Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and

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1 Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

2 SOME ASPECTS OF ARTIFICIAL INSEMINATION IN THE BITCH, USING FROZEN SEMEN A thesis presented in partial fulfilment of the requirements for the degree of Master ofveterinary Science at Massey University Marion Scobbie Wilson 1992

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4 ii Abstract The development of freezing techniques for dog semen allowing long term storage of semen from valuable stud dogs and its use locally or thousands of miles away has opened up exciting new prospects for dog breeding. However, it has not been possible to consistently achieve acceptable pregnancy rates and litter sizes with frozen semen. The reason for this arises from the many factors involved in processing and inseminating frozen canine semen and their complex inter-relationships. In order to successfully use semen prepared in this way it is essential to understand the effect processing has on the fertilising capacity of sperm and the implications this may have regarding the techniques required for semen insemination. The key problems revolve around establishing the period for which such semen remains able to fertilise ova, being able to identify when ovulation takes place so that timing of insemination occurs when ova are ready for fertilisation, and having a technology that will allow placement of the semen in a position from which fertilisation is likely to be achieved. In this study 18 bitches were divided into three groups on a random basis. Group 1 bitches were inseminated twice with four straws of semen (a total insemination dose of 240 to 280 x 10 6 live sperm), the semen being deposited into the uterus using the 'Norwegian' insemination technique. Group 2 bitches received the same insemination dose deposited into the uterus using the 'Endoscopic' technique (a technique developed for this trial), and Group 3 bitches received 25% of the semen dose in Group 1 and 2 (a total insemination dose of 60 to 70 x 10 6 live sperm), inseminated using the 'Endoscopic' technique. The semen all came from one stud dog. Insemination timing was based on blood progesterone concentration determined using a commercial ELISA kit. The results from the kit were compared with RIA determinations of plasma progesterone to validate its accuracy. Visual observations of the bitch, vaginal cytology and vaginal endoscopy observations were also considered in relation to the timing of insemination. The pregnancy rate over all three groups was 83.3% with a mean litter size of 7.5 (range 4-11) pups. There was no difference in pregnancy rate or litter size between the groups. The insemination protocol adopted in respect of semen dose, insemination timing and site of deposition of semen demonstrated that it was possible to achieve good pregnancy rates and litter sizes following the insemination of frozen semen. The new 'Endoscopic' method of depositing the semen into the uterus was shown to provide an effective alternative method to the 'Norwegian' technique. The results of insemination with a significantly lower sperm dose of frozen semen demonstrates that equivalent pregnancy

5 iii rates and litter sizes to those achieved with high doses of semen, can be achieved when the semen used is of high quality. It was also shown that using blood progesterone concentration as the basis for timing insemination provides an alternative and perhaps more appropriate method of ensuring insemination occurs at the optimum time than traditional methods used; the progesterone kits were found to be reliable in this trial and were particularly useful because they were simple and provided results within hours.

6 iv ACKNOWLEDGEMENTS I wish to thank my supervisor Professor Des Fielden for his guidance and review of the manuscript. Thanks also to Dr Alan Alexander, Animal Health Services Centre, who provided many of the bitches for this trial and who as co-supervisor provided the impetus and enthusiasm to undertake this study in the first place and see it to its conclusion. Dr Keith Lapwood provided the progesterone radioimmunassays; Dr Hilary Burb idge performed the bitch spays and Nicky Ryan undertook the radiology - my thanks to them all. I am particularly grateful to have been awarded a Phyllis Irene Grey Fellowship which made this study possible.

7 Table of Contents Page Abstract Acknowledgements List of Figures List oftables ii iv vi vii Chapter 1 Introduction 1 Chapter 2 Literature Review 3 Chapter 3 Materials and Methods 31 Chapter 4 Results 45 Chapter 5 Discussion 55 Chapter 6 Conclusions 69 Appendix A 70 Appendix B 71 Appendix C 90 Appendix D 94 References 95

8 vi LIST OF FIGURES Figure Page 1 Ventrolateral view of anterior vagina 6 2 Dorsal view of vagina and vestibule 6 3 Vaginal smear from a bitch in oestrus 34 4 Vaginal smear from a bitch in early metoestrus 34 5 Endoscopic equipment 35 6 Bitch restrained for endoscopic examination 35 7 Endoscopic appearance of vagina - early proestrus 37 8 Endoscopic appearance of vagina - mid proestrus 37 9 Endoscopic appearance of vagina - late proestrus Endoscopic appearance of vagina - external cervical os Endoscopic appearance of vagina- early oestrus Endoscopic appearance of vagina - mid oestrus Endoscopic appearance of vagina- 48 hours later than Fig Endoscopic appearance of vagina - late oestrus Sowside Rapid ELISA progesterone kit "Norwegian" catheters Ventrolateral view of anterior vagina - ''Norwegian'' catheter in position Ventrolateral view of anterior vagina - endoscope in position External cervical os viewed through endoscope - catheter in position GB 17 /GB30 with her second litter in the trial Comparison of progesterone concentrations: RIA and Sowside kit Comparison of progesterone concentrations: RIA and Premate kit a-d Radiographs demonstrating insemination using the ''Norwegian'' technique a-d Radiographs demonstrating insemination using the ''Endoscopic'' technique 53

9 vii LIST OF TABLES Page I Commonly used extenders for freezing canine semen 22 II Pregnancy rates and litter sizes achieved with frozen semen 30 III Bitch fertility after transcervical insemination of frozen semen 45