Deposition Of Frozen-thawed Semen Research Articles

Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle

BackgroundCervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds).ResultsRNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P < 0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds.ConclusionsThis novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Surgery on cervical folds for transcervical intrauterine artificial insemination with frozen-thawed semen enhances pregnancy rates in the sheep

In sheep industry, genetic progress rate achieved by artificial insemination (AI) is limited by the convoluted anatomy of the cervix, which does not allow the passage of an insemination catheter for uterine semen deposition. The aim of this study was to test, in 98 pregnant at term Sarda ewes, the effects of: Experiment 1) total or partial ablation of cervical folds and Experiment 2) 4 or 2 incisions of cervical folds, on the passage of an insemination catheter, deposition of frozen-thawed semen and pregnancy rates. Surgical procedures were performed within 24 h from parturition providing deep sedation and epidural anaesthesia. Duration of surgeries and post-operatory recovery were carefully monitored. For both experiments, 5 months since surgery, independently of the stage of oestrus cycle, cervical patency was tested through the transcervical passage of a palpation probe. Six months since surgery, in Experiment 1, ewes were naturally mated with fertile rams. In Experiment 2, ewes submitted to incisions of the cervical folds and a control group underwent synchronisation of oestrus and transcervical AI with frozen-thawed semen. Thirty days later, for both experiments, pregnancy rates were assessed by ultrasonography and lambing rates were recorded. Five months after surgery, in Experiment 1, transcervical passage of a palpation probe to reach the uterine lumen was possible in all ewes submitted to total and partial ablation of folds. In Experiment 2, this was achievable in 90.5% ewes with 4 incisions of the folds and in 89.6% ewes with 2 incisions with no significant differences among groups (P = 0.44). In Experiment 1, pregnancy rates in ewes mated to rams after total or partial ablation of the cervical folds was 100%. In Experiment 2, following transcervical AI, pregnancy rates were higher in groups submitted to 4 (63.7%) or 2 (41.4%) incisions of the cervical folds compared to the control group (8%; P<0.05). These data were confirmed at lambing with rates of 56.8% and 41.4% in ewes submitted to 4 or 2 incisions respectively, significantly higher than the control group (4%; P<0.05). Surgical ablation or incision of the cervical folds in post-partum ewes represent valid procedures for transcervical intrauterine deposition of semen for AI, obtaining satisfactory pregnancy rates. These procedures might be useful in programs of genetic selection and MOET.

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.

Cervical penetration and transcervical AI of tropical sheep (Malpura) at natural oestrus using frozen-thawed semen

This study comprising of two trials was undertaken in ewes exhibiting natural oestrus to evaluate a simple transcervical artificial insemination technique suitably modified for use in tropical sheep. Twenty four adult multiparous Malpura ewes which had lambed 6 to 12 months prior to the experiment were utilized for two oestrous cycles during the autumn breeding season. The cervical penetration ( n=47) was conducted in both first ( n=24) and second ( n=23) trial ≤6 to 18 h following onset of oestrus, while lambing rate was assessed in the second trial ( n=23) following deposition of frozen-thawed semen having >70% mean post-thaw motility at os, mid cervix or uterus. The overall success achieved in cervical penetration was 44.7%. There was no difference in cervical penetration rate at ≤6 and 18 h after detection of oestrus (46.1% vs. 42.8%, respectively). A mean lambing rate of 22.7% was recorded in ewes inseminated using this technique. Mid cervical and transcervical insemination with frozen semen resulted in similar lambing rate (28.5 vs. 22.7%, respectively). However, no lambing was achieved on os-cervical insemination. Further efforts are needed to determine factors controlling the cervical penetration and conception rate following artificial insemination with frozen semen.

Control of ovulation, storage of semen, and artificial insemination of fibre-producing goats in Australia: a review

Artificial insemination (AI) allows individual bucks to be exploited widely and so is a potentially useful tool for the rapid genetic improvement of fibre goats. In Australia, where there is a desire by farmers to improve the productivity of their goats, AI may best be adopted under extensive grazing conditions using control of ovulation to allow efficient and accurate timing of the deposition of frozen-stored semen. Although ovulatory activity is influenced by the manipulation of environmental factors, the time of ovulation is synchronised most accurately by the combined use of intravaginal progestagens and pregnant mares' serum gonadotrophin. However, the costs of these exogenous hormones remain high, which justifies investigation of alternative methods to control ovulation. Bucks show strong seasonality in the quality and quantity of their sperm production, and so there is limited time in which semen may be collected for storage and AI, but this can be extended by optimising nutrition and management. There appears to be no improvement in the fertility of stored semen when seminal plasma, which contains egg yolk coagulating enzyme, is removed and an extender containing only a low concentration of egg yolk is used for dilution. Simple methods have been developed for 1-step dilution and freezing of buck semen. However, the post-thawing viability of spermatozoa frozen in pellets on dry ice is higher than for semen frozen in straws in liquid N2 vapour, although straws are preferred for commercial trade. For frozen-thawed semen, fertility after laparoscopic insemination is high, whereas the fertility after cervical insemination is considerably lower but improves by the deeper placement of semen into the reproductive tract. Does are best inseminated 5-10 h before the expected time of ovulation. A dose as low as 1 x 106 motile spermatozoa may be used for laparoscopic insemination of thawed semen that was previously diluted at rates (semen: diluent) of 1:2 to 1:23. However, for the cervical method, a low dilution rate of 1:2 allows a sufficiently small, highly concentrated dose of at least 120 x 106 motile frozen-thawed spermatozoa t o be deposited into the reproductive tract of the doe. Cervical insemination is cheaper and simpler than the laparoscopic method, and this warrants the development of an improved technique for the consistent, deep deposition of frozen-thawed semen through the cervix in a high proportion of does.