperation chosen and the different times of the incubation period analyzed, optimizing
39
through these steps, the final results. These trials allow us to identify the most suitable antigen
retrieval method and primary antibody dilution.
The silane coated slides were hydrated through a series of alcohols with decreasing
concentrations (95%, 80%, 70%). Pre-treatment for maximize the exposition of antigen epitopes
was achieve in the microwave oven, (2 cycles of 5 minutes each, at 750 Watts), with the slides
totally immersed in citrate buffer (pH=6.0). Afterwards, endogenous peroxidases were inhibited
with hydrogen peroxide at 3% during 30 minutes, followed by a passage in PBS with detergent,
for 5 minutes. The objective of this step was to avoid a possible unspecific reaction of the
cromogenic agent, DAB, with other peroxidases, besides those of the complex streptavidinbiotin peroxidase.
Then, the cytologies were incubated with Ultra V Block® to reduce the unspecific
reaction of the primary antibody with the endogenous avidine and biotine of the cytologies.
Subsequently, slides were incubate with the primary HSP70 polyclonal antibody (clone Ab31010; AbCam, UK®), diluted at 1:150, in a humid horizontal chamber, overnight (ca. 18 hours)
in a 4ºC temperature. Thereafter, the slides were incubated with Biotinylated Goat AntiPolyvalent® followed by incubation with Streptavidin Peroxidase®. The reaction was visualized
through a solution of tetra-hidrocloret 3,3’-diaminobenzidine (DAB; SIGMA®) for 7 minutes
and counterstained with Gill’s Hematoxilin . Hematoxilin is a basic nuclear stain that colors the
nuclei of the cells in blue, allowing the evidence of the brown color caused by the
immunocytochemical markers.
The slides were then dehydrated in ethanol of increasing concentrations (95%, 95%,
100% and 100%), diaphanized with xylol and mounted using a synthetic resin, Entellan
(Merck®).
The results from the immunostaining for HSP70 were evaluated under a magnification
lens of 100x. A total of 200 spermatozoa were evaluated for determing both the intensity of the
immunoreaction and the sub-celular pattern of immunostaining. The intensity of immunostaining
was scored according to a three-point scale (mild = 1, moderate = 2 and strong = 3) or as
negative. According to available information in other species, the normal pattern of HSP70
location is the acrosome area. As this molecule is associated with the acrosome envelope,
reinforcement of the intensity of immunostaining in the areas not occupied by the nucleus gives
to the labeling the appearance of a halo around the spz head. Thus, the immunostaining was
evaluated and scored as follows:
40
0 – negative – absence of immunostaining in the acrosome area
1 – The halo is observed but the acrosome membrane presented a faint intensity over the
nucleus
2 - The acrossome membrane over the nucleus showed a finely granular immunostaining
of moderate intensity, with the halo reinforcement well marked
3 – The acrosome membrane over the nuclear area shows strong reaction, with a dense
granular pattern, and also the halo is strongly marked.
Further, changes in the subcellular location of the immunostaining were recorded as
being dislocated to the neck, middle piece or tail, which included the intermediate and terminal
piece of the spermatozoon. In the flagellum and neck, the intensity of immunostaining was not
quantified, as it was not possible to distinguish by visualization, differences in the staining
intensity.
3.2.4 Statistical analysis
Statistical comparisons were performed by using the IBM SPSS Statistics Base 19.0 software
for Windows®. Statistical analysis of the effect of treatment over the intensity and sub-celular
location of the immunoexpression for HSP70 was performed using the chi-square and Fisher
exact tests. A p value ≤ 0.05 was regarded as statistically significant.
3.2.5 Results
In all the samples used, HSP70 immunoreaction was found in the majority of sperm cells
analysed according to the expected acrosomal pattern. This pattern was observed in more than
98% of the cells in control samples, which only rarely showed dislocation of HSP70
immunolabeling to the tail of the cell. Never dislocation of the immunoreaction into the sperm
neck was recorded in control samples. Further, in these samples the intensity score 2 prevailed
over all other intensities scores (Figure 15).
41
Figure 15 – Immunocytochemical evidence of HSP70 in the spermatozoa head from the control group
spermatozoa. On the left, the presence of positive immunolabeling was visible for the large majority of
cells present (Scale bar = 30 m). In the right, in higher magnifications it was clearly visible that the
immonoreaction was limited to the acrosome area, whilst the flagellum remained negative for this
molecule (Scale bar = 10 m).
The normal features for HSP70 protein expression, as determined in this study, were the
predominance of moderate intensity of immunoreaction that was exclusively observed over the
acrosome membrane. A change in the percentage of exhibition of score 2 and the dislocation of
the immunoreaction towards the flagellum was considered as corresponding to a reaction to the
treatment.
Among our control samples, small individual differences were observed, with samples
from ELTON and ELIOS showing slightly higher intensities of immunoreaction than those of
the other males (Graphs 7 and 8; Table 9) Overall intensities and pattern of immunolabeling tend
to be maintained in treatment A/time 3 groups (Graphs 7 and 8; Table 9), although DINGO´s
sample evidence a marked increase in the amount of HSP70 immunoreaction in the sperm cell
tail, clearly distinguishing this sample from the cohorts (Figure 16).
In contrast to the observed in samples from Treatment A, the samples submitted to
freezing/thawed procedures showed an important decreased in the intensity of immunolabeling,
along to a dislocation of the immunoreaction towards the sperm tail (Graphs 7 and 8; Table 9)
those changes were particularly notorious in DINGO´sample, but also ELTON´s and ELIOS´.
The smallest changes were observed for UROCH and ELWOOD´samples (Figure 17).
42
3
2
2
2
1
Figure 16 – Immunocytochemical evidence of HSP70 in chilled spermatozoa (Scale bar = 10m). On the
left, smaller magnification. On the right, examples of the three intensity scores for the Spz head used in
this study: 1- weak, 2- moderate and 3- strong.
Figure 17 - Immunocytochemical staining for HSP70 in frozen/thawed samples (Scale bar = 10 m). On
the left, an increased number of cells with weak intensity were found.On the right, besides the different
intensities of immunoreaction for the Spz head, we also found a clear dislocation of the immunoreactions
for HSP70 into the sperm tail (arrow).
The cold treatment significantly influenced the immunoreactions for HSP70 (p=0.006).
Controls did not differed from the treatment group A (p=1.0), but both these groups significantly
differed from the treatment B group (p=0.09 and p=0.20 respectively).
43
HSP 70
100%
Head
80%
level 3
60%
level 2
40%
level 1
20%
level 0
Controls
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
0%
Chilling T3
Freezing
Graph 7– Distribution of HSP 70 intensity in the spermatozoa’s head
HSP 70
100%
80%
Tail
60%
neg
40%
pos
20%
Controls
Chilling T3
Graph 8 – Distribution of HSP 70 in the spermatozoa’s tail
44
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
DPART
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
0%
Freezing
Table 6 – Immunocytochemical classification of the HSP70 in the spermatozoa
Animals
DPAR
DLIT
ELTON
ELIOS
TECH
DINGO
UROCH
ELWOOD
Group
Control
Treatment A
Treatment B
Control
Treatment A
Treatment B
Control
Treatment A
Treatment B
Control
Treatment A
Treatment B
Control
Treatment A
Treatment B
control
ControlA
Treatment B
Control
Treatment A
Treatment B
Control
Treatment A
Treatment B
0
2
2
4
4
6
4
5
2
3
2
3
5
2
3
Degree Head
1
2
21
174
24
170
41
153
16
178
17
172
39
155
4
189
13
181
62
131
4
190
10
182
50
145
14
183
15
180
3
3
4
2
2
5
2
2
4
4
4
5
0
1
2
5
0
6
5
42
8
15
135
145
188
179
52
14
9
5
14
3
3
0
2
25
9
8
17
Neck
Tail
Tail
2
6
86
7
4
73
5
0
84
7
4
50
3
2
0
2
2
0
0
0
0
0
0
0
0
0
0
0
Midpiece
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Negative
198
192
112
193
196
127
195
200
116
193
196
150
197
198
8
4
0
8
0
0
0
0
0
0
0
0
59
1
27
158
141
199
173
42
178
176
3
1
1
0
0
0
6
6
193
194
170
185
190
180
2
3
2
1
0
0
0
0
0
0
0
0
19
5
3
13
180
195
197
187
4. Discussion
Exposition of semen to cold temperatures, in particular to freezing, may induce some
changes in the fertility of treated spermatozoa that may compromise the fertility potential of
sperm (Quintela, 2010). In this study we compared the behavior of canine sperm samples
submitted to chilling and to freezing/thawing procedures with its own control, the freshly
ejaculated semen. In the experiment 1 assessment of the sperm cells quality was performed
according to classical and advanced technologies, such as CASA and HOST. In experiment 2 we
evaluated the changes in HSP70 expression associated to the cold temperature treatments, as
HSP may be involved in the cell response to temperature stressors.
For all the animals with exception to DINGO, the samples from freshly ejaculated semen
used in this work were within the normal range of values accepted for the species. The collected
45
volume was variable within the stud males used, which was probably related to the volume of the
3rd fraction that was collected. Also, the color, pH and morphology of the samples confirmed
that they were normal and suitable for this study. Only DINGO revealed some parameters that
could compromise its fertility, particularly if a reproductive technology such as artificial
insemination was intent to be used. In the case of this male, a large number of abnormal sperm
forms were found, along with worsen motility traits and HOST results.
Nonetheless, individual variations on the concentration were observed. This in fact agrees
with previous studies that demonstrated that individual variations exist in the sperm
concentration between males and also within collections from the same male (Johnston et al.,
2001).
CASA analysis is now currently used in larger artificial insemination centers, as it allows
a less subjective evaluation of sperm motility and velocity. Assessment of sperm motility is not a
direct measure of the ratios between the number of dead and live spermatozoa, but it gives some
information about its fertilizing ability as it reflects its structural and functional competence
(Peña Martinez, 2004). Though the predictive value of sperm morphology in determining
pregnancy rates is low, it is recognized that sperm motility, particularly sperm progressive
motility and speed are important parameters for sperm fertility. The most regarded parameter to
evaluate the semen quality is the motility, however, very frequently the motility parameters are
directly linked to the increase of abnormal Spz forms (Rijsselaere et al., 2004).
In control samples it was also found the existence of small differences concerning the
total and progressive motility, however not compromising the quality of the samples, taking into
account that most of our samples presented what is considered a good quality semen, over 70%
in total motility (Feldman et al., 2004). Only DINGO´s control sample showed slightly lower
values for motility in comparison to those of the other males.
When chilled, in treatment A, at the times 1, 2 and 3, spermatozoa showed a decrease in
the total and progressive motility for 88% of our sample population. This is in concordance with
other published works, and is considered to be normal and usual. The values found in the study
presented here are within the average for the species (Rota et al., 1995; Bessa, 2005). In contrast,
in samples submitted to treatment B, the results for both progressive and total motility show an
important decrease. Although it coincides with the average described in other published data, one
animal in our experiments showed a very bad performance under freezing conditions (DINGO),
46
evidencing a huge decrease in motility. Such an event might allow the classification of this male
as a “bad freezer” (England et al., 1996; Bessa, 2005), even if it was the sole defect found in this
male. However, DINGO also presented other characteristics, such as the increased number of
abnormal sperm cells, which could be related to the low motility parameters and lower number
of rapid cells whilst the static Spz increased particular after treatment.
The hypo-osmotic swelling test (HOST) allows the identification of spermatozoa which
possess a functional intact membrane. The integrity of the sperm cell membrane is of crucial
importance, as it allows maintaining both biochemical and structural integrity of the spermatozoa
and is essential to gamete interaction at fertilization (Quintela, 2010). When exposed to hypoosmotic solutions, biochemically-active, and membrane intact spermatozoa increase their volume
in order to re-establish the equilibrium between the fluid compartment within the spermatozoa
and the extracellular environment. Hypo-osmotic induced cellular swelling implies changes in
both cell size and shape that can be visualized in a phase contrast microscope. The swelling
process culminates with the spherical expansion of the cell membrane covering the tail that
forces the flagellum to coil inside the membrane (Quintela, 2010).
When evaluating the cold treatment effects on sample responses to the hypo-osmotic
swelling test, it was found a slight decrease on the number of coiled spermatozoa’s tails for
treatment A, although all the samples maintained acceptable values, comparable to those
reported in other available studies (Rota et al., 1995). In contrast, for treatment B, a significant
decrease in the coiled spermatozoa was observed for all the samples, which was more
pronounced for DINGO´s sample. This corresponds to a loss of membrane integrity in
frozen/thawed spermatozoa, which has been described for the dog in the available literature
(England et al., 1996; Pinto et al., 2008).
According to the evaluation of morphology, the CASA parameters and the HOST results,
we could categorize the animals according to levels of quality. Crossing information from all
those tests, in particular comparing reduction in parameters such as progressive motility and
increase in membrane damaged spermatozoa, we propose the classification of the animals as
follow:

Excellent freezers, where DPAR, ELTON and ELWOOD would be included;

Good freezers, which would include ELIOS and UROCH;

Acceptable freezers were DLIT and TECH would be included;
47

We could consider DINGO as being a bad freezer, but indeed, DINGO´samples
presented some bad characteristics since the beginning. To be sure, we should
perform additional tests in this animal to ascertain the existence of an eventual
sub-clinical concurrent disease that could interfere with the male fertility, or a
complete breeding examination to determine its (in)fertility level.
The heat-shock response is a highly conserved mechanism in all the organisms, which
may be mediated by extreme proteotoxic insults, such as heat, oxidative stress, heavy metals,
toxins or even bacterial infections (Åkerfelt 2010). It is now believed that heat-shock response is
essential for survival in a stressful environment.
Spermatozoa’ ability to respond to osmotic, oxidative and cryopreservation induced cell
stress is not completely well understood. The sperm subpopulations survival at cryopreservation,
within a single ejaculate may reflect different maturational states, the activation of cell protective
mechanisms or status of membrane stability at the time of cryopreservation. In order to improve
cryopreservation protocols, it is critical to define the characteristics of surviving sperm in order
to minimize the negative effects of cryopreservation on functions important to sperm function.
In controls, a moderate HSP70 immunoreaction was found in the sperm head, at the
acrosomal level. In freshly ejaculated spermatozoa, tail immunostaining was seldom observed.
This was also found in other species, such as the pig and bull, but also for the dog (Volpe et al,
2008). In cold temperature-stressed samples, some changes in the intensity of immunoreactions
and more or less notorious dislocation of the immunostaining into the flagellum. These changes
were less pronounced in chilled samples (treatment A) than in freeze/thawing samples (treatment
B). This sort of modification in the sub-celular location of the immunostaining that co-existed, in
our study, with a decrease in the intensity score for acrosomal staining has been described in the
pig as being a “capacitated” pattern of HSP70 immunoexpression.
Considering that the variation in the pattern for HSP immunostaining is not equal to all
the animals, we should suggest that it could be associated also with different ability to resist to
cold-induced lesions. In fact, the case with poor fertility potential (DINGO) was also the one
showing the higher decreases in the intensity scores in the acrosome and staining dislocation
after treatment B. Those changes occurred in parallel with reduction in motility and a decrease in
the HOST test. It is possible that those events may be associated. In fact, as spermatozoa has a
48
limited synthetic ability, under stressing conditions the cell is much dependent on the ability of
activating the defense mechanisms available. During the cryopreservation of sperm, both
osmotic and oxidative stressors are thought to contribute to membrane and cellular damage
which leads to the reduction in post-thaw motility and viability (McCarthy, et al., 2010). As
known effects of osmotic stress on the sperm cell, there is the production of oxygen free radicals,
leading to the peroxidation of the membrane lipids (Alvarez et al., 1992; McCarthy et al., 2010),
a decrease in progressive and total motility, an increase in the tyrosine phosphorylation of tail
proteins (Liu et al., 2006) and a change in the actin cytoskeleton (Correa et al., 2007).
Spermatozoa are sensible to various kinds of stresses, such as, the oxidative stress, once they
lack cytoplasmatic defenses (Saleh et al., 2002). The sperm plasma membrane is known to
contain lipids in the form of polyunsaturated fatty acids, which are vulnerable to ROS (reactive
oxygen species), that together with the lipids, triggers a chain of chemical reactions which is
called lipid peroxidation (Eddy, 1999).
Being aware that the heat-shock response is a highly conserved mechanism in all the
organisms, which is induced by extreme proteotoxic insults, such as heat, oxidative stress, heavy
metals, toxins or even bacterial infections, it is believed that this heat-shock response is essential
for survival in a stressful environment.
Spermatozoa’s ability to respond to osmotic, oxidative and cryopreservation induced cell
stress is not completely well understood. The sperm subpopulations survival at cryopreservation,
within a single ejaculate may reflect different maturational states, the activation of cell protective
mechanisms or status of membrane stability at the time of cryopreservation. In order to improve
cryopreservation protocols, it is critical to define the characteristics of surviving sperm in order
to minimize the negative effects of cryopreservation on functions important to sperm maturation.
In our study, after the cryopreservation procedure, more exclusively the chilling part, we
could observe a slight decrease on the marking intensity of the heat shock proteins 70 in the head
of the spermatozoa’s in a general point of view, which correlating with the increase noted in the
presence of the HSP in the tail, can evidence the dislocation of them in response to a stress factor
caused by the cryopreservation procedure. This dislocation is far more evident, when we
evaluate the immunocytochemistry of the frozen samples. All the animals show a relevant
decrease of the level 2 head marking, an increase on the level 1 marking, noting a reduction of
the activity or amount of HSP70 on the acrosome/head of the spermatozoa and consequently an
increase in the amount/activity of the HSP70 on the tail region. Considering that the variation is
49
not equal to all the animals we should take into account a great number of variables and
limitations to evaluate the global equation.
During the cryopreservation of sperm, both osmotic and oxidative stressors are thought to
contribute to membrane and cellular damage which leads to the reduction in post-thaw motility
and viability (McCarthy, et al., 2010).
50
5. Final considerations
As find by other authors, we observed that the preservation processes may cause a
decrease in the quality of the dog semen. This loss of quality parameters is far more evidenced in
the freezing procedure, rather than in the chilling one, making it important to further study the
reasons, in order to improve the methods of cryopreservation.
The heat-shock induces significant changes in the viability, motility and consequently in
the fertilizing capacity of the dog spermatozoa. Heat stress causes an increase in the expression
of the HSP70 and a dislocation of these to the caudal part of the spermatozoa, specifically to the
tail of the spermatozoa. There is a diferential increase in the expression of the HSP70 under
different cold treatments, resulting that in the freezing procedure, the described above is strongly
observed, rather than in the chilling procedure, where subtle changes are duly noted. Further
experiments are being undertaken to evaluate better the distribution and role of the HSP70, along
with other HSP, in order to develop cryopreservation protocols which promote cell survival.
51
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58
ANNEXES
59
Annex 1
Total and progressive motility and consequent treatment losses
Total Motility (%) losses associated with treatment
Treatment A
Time 0
(Fresh)
Time 1
Time 2
Time 3
DPAR
89
-3
-4
-5
-18
DLIT
85
-19
-18
-16
-32
ELTON
68
18
4
13
-15
ELIOS
80
7
0
8
-25
TECH
89
-13
-8
-4
-39
DINGO
61
21
10
20
-45
UROCH
88
-7
-1
-1
-28
ELWOOD
73
8
4
16
-12
Treatment B
Progressive Motility (%) losses associated with treatment
Treatment A
Time 0
(Fresh)
Time 1
Time 2
Time 3
DPAR
61
-12
-9
-2
-22
DLIT
61
-22
-21
-17
-27
ELTON
49
0
4
4
-6
ELIOS
54
-5
-2
-4
-21
TECH
55
-2
-3
-8
-24
DINGO
32
26
18
21
-20
UROCH
52
0
2
-5
-20
ELWOOD
50
1
3
4
-7
60
Treatment B
Annex 2
CASA parameters for sperm cells under cold treatments, when compared to the controls (fresh semen).
Treatment A – Chilling (times 1, 2 and 3 respectively, 1.5, 4 and 24h of refrigeration); treatment B –
Freezing/Thawing.
VAP (μm/s)
Controls
Treatment A
Time 1
Time 2
Time 3
Treatment B
DPAR
155.5
152.5
153.8
148.7
132.8
DLIT
ELTON
ELIOS
160.2
137.4
137.3
134.6
142.3
148.5
134
125.7
144.7
133.9
134.4
148.3
115.7
126.2
123
TECH
111.9
148.3
148.8
146.5
125.8
DINGO
UROCH
ELWOOD
105.7
142.7
139.5
147.8
133.1
150.5
143.9
136
147.7
137.2
140.3
153
111.1
108.3
111.3
DPAR
DLIT
130.7
137.6
123.2
111.5
125.9
110.1
124.6
110.8
106.7
99.8
ELTON
ELIOS
TECH
121.9
116.7
89.7
113.5
117.9
127.9
110.6
120.5
123.7
110
117.4
114
115.2
101.5
107.4
DINGO
83.1
125.7
124.1
112
100.7
UROCH
ELWOOD
114.9
119.9
110.1
124.8
110.2
124.9
108.5
122.3
88
97
DPAR
205.8
208.2
213
231.7
183.5
DLIT
ELTON
212
167.1
187.2
201.5
209.5
156.7
237.6
175.4
162.3
152.5
ELIOS
177.9
210.7
189
205
172.9
TECH
DINGO
186.6
187.7
183.9
195.1
192.6
190.2
209
204.6
161.2
143.9
VSL (μm/s)
VCL (μm/s)
UROCH
193.9
179.2
185.3
206.1
152.4
ELWOOD
191.2
199
194.4
212.5
159
DPAR
6.8
7.6
7.7
9.1
6.8
ALH (μm)
DLIT
7.4
7.6
8.9
10.5
16.6
ELTON
5.9
7.8
6.3
7
5.4
ELIOS
6.5
7.7
6.8
7.3
7.0
TECH
7.7
6.6
7.4
7.9
5.7
DINGO
UROCH
8.1
6.5
7.5
4.9
7
7.1
8.1
7.9
6.1
6
ELWOOD
7.3
7.3
7.8
3.3
6.6
61
STR (%)
DPAR
83
78
79
83
77
DLIT
85
80
80
81
85
ELTON
ELIOS
87
84
78
77
86
81
81
74
90
80
TECH
80
84
81
76
83
DINGO
78
83
84
81
88
UROCH
79
81
79
76
79
ELWOOD
84
80
82
78
86
DPAR
DLIT
65
66
60
60
60
54
56
50
59
63
ELTON
73
59
72
65
76
ELIOS
68
58
65
59
60
TECH
50
70
65
56
67
DINGO
UROCH
46
62
65
63
66
61
57
55
71
59
ELWOOD
64
63
65
59
62
LIN (%)
62