Fertility of ewes after intrauterine insemination with frozen semen.

Abstract

Animal Production in Australia Vol. 15 FERTILITY OF EWES AFTER INTRAUTERINE INSEMINATION W ITH FRO ZEN SEMEN W.M.C. MAXWELL*, H.R. WILSON** and L.G. BUTLER* SUMMARY In an experiment involving the artificial insemination of 312 Merino ewes, fertilization, early (18 day), and late (100 day) pregnancy rates were examined after intrauterine insemination with frozen semen using a laparoscope. Oestrus was synchronized in ewes by progestagen sponge/PMSG treatment. After cervical insemination of fresh semen (control) 55 hrs after sponge removal, the fertilization, early and late pregnancy rates were 24/30, 37/46 and 33/46 respectively. Following intrauterine insemination of frozen-thawed semen at 24, 36, 48 and 60 hr after sponge removal, fertilized eggs were recovered in 7/27, 12/22, 17/25 and 19/25 ewes; the early pregnancy rates were 13/34, 18/34, 20/34 and 23/35; and the late pregnancy rates were 3/34, 6/34, 17/34 and 19/35 for the respective insemination times. INTRODUCTION There have been a number of reports on the frozen storage of ram semen and artificial insemination with frozen semen has been performed on an experimental basis in several countries. Intensive laboratory studies led to considerable progress regarding the freezing and thawing procedures (Salamon and Visser 1972; Colas 1975). However, fertility following insemination tends to be considerably lower than for fresh or chilled semen (Maxwell et al. 1980). During artificial insemination, it is rarely possible to pass the inseminating pipette through the cervix of the ewe. The cervix constitutes an initial barrier to the ascent of spermatozoa, and a relatively small proportion of cells deposited reach the site of fertilization (Lightfoot and Salamon 1970a). A number of attempts have been made to overcome this disadvantage by bypassing the cervix, and placing semen directly in the uterus. To date, these intrauterine insemination techniques have not proved very successful as they have relied either on deposition of semen in the uterine horns after laparotomy (Lightfoot and Salamon 1970a, 1970b), or a forceful entry to the uterus through the cervix (Anderson et al. 1973; Fukui and Roberts 1976). Recently Killeen and Caffrey (1982) described a technique for location of the uterus and intrauterine insemination by laparoscopy. Preliminary results indicated that high levels of egg fertilization could be obtained in ewes following intrauterine insemination with fresh or frozen-thawed semen (Killeen et al. 1982). The experiment reported in this paper was conducted to examine the rates of fertilization, early (18 day), and late (100 day) pregnancy after intrauterine insemination of frozen-thawed semen with the aid of laparoscopy. * ** Anima 1 Breed ing and Research Institu te, Katanning , W.A. 6317. Depar tment 0 f Veter inary Biology, Mu rdoc h Univers ity, W.A. 6150. 448 Animal Production in Australia Vol. 15 MATERIALS AND METHODS Semen collection and storage Semen was collected by artificial vagina from three Merino rams. Only ejaculates of initial motility > 4 (0 - 5 assessment scale) and a minimum concentration of 3~10~ spermatozoa per ml were used. For frozen storage, the individual ejaculates were diluted 1:2 (semen: diluent, v/v) at 30�C with a Tris-based diluent (Visser and Salamon 1973). The diluted semen was cooled to 5OC in 11/2 h and frozen on dry ice in pellet form (0.1 ml/pellet). The frozen pellets were transferred to liquid nitrogen and stored for at least 4 weeks before use. Fresh semen (control) was prepared on the day of insemination. Semen was collected from the same rams as for frozen storage. The individual ejaculates were diluted 1:2 (semen:diluent, v/v) with reconstituted skim milk (Colas et al. 1968). Synchronization of oestrus Mature Merino ewes were treated with intravaginal progestagen-impregnated sponges (30 mg Chronogest, Intervet, Australia, Pty Ltd) for 12 days. At sponge withdrawal the ewes received an injection of 400 i.u. PMSG (Gravamed, Beresford Laboratories, Melbourne) and were joined with 10% vasectomized rams. Insemination Frozen semen was thawed in dry test tubes which were shaken in a water bath at 37OC (2-3 pellets per tube), and used for insemination within 10 minutes. Fresh semen was used for insemination within one hour of collection. Cervical inseminations with fresh semen (control) were carried out 55 h after sponge removal. At each insemination a volume of 0.1 ml was used containing 100~10~ spermatozoa. Intrauterine inseminations with frozen-thawed semen were performed under local anaesthesia (Xylocaine 2%, Astra Pharmaceuticals) following location of the uterine horns with the aid of a laparoscope 24, 36, 48 and 60 h after sponge removal. The semen (0.02 ml, containing 20x106 total spermatozoa) was introduced into each uterine horn half way between the bifurcation and the utero-tubal junction, without exposing the ovaries with a glass pipette (6 mm o.d., soda glass, finely drawn out) attached to a 1 ml tuberculin syringe. The pipette was introduced through a cannula in the abdominal wall (Killeen and Caffrey 1982). Ewes allocated for egg recovery were laparotomized 2-3 days after insemination, the uterine horns and fallopian tubes were flushed with sterile saline, and the eggs were examined for cleavage as the criterion for fertilization. Fertilization rate was defined as the proportion of ewes with fertilized eggs rather than the proportion of eggs fertilized. Early pregnancy diagnosis was obtained in the remaining ewes by plasma progesterone assay (Robertson and Sarda, 1971). Blood was collected into 10 ml heparinized vacutainers from the jugular vein 18 days after insemination for assay by double antibody radioimmunoassay (Martin et al. 1983). Ewes which had a plasma progesterone level>1 ng/ml were considered as pregnant. The same ewes were slaughtered at 100 days after insemination to determine the late pregnancy rate. The data was analysed using generalized linear r egression mode 1s wi th Poisson elrrors as performed by the GENSTAT programme (Nelder 1977). The 449 Animal Production in Australia Vol. 15 significance of the derived deviance statistic was tested by comparison with a Chi-square value. RESULTS AND DISCUSSION The results are presented in Table 1, There was an increase in fertilization and pregnancy rates with time of insemination (linear, p< 0.01). Similar egg fertilization rates have been obtained in superovulated ewes (Killeen et al. 1982), and these authors also reported 18 of 33 (55%) ewes lambing after intrauterine insemination with frozen semen 48-55 h after progestagen sponge/PMSG treatment. In the present study, fertilization and pregnancy rates following intrauterine insemination with frozen semen 60 h after sponge removal were not statistically different from the fresh semen cervical insemination control. Table 1 Fertilization, early (18 day) and late (100 day) pregnancy rates+ after insemination with fresh and frozen semen + rates with the same superscript do not differ significantly (p < 0.05) Considerable embryonic loss occured between 2-3 and 100 days after insemination (pcO.05). The embryonic survival (ES =` ewes pregnant at day 100 per ewes pregnant at day 18) was higher for inseminations at 48 and 60 h than at 24 and 36 h after sponge removal (36/43 v. 9/31, p <O.OOl), indicating that the survival rates of embryos were lower when inseminations were performed too early. Embryonic loss in sheep is relatively high even after natural mating (Edey 1976), and the situation may be aggravated after insemination with spermatozoa subjected to frozen storage and/or ageing (Salisbury et al. 1976). The large embryonic loss obtained in the present study points to the importance of time of semen deposition in order to avoid additional ageing of spermatozoa. Spermatozoa deposited at a relatively early stage (24 and 36 h) were exposed to longer ageing in the female genital tract than spermatozoa deposited closer to the time of ovultion (48 and 60 h after sponge removal). The of frozen injection after the results of the present study confirm that intrauterine insemination semen by laparoscopy at 60 h after progestagen sponge removal/PMSG may be as effective as cervical insemination of fresh semen at 55 h synchronization treatment. Further, the intrauterine insemination 450 Animal Production in Australia Vol. 15 technique is more effic ient than cer vical ins emination because of the relatively F small dose o f semen used (0.02 ml, containing 20x106 sperma tozoa ACKNOWLEDGEMENTS The authors are grateful to Trans Australian Animal Breeding Centre, Gnowangerup, Western Australia for provision of sheep and facilities; to Mr David Suckling, Department of Animal Science and Production, University of Western Australia for conducting the radioimmunoassay; to Mrs Jane Speijers for statistical analysis. REFERENCES ANDERSON, K., AAMDAL, J. and FOUGNER, S.A. COLAS, G. (1975). J. Reprod. Fert. (1973). Zuchthyg. 8: 113. g: 277. (1968). 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