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Introduction: An overview of assisted reproduction in the dog, and assessment of male and female fertility
Lazzaro Spallanzani successfully performed the first artificial insemination (AI) in a dog as early as 1780. In 1885, Millais provided a well-documented report on the insemination of three bitches that were on heat simultaneously, with the divided ejaculate of a single dog. Millais also reported superfecundation induced by inseminating a bloodhound bitch with basset hound semen and then naturally mating her to a bloodhound male (Heape, 1897). Since the early attempts by Spallanzani and Millais, AI in bitches has been carried out with fresh semen, chilled semen and frozen-thawed spermatozoa. The first successful use of frozen-thawed dog semen was, however, not reported until recently by Seager (1969), and it took another 25 years before the number of litters registered as resulting from frozen-thawed semen AI began to increase substantially (Linde-Forsberg, 1995). For assisted reproductive techniques, the use of frozenthawed semen is becoming increasingly common. Frozen dog semen is primarily used because it can be shipped over long distances and therefore allows breeding of male and female dogs that cannot be brought together for natural mating, it also allows the use of semen long after the semen was produced, and even after the dog has lost its fertility or died. Common techniques for artificial insemination with frozen-thawed spermatozoa include vaginal, trans-cervical intrauterine or surgical intrauterine AI. Success rates vary between techniques, and are affected by the timing of insemination and number of spermatozoa used for each insemination. Cryopreserved spermatozoa yield lower conception rates than fresh spermatozoa, presumably because of the shortened life span of the spermatozoa post-thaw and compromise of various properties required for fertilization (Peña et al., 2003b). Even though AI with frozen-thawed semen is a routine procedure in many parts of the world today (Linde-Forsberg et al., 1999), there is substantial room for improvement in areas such as; improving conception rates to match those resulting after natural mating (Farstad and Berg, 1989), decreasing sperm dose to achieve optimum results with the least possible number of valuable spermatozoa (Nöthling et al., 2000; Nöthling et al., 2005; Tsutsui and Ejima, 1988; Tsutsui et al., 1989b; Wilson, 1993) and optimising the timing of insemination to reduce the number of inseminations required (Concannon et al., 1977; Linde-Forsberg, 1995; Tsumagari et al., 2003; Tsutsui, 1989).
In the case of breeding animals, sub-fertile individuals are increasingly allowed to remain in the breeding population and are instead managed more intensively to maximise their fertility. In the case of males, detailed evaluation of semen samples are required to give an accurate indication of the quality, and likely fertility, of the semen. In order to predict fertility of a male animal, laboratory assays would be more useful if they proved to better able correlate with fertility of a given semen sample than the tests currently being used. Conventional light microscopic evaluation has been used to evaluate the principle quality parameters of dog semen including concentration, and the percentages that are motile or morphologically normal. The limitations of light microscopic techniques commonly used in private practice include subjectivity and variability due to evaluator (in)experience, the small number of spermatozoa that can be assessed, and artefacts induced by the handling of the semen sample (Rijsselaere et al., 2005).
During the last decade, several new methods have been developed that may enable more accurate prediction of the fertilising capacity of spermatozoa / an ejaculate / an individual male. Computer Aided Sperm Analysis (CASA) and fluorescent staining techniques analysed by flow cytometry can objectively assess various different attributes that a spermatozoon must possess in order to fertilize an oocyte, and do so rapidly in large numbers of spermatozoa. Each of the attributes examined should relate to the fertilizing capacity of a spermatozoon and, by looking at large numbers of sperm, the population destined for insemination. Sperm attributes required for fertilization include motility, the ability to capacitate, an intact acrosomal membrane, progesterone receptors, and condensed, intact, and resistant sperm chromatin (Graham and Mocé, 2005). However, neither light microscopic, nor modern laboratory methods seem to consistently correlate with fertility in vivo (Graham and Mocé, 2005; Rijsselaere et al., 2005).
This inconsistency relates to the fact that every spermatozoon must possess a wide range of different attributes to complete its role in fertilizing an oocyte. A single assay that measures only a single attribute is therefore unlikely to be able to evaluate the fertilising potential accurately. Theoretically, the probability that a single assay or a combination of a number of assays will correlate to actual fertility will be higher if the assay(s) permits the simultaneous identification of sub-populations of spermatozoa that satisfy a wide range of properties required. Due to the more sophisticated and broader array of assays now available, modern assay combinations are expected to give a better indication of the fertilising potential of spermatozoa than conventional light microscopic methods.
Chapter 1 General introduction and literature review
1.1 Introduction: An overview of assisted reproduction in the dog, and assessment
of male and female fertility
1.2 The female: Selected aspects of the reproductive biology of the bitch
1.2.1 Gross anatomy of the female reproductive organs
1.2.2 Overview of the bitches’ reproductive cycle
1.2.3 Measuring fertility in the bitch
1.2.4 Artificial insemination with frozen-thawed semen in the bitch
1.2.5 Effect of the number of spermatozoa per insemination on fertility
1.2.6 Heterospermic insemination (HI)
1.2.7 Embryonic developmen
1.2.8 Stages of reproductive failure in the bitch prior to parturitio
1.2.9 Paternity testing in the dog
1.3 The Male: Selected aspects of the biology of spermatozoa and semen
1.3.1 The spermatozoon from formation to fertilization
1.3.2 Sperm motility
1.3.3 Sperm membrane integrity and viabilit
1.3.4 Sperm morphology
1.3.5 Sperm capacitation
1.3.6 Acrosome reaction (AR)
1.3.7 Sperm chromatin integrity
1.3.8 The role of prostatic fluid in the domestic dog
1.3.9 Interaction between the spermatozoa of dogs and the reproductive tract of the
bitch
1.3.10 Semen evaluation
1.3.11 Sperm functions and available evaluation techniques
1.4 The relationship between sperm evaluation results and in vivo fertility
1.5 Research questions addressed in this thesis
1.6 Hypothese
1.7 Objectives
Chapter 2 The probability of more than one concept deriving from one ovulatory
follicle in the bitch
2.1 Introduction
2.2 Materials and methods
2.2.1 Experiment 1
2.2.2 Data recording
2.2.3 Data analysis
2.2.4 Experiment 2
2.3 Results
2.3.1 Experiment 1
2.3.2 Experiment 2
2.4 Discussion
2.5 Conclusion
Chapter 3 Prediction of the optimal time for insemination using frozen-thawed semen in a multi-sire insemination trial in bitches
3.1 Introduction
3.2 Materials and Methods
3.2.1 Experimental animals
3.2.2 Semen processing, thawing and evaluation
3.2.3 Bitches
3.2.4 DNA sampling
3.2.5 DNA analysis
3.2.6 Data and Statistical Analysis
3.3 Results
3.3.1 DNA and parentage analysis
3.3.2 Comparison of fertility between insemination days
3.3.3 The effect of day of insemination on gender of offspring
3.4 Discussion
3.4.1 Main finding
3.4.2 The model used
3.4.3 The poor fertility of Day 5 inseminations
3.4.4 The poor fertility of Day 7 inseminations
3.4.5 The best days on which to inseminate a bitch with frozen-thawed spermatozoa
3.4.6 The effect of day of insemination on gender
3.4.7 Conclus
3.4.8 Acknowledgments
Chapter 4 Validation of Merocyanine 540 staining as a technique for assessing
capacitation-related membrane destabilization of fresh dog sperm
4.1 Introduction
4.2 Materials and methods
4.2.1 Experimental animals
4.2.2 Reagents
4.2.3 Media used to inhibit or stimulate capacitation
4.2.4 Semen collection and processing
4.2.5 Staining
4.2.6 Statistical analysis
4.3 Results
4.3.1 Staining patterns observed in dog sperm with M540-YP and EH-TP
4.3.2 Comparison of viability assessed using EH versus YP
4.3.3 Comparison of capacitation in viable sperm assessed with M540-YP and
TP-EH
4.3.4 The effect of incubation in various media with known effects on capacitation
over time on the percentage of viable sperm that are capacitated as detected b
M540-YP
4.4 Discussion
4.4.1 Conclusion
4.4.2 Acknowledgements
Chapter 5 Relationship between in vitro characteristics of frozen-thawed dog sperm and in vivo fertility in a multi-sire insemination trial
5.1 Introduction
5.2 Materials and Methods
5.3 Results
5.4 Discussion
Chapter 6 Summarizing discussion
Reference List
