Using transrectal ultrasound to examine the effect of exogenous progesterone on early embryonic loss in sheep

Abstract

The financial impact of early embryonic loss in Australia may be as high as $137 million AUD/year. Embryos may be lost due to environmental conditions, or maternal factors such as nutrition or progesterone (P4) profiles. However, studies on the supplementation of P4 during early pregnancy have returned contradictory results, partly as a reliable method of detecting embryos in the early stages of gestation (<day 20) has yet be established. As such, Merino ewes (n = 62) were either not supplemented (control) or were given exogenous P4 at the time of insemination (day 0) or 3 days later (day 3). Transrectal ultrasound (TRUS) was performed on day 10, 12, 14, 17, 19 and 29 following laparoscopic artificial insemination. Transcutaneous ultrasound (TCUS) was performed on day 54 to confirm pregnancy and peripheral blood was collected for hormone analysis on day 19 to compare the accuracy of all three pregnancy diagnosis methods. Data were then analysed in developmental periods. The percentage of ewes detected as pregnant by TRUS during pre-, peri- and post implantation was 66% (41/62; day 12 and 14), 61% (38/62; day 17 and 19) and 58% (36/62; day 29), respectively. TCUS during established gestation recorded a pregnancy rate of 60% (37/62). The sensitivity of TRUS to correctly diagnose ewes as pregnant during pre-, peri- and post implantation was 68% (25/37), 89% (33/37) and 100% (36/36), respectively, while the sensitivity to correctly identify multiples was 49% (16/33), 60% (21/35) and 97% (34/35), respectively (P<0.05). The majority of embryonic loss occurred between pre- and peri- implantation (0.9±0.15 per ewe; P<0.001). No further loss was recorded after this point. Ewes that were given P4 at day 0 had significantly higher embryonic loss (77%) compared to the control (52%) and day 3-ewes (56%; P<0.05). These results show TRUS is a viable tool for investigating early embryonic loss and that the variability noted in previous P4 supplementation studies may be due to variation in time and length of treatment.

Fig 1. Transrectal ultrasound (Esaote Germany, MyLab™One VET, equipped with electronic linear array 10–5 MHz transducer (SV3513 Vet)) images of the ovary, uterus and developing conceptus during early pregnancy in sheep (oestrus = day 0).

A Day 10: The coiled uterus (circle) is located cranial to the bladder (b). Within the coiled uterine horns, the lower echogenic endometrium (arrows) is well distinguished. B Day 10: Two corpora lutea (arrows) are present on the ovary (circle), documenting recent ovulation. C Day 12: Trophoblastic expansion (within the circle): At the site of trophoblastic expansion the endometrium appeared enlarged. A dark oval shaped structure expanded within the uterine lumen. Boundaries between endometrium and trophoblast could not be distinguished at this stage. Yet the trophoblast expanded the uterine lumen exceeding the regular endometrial height by 2-fold. D Day 14: Trophoblastic expansion (arrow): Low echogenic oval-shaped trophoblast within the uterine lumen, further expands in length but not in height. E Day 17: Embryonic vesicle (circle) with embryo (arrow) within. F Day 19: Embryo (arrow), distinctly present inside the embryonic vesicle (circle). G Day 19: Power Doppler of the embryo (arrow) heartbeat. H Day 29: Two embryo’s (e) surrounded by the amniotic membranes (arrows). A cotyledon is in close proximity of the embryo (Co). Crown rump length of the embryo measures 14 mm. Fluids of the embryonic cavity (ec) expand further into the adjacent uterine horn. I Day 29: Embryo (e), situated below a cotyledon (Co). Above this the fluid filled embryonic cavity (ec), which expands into the uterine horn. J Day 29: Power Doppler of the embryo heart. Embryo (e) is surrounded by the amnion (a) and the fluid filled embryonic cavity (arrow).

Fig 2

The a) percentage of ewes detected as pregnant by TRUS and TCUS (established gestation only and b) sensitivity of TRUS (TP% = #TP/#TP+#FP; [35]) to correctly detect pregnancy (black bars) and number of conceptuses per ewe (grey bars) during pre-implantation (day 12/14), peri-implantation (day 17/19), post implantation (day 29) and established gestation (day 54).

Fig 3

Predicted ratios of each category; true positive (black segment), true negative (dark grey segment), false negative (white segment) and false positive (light grey segment), generated from an Ordinal Logistic Regression for the accuracy of using TRUS to detect pregnancy (a) and multiples per ewe (b) during pre-implantation (day 12/14), peri-implantation (day 17/19), post-implantation (day 28) in reference to results obtained by TCUS during established gestation (day 54).

Fig 4. The number of embryos lost per ewe between each developmental period; ovulation v pre-implantation, pre v peri-implantation, peri v post-implantation and post implantation and established gestation.

Values are mean ±SEM. Columns without common subscripts differ significantly (P<0.05).

Fig 5. Average length of embryonic vesicles measured on day 12, 14, 17 and 19 (dark grey bars) and embryos measured on day 17, 19 and 28 (light grey bars).

Values are averaged over ewe ±SEM. Columns without common superscripts differ significantly; lowercase indicates significance in relation to embryonic vesicle size, while capitalised letters indicate significance in relation to embryo size (P<0.05).