Ultrasoundguided Ai And Postbreeding Monitoring In The Female Giant Panda

The Berlin Zoo

At no time during the eight years that the giant pandas were together at the Berlin Zoo did natural breeding occur. Therefore, AI was developed, and from 1997 to 2003, six ultrasound-guided inseminations were conducted (see Table 17.1).

Follicular development and ovulation were induced in the female with exogenous gonadotrophins for four of the six inseminations. Ovarian responses were relatively uniform, with one to three graafian follicles (>6 mm in diameter) and more than ten smaller follicles (1-3 mm) produced after each treatment (eCG with or without FSH). There was also a urinary oestrogen metabolite rise associated with follicular stimulation, but no oestrous behaviour. Ovulation was induced with hCG given on the day of AI and confirmed by ultrasonography and urinary progestin analysis. For each insemination, a disposable catheter (2 mm diameter) was guided into the external cervical os using an endoscope and then directed into the uterus by transrectal ultrasound.

the prostate (Pr, arrow heads); (g) central region of the left ductal gland (DG) of the ductus deferens located dorsal to the urinary bladder (Bl); (h) transrectal sonogram (7.5 MHz) of the ductus deferens (DD, arrow heads) running into the cranial end of a ductal gland (DG) situated partly above the urinary bladder (Bl).

Panda Fetus
Figure 17.10. Transcutaneous ultrasound image illustrating several hyperechoic foci in the left testis of the adult male giant panda SB 381 at the San Diego Zoo.

Fresh or frozen semen from SB 208 (1-2 ml; 0.35-7.5 x 109 spermatozoa ml-1, 85-95% sperm motility) was deposited after the end of eCG treatment. Although no full-term pregnancies resulted, there was ultrasonographic evidence of a pregnancy and embryonic resorption after the 1998 AI.

Images in Figure 17.11 (Plate XXVI) illustrate the concurrent use of endoscopy and transrectal ultrasound to facilitate AI. After gonado-trophin treatment, the vulva enlarged under the influence of oestrogen (see Fig. 17.11a). Near the time of ovulation, ultrasound revealed a thickened uterus, including a proliferated endometrium (see Fig. 17.11b). A Graafian follicle with a diameter of 5 mm on the active ovary was observed shortly before ovulation (see Fig. 17.11c). Although several small follicles were often observed, there were never more than three Graafian follicles present. A colour-flow Doppler image of the same ovary illustrates Graafian follicle characteristics, including the large size (>5 mm in diameter), location within the ovarian periphery and independent blood supply (see Fig. 17.11d).

Giant Panda Ultrasound

Figure 17.11. Ultrasound examination and AI of the female giant panda SB 378 at the Berlin Zoo. (a) Mildly swollen vulva (arrow heads) near oestrus; (b) transrectal sonogram (TR-500, 7.5 MHz) of the uterus (Ut, arrow heads) and endometrium (En) at the time of ovulation; (c) transrectal sonogram (TR-500, 7.5 MHz) of a Graafian follicle (Fo) on the ovary (Ov, arrow heads) shortly before ovulation; (d) colour-flow Doppler image (TR-500, 5.0-9.0 MHz) of the same ovary (Ov, arrow heads) showing the independent blood supply of the Graafian follicle (Fo); (e) endoscopic image of the transcervical passage of the AI catheter (ca, 2 mm in diameter) showing the swollen portio (Po) containing opaque viscous

Figure 17.11. Ultrasound examination and AI of the female giant panda SB 378 at the Berlin Zoo. (a) Mildly swollen vulva (arrow heads) near oestrus; (b) transrectal sonogram (TR-500, 7.5 MHz) of the uterus (Ut, arrow heads) and endometrium (En) at the time of ovulation; (c) transrectal sonogram (TR-500, 7.5 MHz) of a Graafian follicle (Fo) on the ovary (Ov, arrow heads) shortly before ovulation; (d) colour-flow Doppler image (TR-500, 5.0-9.0 MHz) of the same ovary (Ov, arrow heads) showing the independent blood supply of the Graafian follicle (Fo); (e) endoscopic image of the transcervical passage of the AI catheter (ca, 2 mm in diameter) showing the swollen portio (Po) containing opaque viscous

The uterus was located just dorsal to the urinary bladder. For AI, the passage of the catheter (2 mm in diameter) through the vagina and into the cervix was visualised by endoscopy (see Fig. 17.11e). The vagina contained mucus of a deep pink colour and was highly oedematised, whereas the cervical portio appeared swollen and contained opaque viscous mucus. Transrectal ultrasound was subsequently used to verify catheter placement for semen deposition within the caudal part of the uterine body (see Fig. 17.11f). After the AI in 1998, the sonogram at one month post insemination revealed evidence of a non implanted embryonic vesicle (see Fig. 17.11g). The hatched blastocyst was less than 1 mm in size and had no visible effect on the uterus at this stage. Six months after AI, an embryonic resorption site was visualised in the sonogram, which caused a temporary cystic degeneration of the endometrium (see Fig. 17.11h).

The San Diego Zoo

Artificial insemination of the female SB 371 was performed under anaesthesia during oestrus in 1998,1999, 2001 and 2002. Ultrasound was used to confirm the placement of the AI rod within the cervix during each procedure. Transabdominal ultrasound without the use of anaesthesia was used to monitor postbreeding changes in the reproductive tract. The latter examination of the ventral abdomen was facilitated by training this female to lie in dorsal recumbency in a cage. Designated bars were removed from the cage, and a thick fabric sleeve was used to protect the ultrasonographer's arm and ultrasound transducer cord (see Chapter 15 and Fig. 17.1). Since 2001, real-time B-mode ultrasound examinations using the Aloka SSD-900V ultrasound machine with 3.5- and 7.5-MHz sector transducers have been performed. Examinations were performed

mucus, and the mucosa of the vagina (Va); (f) sonographic verification (TR-250, 7.5 MHz) of the intrauterine position of the insemination catheter (Ca, arrow heads). Semen was deposited in the caudal part of the uterus (Ut), which was located dorsal to the urinary bladder (Bl); (g) sonographic evidence (TR-250, 7.5 MHz) of a nonimplanted embryonic vesicle (EV) in the enlarged uterus (Ut) one month after AI; (h) sonogram (TR-250, 7.5 MHz) showing the embryonic resorption site (ER, arrow heads) six months after AI. (See also Plate XXVI.)

1.2 ■ ■ 1 ■ i 1 ■ 1 • i 1 ■ ■ 1 i 1 1 1 1 i 1 1 • 1 i • 1 ■ 1 i ■ 1 ■ ■ i 1 ■ 1 ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i

50 60 70 80 90 100 110 120 130 140 150 Days post-artificial insemination

Figure 17.12. Change in uterine diameter over time (in 2002) as assessed by transabdominal ultrasound after AI of the giant panda SB 371 at the San Diego Zoo. •, right uterine horn; o, left uterine horn.

outdoors in the early morning, which afforded better visualisation of the ultrasound monitor due to low ambient light, and improved viewing of abdominal structures due to less food in the gastrointestinal tract. These examinations, which lasted about 15 minutes each, were generally conducted every one to three weeks beginning in May and continued until the average gestation length had passed, and the female's behaviour and appetite had returned to normal. In 2002, an enlarged uterine diameter was observed (Fig. 17.12); however, foetal structures were never imaged. The increase in uterine diameter combined with the presence of an amorphic structure (30 mm by 15 mm) surrounded by uterine fluid indicated an early embryonic loss and was most likely the resorption site.

In 2003, female SB 371 bred naturally on 22 March (Day 0) with male SB 415. Weekly ultrasound examinations began on Day 32. An increase in the diameter of the right uterine horn was noted by Day 109 (Fig. 17.13), with the left horn not as consistently visualised. At 134 days (16 days before parturition), a twin pregnancy was diagnosed via

Figure 17.13. Change in uterine diameter over time (in 2003) as assessed by transabdominal ultrasound after natural breeding of the giant panda SB 371 at San Diego Zoo. •, right uterine horn; o, left uterine horn.

ultrasound. An anechoic gestational sac containing an elongated echo-genic foetus was seen in both the right and left uterine horns, and the placenta was visible. A heartbeat was evident in each foetus; however, other foetal structures were not well developed. The uterine wall, foetus and placenta were isoechoic to slightly hyperechoic relative to the surrounding tissues. The foetuses were located just proximal to the inguinal regions at a depth of 3 to 4 cm. Subsequent ultrasound examinations were performed on Days 137,138,140 and 142 (13,12,10 and 8 days before parturition).

Metrics associated with the gestational sac, foetus and placenta are provided in Table 17.2. The gestational sac measure was taken from the innermost uterine layers. Foetal length was assessed as the longest length that could be imaged. The appearance of the placenta was discoidal, an elliptical homogeneous mass protruding into the gestational sac. Although it was not possible to image the complete placenta in a single view, it was possible to measure the largest placental dimensions

Table 17.2 Foetal, gestational sac and placenta measurements in a giant panda at 134, 137, 138, 140 and 142 days post-mating (adapted with permission from Sutherland-Smith et al., 2004)

Foetal length (cm) Gestational sac (cm) Placenta (cm) Days post- _ _ _

mating Left Right Left Right Left Right

134 1.42 0.93 2.58 X 2.14 1.53 X 1.13 a 1.28 X 0.93

137 2.32b 1.76b 3.19 X 3.07 2.99b 2.35 X 1.2 1.8 X 1.06b

138 c 1.79b 3.71 X 2.81 2.81 X 2.67 2.1 X 1.19 1.64 X 0.96b 140 3.32 2.92 5.33 X 2.99 3.85 X 3.44 3.4 X 1.6 d

142 4.8 e 4.0 X 6.6 e 4.3 X 2.6 e a Placenta was visualised, but not measured. b Represents an average measurement. c Measurement was inaccurate so was excluded. d Placenta was difficult to visualise and not measured. e Ultrasound evaluation of the right uterine horn was not conducted.

visualised. The foetuses were located at a depth of 3 to 6 cm within the abdomen.

Figure 17.14 depicts the ultrasound images of the developing foetuses (Sutherland-Smith et al, 2004), which were clearly visible by Day 134 and 137 (see Fig. 17.14a,b). Linear hyperechoic areas along the spine compatible with skeletal ossification were evident at Days 138 and 140 (12 and 10 days before parturition) for the left foetus (see Fig. 17.14c,d). At Day 140, similar changes consistent with ossification of the limb buds and cranium in the left fetus were visible (see Fig. 17.14d). Thin hyperechoic lines consistent with foetal membranes were observed in the left uterine horn at Day 140. Foetal movement within both gestational sacs was noted on Day 140. Heartbeats were evident in both foetuses, but could not be accurately measured because of interference from the dam's respiratory motions. Only the left fetus and placenta were visualised on Day 142 (8 days before parturition), showing both further size development and heartbeats (see Fig. 17.14e, f).

A hypoechoic area was noted adjacent to the right placenta and within the right myometrium on Days 134 and 138 (12 days prior to parturition) (see Fig. 17.14g,h). A hypoechoic area was also evident in

Figure 17.14. Ultrasound images of the right and left gestational sacs, foetus and placenta from the giant panda SB 371 at the San Diego Zoo (adapted with permission from Sutherland-Smith et al., 2004). (a) Day 134, left uterine horn and foetus; (b) Day 137, left uterine horn and foetus; (c) Day 138, left uterine horn and foetus with ossification of the spine evident; length measurement inaccurate due to curled foetal position; (d) Day 140, left uterine horn containing foetus with ossification of the spine and cranium evident; note tent-like projections (arrows); (e) Day 142, left uterine horn and foetus with progressive spinal ossification; tent-like projections still visible (arrow); (f) Day 142, left placenta; (g) Day 134, right uterine horn, foetus and placenta; abdominal fluid is present; (h) Day 138, right uterine horn and foetus; hypoechoic areas adjacent to the placenta and within the uterine wall (arrows); (i) Day 140, right uterine horn and foetus with ossification of the spine and cranium apparent as well as free abdominal fluid; (j) Day 140, right uterine horn and foetus; hypoechoic

Figure 17.14. Ultrasound images of the right and left gestational sacs, foetus and placenta from the giant panda SB 371 at the San Diego Zoo (adapted with permission from Sutherland-Smith et al., 2004). (a) Day 134, left uterine horn and foetus; (b) Day 137, left uterine horn and foetus; (c) Day 138, left uterine horn and foetus with ossification of the spine evident; length measurement inaccurate due to curled foetal position; (d) Day 140, left uterine horn containing foetus with ossification of the spine and cranium evident; note tent-like projections (arrows); (e) Day 142, left uterine horn and foetus with progressive spinal ossification; tent-like projections still visible (arrow); (f) Day 142, left placenta; (g) Day 134, right uterine horn, foetus and placenta; abdominal fluid is present; (h) Day 138, right uterine horn and foetus; hypoechoic areas adjacent to the placenta and within the uterine wall (arrows); (i) Day 140, right uterine horn and foetus with ossification of the spine and cranium apparent as well as free abdominal fluid; (j) Day 140, right uterine horn and foetus; hypoechoic

area in uterine wall visible (arrow); free abdominal fluid present; (k) Day 140, right uterine horn and foetus; increased heterogeneity in the placenta and uterine wall present (arrows). c, cranium; f, foetus; fl, abdominal fluid; p, placenta; s, spine; u, uterus.

area in uterine wall visible (arrow); free abdominal fluid present; (k) Day 140, right uterine horn and foetus; increased heterogeneity in the placenta and uterine wall present (arrows). c, cranium; f, foetus; fl, abdominal fluid; p, placenta; s, spine; u, uterus.

the right myometrium on Day 140, as well as a foetus with ossification of the spine and cranium (see Fig. 17.14i,j). The placenta was difficult to discern in the right gestational sac at Day 140 and appeared more heterogeneous than on previous evaluations (see Fig. 17.14k). Similar changes were not observed in the left myometrium or placenta. Tent like projections were observed within the left gestational sac protruding into the amniotic fluid on Days 140 and 142 (see Fig. 17.14d,e). These were not apparent within the right gestational sac. The female did not cooperate for additional ultrasound examinations, and on 19 August (8 days after the last ultrasound and 150 days post mating) the female gave birth to a single cub. Neither a second cub nor foetal remnants were observed. The first post-partum ultrasound was conducted opportunistically 45 days after birth but the uterus was not identifiable. Evidence of retained foetal structures was not apparent on subsequent ultrasound examinations, and the uterus remained difficult to visualise.

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