- Email: [email protected]
Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen
Theriogenology
TRANSCERVICAL
42:147-l
57,1994
ARTIFICIAL INSEMINATION OF AUSTRALIAN MERINO EWES WlTH FROZEN-THAWED SEMEN
D.P. Windsor,1 A.Z. Szell,l C. Buschbeck, A.Y. Edward,1 J.T.B. Milton3 and B.C. Buckrell2 lSheep Industries Branch, Great Southern Agricultural PO Box 757 Katanning, Western Australia 2Department of Population Medicine, University Guelph, Ontario NlG 2Wl Canada 3School of Agriculture, University of Western Nedlands, Western Australia 6009
Research Institute 6317 of Guelph Australia
Received for publication: November 2, 1993 Accepted: April 24, 1994 ABSTRACT We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical Al; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment l(771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120/251) was higher (P
Acknowledgements Financial assistance was provided by the Wool Research and Development Corporation and the Meat Research Corporation. Pregnecol and Ovaluon used in Experiment 1 were donated by the manufacturers. We thank S. Dorman (Dale River WA) and 3. Caldwell (Mt Barker, WA) for the use animals, and their staff for valuable assistance. Technical assistance was provided by DAWA and UWA staff. We thank J. Dhaliwal for advice and assistance with the statistical analyses and M. Johns for pregnancy scanning.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
148
INTRODUCTION Fertility following artificial insemination (AI) with frozen-thawed ram semen is limited by the inability of current cryopreservation procedures to maintain the capacity of spermatozoa to transit the cervix. Despite maintaining their ability to fertilize (19), most frozen-thawed ram spermatozoa are unable to colonize or traverse the length of the cervix (20,21) and are rapidly lost from the female reproductive tract (16). This results in an inadequate number of spermatozoa reaching the oviducts to ensure fertilization. Vaginal or cervical insemination with frozen-thawed semen in Merino ewes often results in conception rates below 20% under Australian conditions (23). Consequently, the commercial use of AI with frozen-thawed ram semen was extremely limited until the advent of laparoscopic insemination (X3), which permits deposition of semen directly into the uterine lumen, thus bypassing the cervical barrier and restoring fertility (2,22). An alternative to intrauterine AI by laparoscopy is the passage of an insemination pipette into the uterus via the cervix (as commonly practised in the cow). Despite a number of reports of successful transcervical AI in the ewe with both fresh and frozen semen (l, lO-13), such techniques have not been adopted by the Australian sheep industry. The recently described Guelph System for Transcervical Artificial Insemination (GST-AI) has been reported to be capable of achieving intrauterine insemination via the cervix in a large proportion of ewes, and to produce commercially acceptable conception rates in a number of breeds (3,4, 14, 15). This paper describes the first study to evaluate the GST-AI technique for insemination of Australian Merino ewes with frozen semen under commercial conditions. The pregnancy rates achieved by GST-AI were compared with those achieved using standard techniques for cervical or laparoscopic insemination. MATERIALS AND METHODS Experimental Design Experiments 1 and 2 were both a single factor design using 3 different AI procedures (laparoscopic, cervical or transcervical AI). Ewes from the transcervical AI groups were scored according to depth of insemination at the time of AI. The GST-AI technique was performed by an experienced operator in Experiment 1 and by 2 inexperienced operators in Experiment 2 Livestock Experiment 1. Ewes (5 to 7 yr old) were obtained from 3 commercial Merino flocks run separately on the same property until 2 wk before es&us synchronization. Ewes from each of the flocks were randomly allocated to 3 equal sized groups and ear tagged for individual identification. The mean liveweights (4 wk prior to insemination) of a sample of 50 ewes from
Theriogenology
each of the 3 treatment groups did not differ. The 3 treatment groups were managed as a single flock (77 1 ewes) from 2 wk prior to sponge insertion until pregnancy diagnosis. Exneriment 2. Individually identified Merino ewes (3rh yr of age) were managed as a single flock (555 ewes) throughout the study. These ewes were randomly divided into 3 experimental groups of equal size prior to insemination. The mean liveweights (at sponge withdrawal) of a sample of ewes from each of the treatment groups did not differ. The postpartum interval for the ewes in both experiments was at least 7 mo, and an unknown proportion of ewes had not lambed within the previous 19 mo. The lambing histories of individual ewes were also not known. Source of Semen All ewes were inseminated with commercially frozen semen from 1 of 2 American Suffolk rams (Experiment 1) or a single Poll Merino ram (Experiment 2). The semen from each ram and ejaculate was allocated across aI1 treatments. Semen had been diluted threefold in Trisyolk-glycerol extender, frozen as pellets (9) and stored at -196°C until required for insemination. Thawed samples were examined randomly during the course of the inseminations. Post-thaw motilities ranged from 40% to 60%. Estrus Synchronization Ewes were hormonally synchronized by treatment for 12 to 14 d with 30 mg flugestone acetate intravaginal spongesa and 350 IU PMSGb at the time of sponge withdrawal. Artificial insemination was started at fixed times after sponge removal. Only those ewes detected in estrus (marked by testosterone treated teaser wethers) prior to the scheduled time of insemination were inseminated in Experiment 1. All ewes were inseminated in Experiment 2.
Semen pellets were thawed in dry glass test tubes in a water bath at 37°C . Ewes were inseminated using one of the following 3 regimens: 1) Laparoscopic AI was performed by an experienced operator, as described by Killeen and Cafferey (18), with ewes restrained in a laparotomy cradle. The reproductive tract was located by laparoscopy and 30 pL of undiluted semen were injected into the lumen of each uterine horn (approximately 60 million spermatozoa). Local anaesthesia (2 mL of 2% lignocainec) was provided at the points of cannula insertion. Inseminations were performed at 5 1 to 54 h after sponge withdrawal. 2) Cervical AI was performed by an experienced operator with the ewes restrained over the rail (9). Semen was deposited at the entrance of the cervical OS. No attempt was made to increase the depth of semen deposition within the cervix. Ewes were inseminated with either 200 pL aExperiment 1, Ovakron, Essential Sterolin Products, Johannesburg, South Africa; Experiment 2, Chronogest, Intervet, Lane Cove NSW, Australia. bPregnecol, Horizon Animal Reproduction, Manly NSW, Australia. cIllium lignocaine, Troy Laboratories, Smithfield NSW, Australia.
150
Theriogenology
(Experiment 1) or 100 pL (Experiment 2) of undiluted semen (containing approximately 200 or 100 million spermatozoa, respectively). Inseminations were performed at 54 to 56 h after sponge withdrawal. 3) Transcervical insemination (GST-AI) was performed at 51 to 56 h after sponge withdrawal as described by Halbert et al. (14). Ewes were restrained in dorsal recumbencyd, and the cervix was located with the aid of a tubular perspex speculum illuminated by a fibre optic light source. The cervix was stabilized by grasping the surrounding tissue with a pair of Bozman forceps. A fine (18 G) stainless steel insemination pipette (attached to a modified IMV insemination gun) was introduced into the cervical opening. Gentle probing was used to locate the openings in the cervical rings and to pass the pipette into the uterine lumen. Thawed semen pellets (200 pL) were diluted six-fold with Dulbecco’s PBSe and loaded into 0.5 mL straws for insemination. The dead space in the pipette meant that only about 0.4 mL of semen could be expelled. Each ewe, therefore, received the equivalent of approximately 80 pL of undiluted (post-thawing) semen (containing approximately 80 million spermatozoa). Insemination was performed by an experienced operator in Experiment 1 and by 2 newly trained operators in Experiment 2. The ewes were mated to backup rams 14 d after the completion of AI. Estimation of Cervical Penetration The depth of insemination achieved using the GST-AI technique was estimated by the given inseminator and recorded for each ewe using to the following scale: 1) Uterine insemination was judged to have been accomplished when the insemination pipette could be inserted into the cervix to its full length (about 8 cm). 2) In deep iutracervical insemination the AI pipette was estimated to have been inserted at least 2.5 cm into the cervix, and little semen back flow was apparent. 3) Shallow intracervical insemination was scored when pipette insertion was judged to be less than 2.5 cm deep, or when substantial semen back flow occurred after insemination. 4) Semen was deposited at the cervical OSor iu the vagina. Pregnancy Diagnosis Pregnancy to AI was determined by real-time ultrasonographyf 65 to 75 d after insemination. Ewes were scanned using a 5MHz external linear array and fetuses attributable to AI or to backup rams distinguished on the basis of fetal size and cotyledon development as described by Johns (17). Statistical Analyses The effect of insemination technique and depth of transcervical AI on the pregnancy rate was determined by logit analyses using Genstat 5 (24). Predicted proportions for different treatments were compared using Z-tests. Day, operator and ram effects were compared by Chi-square analyses.
dcommodore turning cradel, Poldenvale, Taunton UK. eCommonwealtl~ Serum Laboratories, Melbourne Victoria, Australia. fToshiba Sonolayer, Shimoshigami, Japan.
Theriogenology
151
RESULTS Experiment 1 Wrthin the GST-AI treatment group, transcervical uterine insemination was judged to have been successfully performed in 76% of the ewes. The proportion of ewes falling into each GST-AI depth of insemination category is shown in Figure 1.
i
2 GST-AI
3
4
penetration score
Figure 1. Cervical penetration rates using Guelph system for transcervical AI (GST-AI, Experiment 1).
The overall pregnancy rate resulting from GST AI (26%, 68/264) was greater (P~0.01) than for cervical AI (9%) but lower (P
Theriogenology
152
Cervical
GST-AI (4)
GST Al (3)
GSTAI (2)
GST-AI (1)
Laparoscopic
Insemination technique
Figure 2. Effect of AI technique and depth of insemination (in parentheses) by Guelph system for transcervical AI (GST-Al) on the pregnancy rate (Experiment 1).
Experiment 2 The rates of cervical penetration achieved by the 2 less experienced operators performing the GST-AI inseminations in Experiment 2 were lower than those achieved in Experiment 1 by the experienced technician. The proportion of ewes falling into each insemination category was influenced by operator (P
Theriogenology
153
19136
761146
10136 281146
Cervical penetration score
Figure 3. Depth of insemination into the cervix by the Guelph system for transcervical AI (GST-AI, Experiment 2).
31/78 40-
Cervical
GST-Al(4)
GST-AI (3)
GST-AI (2)
721187
GST-Al (1) Laparoscopic
Inseminationtechnique
Figure 4. Effect of AI technique and depth of insemination (in parentheses) by Guelph system for transervical AI (GST-AI) on ewe pregnancy rates (Experiment 2).
154
Theriogenology
Cervical
Laparoecopic
GST-AI
Insemination technique
Figure 5. The effect of insemination technique on the proportion of ewes becoming pregnant to a subsequent backup mating (Experiment 2).
DISCUSSION The rates at which a pipette could be passed through the cervix to effect uterine insemination in this study represent a substantial improvement over those previously described for Australian Merino ewes. This may be attributable to the improved pipette design and modified method of ewe restraint incorporated in the GST-AI technique. Salamon and Lightfoot (26) found that although the depth of insemination could be increased by the application of cervical traction, a conventional AI pipette could be inserted to a depth greater than 2 cm in only 13% of the ewes. In a more recent study, Eppleston (7) reported that insemination beyond 2 cm was achievable in 6 to 38% of the ewes, but that cervical penetration beyond 3 cm was only possible in 3% of the ewes. Cervical penetration or subsequent fertility was not increased significantly by reducing the AI pipette tip diameter (from 2 to 1 mm), or by the use of a helicoid pipette (7). By contrast, in Experiment 1 an experienced operator performed succussful tmnscervical intrauterine insemination in 76% of the ewes. Fukui and Roberts (lo- 13) reported that a fine pipette (similar to the one used for the GST-AI procedure) could be passed through the cervix of 35 to 50% of estrous ewes when the animals are restrained in a standing position. One of the two inexperienced operators (Operator 1) achieved intrauterine insemination in 52% of the ewes in Experiment 2. While this figure (and that for Experiment 1) is somewhat lower than those recently reported for GST-AI in North America (>90%; 3,4), it is comparable to cervical penetration rates reported following the first field trials using the technique (54%; 15). This suggests that transcervical AI in the Merino may be no more
Theriogenology
155
difficult than in the larger North American meat sheep breeds. The very high penetration rates achieved in Canada may be associated with insemination after a shorter postpartum interval (3 to 4 mon; 4) than is generally possible under most Australian flock management systems. Preliminary results indicate that higher penetration rates may be achieved in older Merino ewes with a shorter post-lambing interval (28). The overall GST-AI conception rates (26 and 19%, respectively, for Experiments 1 and 2), while higher than those achieved by insemination at the cervical OS,were lower than those for laparoscopic insemination. In the case of Experiment 1, intrauterine insemination by GST-AI also produced lower pregnancy rates than intrauterine insemination by laparoscopy. Similar results have been reported elsewhere (25) for the goat doe, with transcervical uterine insemination resulting in lower conception rates than laparoscopic insemination, even when larger doses of spermatozoa were used for the former. The factors controlling this phenomenon are poorly understood, and may need to be fully elucidated if transcervical AI is to have wide application. Cervical traction (as used with the GST-AI technique) is a potential source of reduced fertility (26) and may have to be carefully controlled. The use of pelleted semen with equipment originally designed for straws, and the consequent need for an additional semen dilution before insemination may also have contributed to lower pregnancy rates. There is also little information available on the optimum time for GST-AI relative to sponge withdrawal. It may well vary from that used in our experiments. The pregnancy rate following intrauterine transcervical AI in Experiment 2 (39%) did not differ from that achieved by laparoscopy (38%) While somewhat low, this pregnancy rate is well within the range experienced by Australian operators (8). The lower overall pregnancy rates in Experiment 2 may have been due to the use of a single ram of unknown fertility, as the fertility of frozen semen from different rams varies substantially when used for intrauterine insemination (5,6). Any external factors (including rainfall at sponge withdrawal) acting to reduce ewe fertility at laparoscopic insemination will presumably have had a consistent effect across all treatments, because ewe management was identical throughout. The results of this experiment are consistent with previous reports that intrauterine GST-AI is capable of producing similar results to laparoscopy (4, 15). Fukui and Roberts (10) found no difference in lambing rate between Merino ewes inseminated cervically with fresh semen (64%) or with frozen semen using transcervical AI (51%). The difference in relative conception rates to laparoscopy and GST-Al between Experiments 1 and 2 is difficult to explain, particularly because the different locations and sources of sheep and semen prevent direct comparisons. Further investigation will be required to determine whether intrauterine GST-AI can be expected to consistently produce similar results to laparoscopic AI. Neither Experiment 1 nor 2 showed evidence for increased fertility as the depth of deposition of semen within the cervix by GST-AI increased, despite the strong relationship between depth of AI with frozen semen and subsequent ewe fertility described elsewhere. The relative fertility achieved by deep cervical AI was lower than that described in other studies (7, 26), possibly due to the use of lower spermatozoa doses than would normally be recommended (9) for cervical AI with frozen-thawed semen. The use of reduced spermatozoal numbers (to allow direct comparison with GST-AI) for cervical AI iu Experiment 2 also produced an extremely low conception rate, which did not differ from that for shallow GST-AL The very
156
Theriogenology
small numbers of pregnant ewes in the intracervical insemination categories may also have prevented the statistical detection of any depth effects on fertility. The likelihood of ewes conceiving to backup ram mating at the estrus periods immediately following the inseminations in Experiment 2 was unaffected by insemination treatment. This indicates that the manipulations of the cervix associated with GST-AI did not compromise subsequent ewe fertility to a greater extent than either cervical or laparoscopic insemination, even when performed by inexperienced practitioners. In conclusion, the results of Experiment 2 indicate that intrauterine insemination by GST-AI has the potential to provide conception rates comparable to those for laparoscopic insemination when used with frozen semen in Merino ewes. However, further research will be needed into the factors controlling the rates of cervical penetration. A previous study has suggested that the repeatability of cervical penetration in Merinos is quite high and that it may be possible to screen ewes for their suitability for manscervical AI (11). The causes of reduced transcervical conception rates observed in Experiment 1 and those previously reported in the goat may also need to be elucidated before overall pregnancy rates become commercially acceptable. REFERENCES 1. Andersen V, Aamdal J, Fougner J. Interuterine and deep cervical insemination with frozen semen in sheep. Zuchthygiene 1973;8:113-118. 2. Armstrong DT, Evans G. Intrauterine insemination enhances fertility of frozen semen in superovulated ewes. J Reprod Fertil1984;71:89-94. 3. Buckrell B, Buschbeck C, Gartley C, Kmetsch T, McCutcheon W, Martin J, Penner, Walton J. A breeding trial using a transcervical technique for artificial insemination in sheep. Proc 12th Int Cong Anim Reprod Art Insem 1992;12:1531-1533 ). 4. Bucknell BC, Buschbeck C, Gartley C, Rroetsch T, Walton J.S. Further development and testing of a transcervical technique for AI in sheep. Theriogenology 1994 (accepted for publication). 5. Butler LG, Maxwell WMC. The effect of site and location on results of on-farm intrauterine insemination with frozen-thawed semen. Proc Aust Assoc Anim Breed Genet 1988;7:394-397. 6. Eppleston J, Maxwell WMC. Between ram variation in the reproductive performance of ewes inseminated with frozen semen. Proc Aust Sot Reprod Biol 1991;23:130. 7. Eppleston J. Studies on the Fertility of Frozen-ThawedRam Semen. Ph.D. Thesis, The University of New South Wales, Sydney, 1992. 8. Evans G. Application of reproductive technology to the Australian livestock industries. Reprod Fertil Dev 1991;3:627-650. 9. Evans G, Maxwell WMC. Salamons’ Artificial Insemination of Sheep and Goats. Butterworths, Sydney, 1987. 10. Fukui Y, Roberts EM. Fertility of non-surgical intra-uterine insemination with frozen-pelleted semen in ewes treated with prostaglandin F20: In: Tomes, GJ, Robertson, DE and Lightfoot, RJ (eds) Proc Int Congr Sheep Breed., Western Australian Institute of Technology, Perth, 1976;482-49. 11. Fukui Y, Roberts EM. Repeatability of non-surgical intrauterine technique for artificial insemination of the ewe. Theriogenology 1977;8:77-81.
Theriogenology
157
12. Fukui Y, Roberts E.M. Sperm transport after non-surgical intrauterine insemination with frozen semen in ewes treated with prostaglandin F20, J Reprod Fertil. 1977;51:141-143. 13. Fukui Y, Roberts EM. Further studies on non-surgical interuterine technique for artificial insemination in the ewe. Theriogenology 1978;10:381-393. 14. Halbert GW, Dobson H, Walton JS, Buckrell BC. A technique for transcervical intrauterine insemination of ewes. Theriogenology 1990;33:993-1010. 15. Halbert GW, Dobson H, Walton, JS, Sharpe P, Buckrell BC. Field evaluation of a technique for transcervical intrauterine insemination of ewes. Theriogenology 1990;33:1231-1243 . 16. Hawk HW. Sperm survival and transport in the female reproductive tract. J Dairy Sci 1983;66:26452660. 17. Johns MA. Estimation of the week of conception in Merino ewes using real-time ultrasonic imaging. Aust .I Exp Agric 1993;33:839-841. 18. Killeen ID, Caffery GJ. Uterine insemination of ewes with the aid of a laparoscope. Aust Vet J 1982;59:95. 19. Lightfoot RJ, Salamon S. Fertility of ram spermatozoa tiozen by the pellet method. I. Transport and viability of spermatozoa within the genital tract of the ewe. J Reprod Fertil1970;22:385-398 . 20. Lightfoot RJ, Salamon S. Fertility of ram spermatozoa frozen by the pellet method. II. The effects of method of insemination on fertilization and embryonic mortality. J Reprod Fertil1970;22:399-408. 21. Mattner PE, Entwistle KW, Martin ICA. Passage, survival and fertility of deep-frozen ram semen in the genital tract of the ewe. Aust J Biol Sci 1969;22:181-187. 22. Maxwell WMC, Butler LG, Wilson HR. Intrauterine insemination of ewes with frozen semen. J Agric Sci Camb. 1984;102:233-235. 23. Maxwell WMC, Hewitt LJ. A comparison of vaginal, cervical and intrauterine insemination of sheep. J Agric Sci Cambridge 1986;106:191-193. 24. Payne RW, Lane PW, Ainsley AE, Bicknell KE, Digby PGN, Harding SA, Leech PK, Simpson HR. Todd AD, Verrier PJ, White RP, Gower JP, Tunnicliffe Wilson G, Paterson LJ. Genstat 5 Reference Manual. Clarendon Press, Oxford, 1987. 25. Ritar AJ, Ball PD, G’May PJ. Artificial insemination of cashmere goats: effects on fertility and fecundity of intravaginal treatment, method and time of insemination, semen freezing process, number of motile spermatozoa and age of females. Reprod Fertil Dev 1990;2:377-384. 26. Salamon S, Lightfoot RJ. Fertility of ram spermatozoa frozen by the pellet method. IlI The effects of insemination technique, oxytocin and relaxin on lambing. J Reprod Fertil 1970;22:409-423. 27. Steel RGD, Torrie JH. Principals and Proceedures of Statistics: A Biometrical Approach. McGrawHill Inc., New York, 1981. 28. Windsor DP. Does postpartum interval influence the success of transcervical uterine insemination in Merino ewes? A preliminary study. hoc Aust Sot Anim Prod 1994;20: (in press).
TRANSCERVICAL
42:147-l
57,1994
ARTIFICIAL INSEMINATION OF AUSTRALIAN MERINO EWES WlTH FROZEN-THAWED SEMEN
D.P. Windsor,1 A.Z. Szell,l C. Buschbeck, A.Y. Edward,1 J.T.B. Milton3 and B.C. Buckrell2 lSheep Industries Branch, Great Southern Agricultural PO Box 757 Katanning, Western Australia 2Department of Population Medicine, University Guelph, Ontario NlG 2Wl Canada 3School of Agriculture, University of Western Nedlands, Western Australia 6009
Research Institute 6317 of Guelph Australia
Received for publication: November 2, 1993 Accepted: April 24, 1994 ABSTRACT We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical Al; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment l(771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120/251) was higher (P
Acknowledgements Financial assistance was provided by the Wool Research and Development Corporation and the Meat Research Corporation. Pregnecol and Ovaluon used in Experiment 1 were donated by the manufacturers. We thank S. Dorman (Dale River WA) and 3. Caldwell (Mt Barker, WA) for the use animals, and their staff for valuable assistance. Technical assistance was provided by DAWA and UWA staff. We thank J. Dhaliwal for advice and assistance with the statistical analyses and M. Johns for pregnancy scanning.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
148
INTRODUCTION Fertility following artificial insemination (AI) with frozen-thawed ram semen is limited by the inability of current cryopreservation procedures to maintain the capacity of spermatozoa to transit the cervix. Despite maintaining their ability to fertilize (19), most frozen-thawed ram spermatozoa are unable to colonize or traverse the length of the cervix (20,21) and are rapidly lost from the female reproductive tract (16). This results in an inadequate number of spermatozoa reaching the oviducts to ensure fertilization. Vaginal or cervical insemination with frozen-thawed semen in Merino ewes often results in conception rates below 20% under Australian conditions (23). Consequently, the commercial use of AI with frozen-thawed ram semen was extremely limited until the advent of laparoscopic insemination (X3), which permits deposition of semen directly into the uterine lumen, thus bypassing the cervical barrier and restoring fertility (2,22). An alternative to intrauterine AI by laparoscopy is the passage of an insemination pipette into the uterus via the cervix (as commonly practised in the cow). Despite a number of reports of successful transcervical AI in the ewe with both fresh and frozen semen (l, lO-13), such techniques have not been adopted by the Australian sheep industry. The recently described Guelph System for Transcervical Artificial Insemination (GST-AI) has been reported to be capable of achieving intrauterine insemination via the cervix in a large proportion of ewes, and to produce commercially acceptable conception rates in a number of breeds (3,4, 14, 15). This paper describes the first study to evaluate the GST-AI technique for insemination of Australian Merino ewes with frozen semen under commercial conditions. The pregnancy rates achieved by GST-AI were compared with those achieved using standard techniques for cervical or laparoscopic insemination. MATERIALS AND METHODS Experimental Design Experiments 1 and 2 were both a single factor design using 3 different AI procedures (laparoscopic, cervical or transcervical AI). Ewes from the transcervical AI groups were scored according to depth of insemination at the time of AI. The GST-AI technique was performed by an experienced operator in Experiment 1 and by 2 inexperienced operators in Experiment 2 Livestock Experiment 1. Ewes (5 to 7 yr old) were obtained from 3 commercial Merino flocks run separately on the same property until 2 wk before es&us synchronization. Ewes from each of the flocks were randomly allocated to 3 equal sized groups and ear tagged for individual identification. The mean liveweights (4 wk prior to insemination) of a sample of 50 ewes from
Theriogenology
each of the 3 treatment groups did not differ. The 3 treatment groups were managed as a single flock (77 1 ewes) from 2 wk prior to sponge insertion until pregnancy diagnosis. Exneriment 2. Individually identified Merino ewes (3rh yr of age) were managed as a single flock (555 ewes) throughout the study. These ewes were randomly divided into 3 experimental groups of equal size prior to insemination. The mean liveweights (at sponge withdrawal) of a sample of ewes from each of the treatment groups did not differ. The postpartum interval for the ewes in both experiments was at least 7 mo, and an unknown proportion of ewes had not lambed within the previous 19 mo. The lambing histories of individual ewes were also not known. Source of Semen All ewes were inseminated with commercially frozen semen from 1 of 2 American Suffolk rams (Experiment 1) or a single Poll Merino ram (Experiment 2). The semen from each ram and ejaculate was allocated across aI1 treatments. Semen had been diluted threefold in Trisyolk-glycerol extender, frozen as pellets (9) and stored at -196°C until required for insemination. Thawed samples were examined randomly during the course of the inseminations. Post-thaw motilities ranged from 40% to 60%. Estrus Synchronization Ewes were hormonally synchronized by treatment for 12 to 14 d with 30 mg flugestone acetate intravaginal spongesa and 350 IU PMSGb at the time of sponge withdrawal. Artificial insemination was started at fixed times after sponge removal. Only those ewes detected in estrus (marked by testosterone treated teaser wethers) prior to the scheduled time of insemination were inseminated in Experiment 1. All ewes were inseminated in Experiment 2.
Semen pellets were thawed in dry glass test tubes in a water bath at 37°C . Ewes were inseminated using one of the following 3 regimens: 1) Laparoscopic AI was performed by an experienced operator, as described by Killeen and Cafferey (18), with ewes restrained in a laparotomy cradle. The reproductive tract was located by laparoscopy and 30 pL of undiluted semen were injected into the lumen of each uterine horn (approximately 60 million spermatozoa). Local anaesthesia (2 mL of 2% lignocainec) was provided at the points of cannula insertion. Inseminations were performed at 5 1 to 54 h after sponge withdrawal. 2) Cervical AI was performed by an experienced operator with the ewes restrained over the rail (9). Semen was deposited at the entrance of the cervical OS. No attempt was made to increase the depth of semen deposition within the cervix. Ewes were inseminated with either 200 pL aExperiment 1, Ovakron, Essential Sterolin Products, Johannesburg, South Africa; Experiment 2, Chronogest, Intervet, Lane Cove NSW, Australia. bPregnecol, Horizon Animal Reproduction, Manly NSW, Australia. cIllium lignocaine, Troy Laboratories, Smithfield NSW, Australia.
150
Theriogenology
(Experiment 1) or 100 pL (Experiment 2) of undiluted semen (containing approximately 200 or 100 million spermatozoa, respectively). Inseminations were performed at 54 to 56 h after sponge withdrawal. 3) Transcervical insemination (GST-AI) was performed at 51 to 56 h after sponge withdrawal as described by Halbert et al. (14). Ewes were restrained in dorsal recumbencyd, and the cervix was located with the aid of a tubular perspex speculum illuminated by a fibre optic light source. The cervix was stabilized by grasping the surrounding tissue with a pair of Bozman forceps. A fine (18 G) stainless steel insemination pipette (attached to a modified IMV insemination gun) was introduced into the cervical opening. Gentle probing was used to locate the openings in the cervical rings and to pass the pipette into the uterine lumen. Thawed semen pellets (200 pL) were diluted six-fold with Dulbecco’s PBSe and loaded into 0.5 mL straws for insemination. The dead space in the pipette meant that only about 0.4 mL of semen could be expelled. Each ewe, therefore, received the equivalent of approximately 80 pL of undiluted (post-thawing) semen (containing approximately 80 million spermatozoa). Insemination was performed by an experienced operator in Experiment 1 and by 2 newly trained operators in Experiment 2. The ewes were mated to backup rams 14 d after the completion of AI. Estimation of Cervical Penetration The depth of insemination achieved using the GST-AI technique was estimated by the given inseminator and recorded for each ewe using to the following scale: 1) Uterine insemination was judged to have been accomplished when the insemination pipette could be inserted into the cervix to its full length (about 8 cm). 2) In deep iutracervical insemination the AI pipette was estimated to have been inserted at least 2.5 cm into the cervix, and little semen back flow was apparent. 3) Shallow intracervical insemination was scored when pipette insertion was judged to be less than 2.5 cm deep, or when substantial semen back flow occurred after insemination. 4) Semen was deposited at the cervical OSor iu the vagina. Pregnancy Diagnosis Pregnancy to AI was determined by real-time ultrasonographyf 65 to 75 d after insemination. Ewes were scanned using a 5MHz external linear array and fetuses attributable to AI or to backup rams distinguished on the basis of fetal size and cotyledon development as described by Johns (17). Statistical Analyses The effect of insemination technique and depth of transcervical AI on the pregnancy rate was determined by logit analyses using Genstat 5 (24). Predicted proportions for different treatments were compared using Z-tests. Day, operator and ram effects were compared by Chi-square analyses.
dcommodore turning cradel, Poldenvale, Taunton UK. eCommonwealtl~ Serum Laboratories, Melbourne Victoria, Australia. fToshiba Sonolayer, Shimoshigami, Japan.
Theriogenology
151
RESULTS Experiment 1 Wrthin the GST-AI treatment group, transcervical uterine insemination was judged to have been successfully performed in 76% of the ewes. The proportion of ewes falling into each GST-AI depth of insemination category is shown in Figure 1.
i
2 GST-AI
3
4
penetration score
Figure 1. Cervical penetration rates using Guelph system for transcervical AI (GST-AI, Experiment 1).
The overall pregnancy rate resulting from GST AI (26%, 68/264) was greater (P~0.01) than for cervical AI (9%) but lower (P
Theriogenology
152
Cervical
GST-AI (4)
GST Al (3)
GSTAI (2)
GST-AI (1)
Laparoscopic
Insemination technique
Figure 2. Effect of AI technique and depth of insemination (in parentheses) by Guelph system for transcervical AI (GST-Al) on the pregnancy rate (Experiment 1).
Experiment 2 The rates of cervical penetration achieved by the 2 less experienced operators performing the GST-AI inseminations in Experiment 2 were lower than those achieved in Experiment 1 by the experienced technician. The proportion of ewes falling into each insemination category was influenced by operator (P
Theriogenology
153
19136
761146
10136 281146
Cervical penetration score
Figure 3. Depth of insemination into the cervix by the Guelph system for transcervical AI (GST-AI, Experiment 2).
31/78 40-
Cervical
GST-Al(4)
GST-AI (3)
GST-AI (2)
721187
GST-Al (1) Laparoscopic
Inseminationtechnique
Figure 4. Effect of AI technique and depth of insemination (in parentheses) by Guelph system for transervical AI (GST-AI) on ewe pregnancy rates (Experiment 2).
154
Theriogenology
Cervical
Laparoecopic
GST-AI
Insemination technique
Figure 5. The effect of insemination technique on the proportion of ewes becoming pregnant to a subsequent backup mating (Experiment 2).
DISCUSSION The rates at which a pipette could be passed through the cervix to effect uterine insemination in this study represent a substantial improvement over those previously described for Australian Merino ewes. This may be attributable to the improved pipette design and modified method of ewe restraint incorporated in the GST-AI technique. Salamon and Lightfoot (26) found that although the depth of insemination could be increased by the application of cervical traction, a conventional AI pipette could be inserted to a depth greater than 2 cm in only 13% of the ewes. In a more recent study, Eppleston (7) reported that insemination beyond 2 cm was achievable in 6 to 38% of the ewes, but that cervical penetration beyond 3 cm was only possible in 3% of the ewes. Cervical penetration or subsequent fertility was not increased significantly by reducing the AI pipette tip diameter (from 2 to 1 mm), or by the use of a helicoid pipette (7). By contrast, in Experiment 1 an experienced operator performed succussful tmnscervical intrauterine insemination in 76% of the ewes. Fukui and Roberts (lo- 13) reported that a fine pipette (similar to the one used for the GST-AI procedure) could be passed through the cervix of 35 to 50% of estrous ewes when the animals are restrained in a standing position. One of the two inexperienced operators (Operator 1) achieved intrauterine insemination in 52% of the ewes in Experiment 2. While this figure (and that for Experiment 1) is somewhat lower than those recently reported for GST-AI in North America (>90%; 3,4), it is comparable to cervical penetration rates reported following the first field trials using the technique (54%; 15). This suggests that transcervical AI in the Merino may be no more
Theriogenology
155
difficult than in the larger North American meat sheep breeds. The very high penetration rates achieved in Canada may be associated with insemination after a shorter postpartum interval (3 to 4 mon; 4) than is generally possible under most Australian flock management systems. Preliminary results indicate that higher penetration rates may be achieved in older Merino ewes with a shorter post-lambing interval (28). The overall GST-AI conception rates (26 and 19%, respectively, for Experiments 1 and 2), while higher than those achieved by insemination at the cervical OS,were lower than those for laparoscopic insemination. In the case of Experiment 1, intrauterine insemination by GST-AI also produced lower pregnancy rates than intrauterine insemination by laparoscopy. Similar results have been reported elsewhere (25) for the goat doe, with transcervical uterine insemination resulting in lower conception rates than laparoscopic insemination, even when larger doses of spermatozoa were used for the former. The factors controlling this phenomenon are poorly understood, and may need to be fully elucidated if transcervical AI is to have wide application. Cervical traction (as used with the GST-AI technique) is a potential source of reduced fertility (26) and may have to be carefully controlled. The use of pelleted semen with equipment originally designed for straws, and the consequent need for an additional semen dilution before insemination may also have contributed to lower pregnancy rates. There is also little information available on the optimum time for GST-AI relative to sponge withdrawal. It may well vary from that used in our experiments. The pregnancy rate following intrauterine transcervical AI in Experiment 2 (39%) did not differ from that achieved by laparoscopy (38%) While somewhat low, this pregnancy rate is well within the range experienced by Australian operators (8). The lower overall pregnancy rates in Experiment 2 may have been due to the use of a single ram of unknown fertility, as the fertility of frozen semen from different rams varies substantially when used for intrauterine insemination (5,6). Any external factors (including rainfall at sponge withdrawal) acting to reduce ewe fertility at laparoscopic insemination will presumably have had a consistent effect across all treatments, because ewe management was identical throughout. The results of this experiment are consistent with previous reports that intrauterine GST-AI is capable of producing similar results to laparoscopy (4, 15). Fukui and Roberts (10) found no difference in lambing rate between Merino ewes inseminated cervically with fresh semen (64%) or with frozen semen using transcervical AI (51%). The difference in relative conception rates to laparoscopy and GST-Al between Experiments 1 and 2 is difficult to explain, particularly because the different locations and sources of sheep and semen prevent direct comparisons. Further investigation will be required to determine whether intrauterine GST-AI can be expected to consistently produce similar results to laparoscopic AI. Neither Experiment 1 nor 2 showed evidence for increased fertility as the depth of deposition of semen within the cervix by GST-AI increased, despite the strong relationship between depth of AI with frozen semen and subsequent ewe fertility described elsewhere. The relative fertility achieved by deep cervical AI was lower than that described in other studies (7, 26), possibly due to the use of lower spermatozoa doses than would normally be recommended (9) for cervical AI with frozen-thawed semen. The use of reduced spermatozoal numbers (to allow direct comparison with GST-AI) for cervical AI iu Experiment 2 also produced an extremely low conception rate, which did not differ from that for shallow GST-AL The very
156
Theriogenology
small numbers of pregnant ewes in the intracervical insemination categories may also have prevented the statistical detection of any depth effects on fertility. The likelihood of ewes conceiving to backup ram mating at the estrus periods immediately following the inseminations in Experiment 2 was unaffected by insemination treatment. This indicates that the manipulations of the cervix associated with GST-AI did not compromise subsequent ewe fertility to a greater extent than either cervical or laparoscopic insemination, even when performed by inexperienced practitioners. In conclusion, the results of Experiment 2 indicate that intrauterine insemination by GST-AI has the potential to provide conception rates comparable to those for laparoscopic insemination when used with frozen semen in Merino ewes. However, further research will be needed into the factors controlling the rates of cervical penetration. A previous study has suggested that the repeatability of cervical penetration in Merinos is quite high and that it may be possible to screen ewes for their suitability for manscervical AI (11). The causes of reduced transcervical conception rates observed in Experiment 1 and those previously reported in the goat may also need to be elucidated before overall pregnancy rates become commercially acceptable. REFERENCES 1. Andersen V, Aamdal J, Fougner J. Interuterine and deep cervical insemination with frozen semen in sheep. Zuchthygiene 1973;8:113-118. 2. Armstrong DT, Evans G. Intrauterine insemination enhances fertility of frozen semen in superovulated ewes. J Reprod Fertil1984;71:89-94. 3. Buckrell B, Buschbeck C, Gartley C, Kmetsch T, McCutcheon W, Martin J, Penner, Walton J. A breeding trial using a transcervical technique for artificial insemination in sheep. Proc 12th Int Cong Anim Reprod Art Insem 1992;12:1531-1533 ). 4. Bucknell BC, Buschbeck C, Gartley C, Rroetsch T, Walton J.S. Further development and testing of a transcervical technique for AI in sheep. Theriogenology 1994 (accepted for publication). 5. Butler LG, Maxwell WMC. The effect of site and location on results of on-farm intrauterine insemination with frozen-thawed semen. Proc Aust Assoc Anim Breed Genet 1988;7:394-397. 6. Eppleston J, Maxwell WMC. Between ram variation in the reproductive performance of ewes inseminated with frozen semen. Proc Aust Sot Reprod Biol 1991;23:130. 7. Eppleston J. Studies on the Fertility of Frozen-ThawedRam Semen. Ph.D. Thesis, The University of New South Wales, Sydney, 1992. 8. Evans G. Application of reproductive technology to the Australian livestock industries. Reprod Fertil Dev 1991;3:627-650. 9. Evans G, Maxwell WMC. Salamons’ Artificial Insemination of Sheep and Goats. Butterworths, Sydney, 1987. 10. Fukui Y, Roberts EM. Fertility of non-surgical intra-uterine insemination with frozen-pelleted semen in ewes treated with prostaglandin F20: In: Tomes, GJ, Robertson, DE and Lightfoot, RJ (eds) Proc Int Congr Sheep Breed., Western Australian Institute of Technology, Perth, 1976;482-49. 11. Fukui Y, Roberts EM. Repeatability of non-surgical intrauterine technique for artificial insemination of the ewe. Theriogenology 1977;8:77-81.
Theriogenology
157
12. Fukui Y, Roberts E.M. Sperm transport after non-surgical intrauterine insemination with frozen semen in ewes treated with prostaglandin F20, J Reprod Fertil. 1977;51:141-143. 13. Fukui Y, Roberts EM. Further studies on non-surgical interuterine technique for artificial insemination in the ewe. Theriogenology 1978;10:381-393. 14. Halbert GW, Dobson H, Walton JS, Buckrell BC. A technique for transcervical intrauterine insemination of ewes. Theriogenology 1990;33:993-1010. 15. Halbert GW, Dobson H, Walton, JS, Sharpe P, Buckrell BC. Field evaluation of a technique for transcervical intrauterine insemination of ewes. Theriogenology 1990;33:1231-1243 . 16. Hawk HW. Sperm survival and transport in the female reproductive tract. J Dairy Sci 1983;66:26452660. 17. Johns MA. Estimation of the week of conception in Merino ewes using real-time ultrasonic imaging. Aust .I Exp Agric 1993;33:839-841. 18. Killeen ID, Caffery GJ. Uterine insemination of ewes with the aid of a laparoscope. Aust Vet J 1982;59:95. 19. Lightfoot RJ, Salamon S. Fertility of ram spermatozoa tiozen by the pellet method. I. Transport and viability of spermatozoa within the genital tract of the ewe. J Reprod Fertil1970;22:385-398 . 20. Lightfoot RJ, Salamon S. Fertility of ram spermatozoa frozen by the pellet method. II. The effects of method of insemination on fertilization and embryonic mortality. J Reprod Fertil1970;22:399-408. 21. Mattner PE, Entwistle KW, Martin ICA. Passage, survival and fertility of deep-frozen ram semen in the genital tract of the ewe. Aust J Biol Sci 1969;22:181-187. 22. Maxwell WMC, Butler LG, Wilson HR. Intrauterine insemination of ewes with frozen semen. J Agric Sci Camb. 1984;102:233-235. 23. Maxwell WMC, Hewitt LJ. A comparison of vaginal, cervical and intrauterine insemination of sheep. J Agric Sci Cambridge 1986;106:191-193. 24. Payne RW, Lane PW, Ainsley AE, Bicknell KE, Digby PGN, Harding SA, Leech PK, Simpson HR. Todd AD, Verrier PJ, White RP, Gower JP, Tunnicliffe Wilson G, Paterson LJ. Genstat 5 Reference Manual. Clarendon Press, Oxford, 1987. 25. Ritar AJ, Ball PD, G’May PJ. Artificial insemination of cashmere goats: effects on fertility and fecundity of intravaginal treatment, method and time of insemination, semen freezing process, number of motile spermatozoa and age of females. Reprod Fertil Dev 1990;2:377-384. 26. Salamon S, Lightfoot RJ. Fertility of ram spermatozoa frozen by the pellet method. IlI The effects of insemination technique, oxytocin and relaxin on lambing. J Reprod Fertil 1970;22:409-423. 27. Steel RGD, Torrie JH. Principals and Proceedures of Statistics: A Biometrical Approach. McGrawHill Inc., New York, 1981. 28. Windsor DP. Does postpartum interval influence the success of transcervical uterine insemination in Merino ewes? A preliminary study. hoc Aust Sot Anim Prod 1994;20: (in press).