Borges, Paulo Alexandre Paulos Comparison of canine sperm quality under different temperature ...

72 Pages·2011·2.11 MB·English

by  Paulo Borges

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
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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.
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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.
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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
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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

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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

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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
PGF – prostaglandin F
2α 2α
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
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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.
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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!
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