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

72 Pages·2011·2.11 MB·English
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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 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 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

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3.1 EXPERIMENT 1 - EVALUATION OF DOG SEMEN QUALITY AFTER CHILLING AND FREEZING . 1996; 46 (1): 165-171. Ettinger SJ and Feldman EC, Textbook of veterinary internal medicine : diseases of the dog and the cat. In: Book Textbook of veterinary internal medicine : diseases of the dog
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Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.