Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination.

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.

Fair S ，Hanrahan JP ，O'Meara CM ，Duffy P ，Rizos D ，Wade M ，Donovan A ，Boland MP ，Lonergan P ，Evans AC ... -  《THERIOGENOLOGY》

The difference in embryo quality between Belclare and Suffolk ewes is not due to differences in oocyte quality.

Fair S ，Hanrahan JP ，Ward F ，O'Meara CM ，Duffy P ，Donovan A ，Lonergan P ，Evans AC ... -  《THERIOGENOLOGY》

Transcervical artificial insemination in sheep: effects of a new transcervical artificial insemination instrument and traversing the cervix on pregnancy and lambing rates.

Wulster-Radcliffe MC ，Wang S ，Lewis GS 《THERIOGENOLOGY》

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen.

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.

O'Meara CM ，Hanrahan JP ，Donovan A ，Fair S ，Rizos D ，Wade M ，Boland MP ，Evans AC ，Lonergan P ... -  《THERIOGENOLOGY》

Resuspending ram spermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervically inseminated ewes.

The role of seminal plasma (SP) components on the maintenance of motility, viability and fertilising ability of frozen-thawed spermatozoa is of considerable interest. However, differences observed in constituents of SP among males could explain differences in fertility obtained in vivo. Two experiments were designed to examine the effects of seminal plasma on fertility from cervically inseminated frozen-thawed semen. The objective of Experiment 1 was to investigate if source or type of SP influences pregnancy rate. Seminal plasma was collected from rams previously classified as having either High (HSP; n=3) or Low (LSP; n=3) fertility in vivo. Artificial SP (fructose/sodium solution with 10% BSA; ASP) was made. Frozen semen from the same 6 rams was thawed and inseminated (Control) or resuspended either in HSP, LSP or ASP (20% in semen) prior to insemination of ewes (n=284, over 2 farms). The overall pregnancy rate was 28.1%. Treatments (Control, ASP, HSP and LSP) were not significantly different (P>0.3). There was no difference between HSP and LSP (P>0.5), and no effect of using ASP compared to ram SP (P>0.7), on pregnancy rate. As there was no effect of SP on pregnancy rate a repeat experiment (Experiment 2) was designed to test the effect of washing and selecting motile sperm prior to resuspending in phosphate-buffered saline (PBS) containing SP on pregnancy rate. Frozen-thawed semen from each of 2 rams was centrifuged through a density gradient, pellets were centrifuged through a wash medium and the sperm concentration/ram was counted. Sperm cells were resuspended in: (1) control PBS, (2) PBS containing 30% HSP or (3) PBS containing 30% LSP to give 100 x 10(6) motile sperm in 0.25 mL. Control straws were thawed and inseminated directly. Ewes (n=223 over 2 farms) were inseminated 57 h post-sponge withdrawal and those not returning to oestrus were slaughtered 29-50 days post-insemination for pregnancy determination. In Experiment 2, the pregnancy rate for Control, PBS, HSP and LSP were 15.4%, 2.3%, 0% and 0%, respectively, for Farm 1 (P>0.05) and 17.8%, 11.0%, 3.9% and 12.4%, respectively, for Farm 2. Under the conditions of the current study, addition of SP from different donors of either High or Low fertility status to frozen-thawed ram semen post-thawing did not improve pregnancy rate in ewes. ASP had no effect on pregnancy rate in ewes when added to frozen-thawed semen. Washing and selection of motile sperm prior to resuspension in PBS with or without SP (30%) before insemination had a negative effect on pregnancy rate in cervically inseminated ewes. Hence, the addition of seminal plasma or some of its constituents to semen does not appear to improve pregnancy rate in cervically inseminated ewes.

O'Meara CM ，Donovan A ，Hanrahan JP ，Duffy P ，Fair S ，Evans AC ，Lonergan P ... -  《THERIOGENOLOGY》