Combined treatment with oestradiol benzoate, d‐cloprostenol and oxytocin permits cervical dilation and nonsurgical embryo recovery in ewes

Abstract

This study examined the feasibility of transcervical embryo recovery after the hormonal treatment to induce cervical dilation, following the 7-day oestrous synchronization protocol in multiparous Santa Inês ewes. A total of 23 cyclic ewes received two doses of 37.5 μg of d-cloprostenol by latero-vulvar route 7 days apart. After the second injection of d-cloprostenol, the ewes were checked for oestrus (every 12 hr) and then mated by fertile rams throughout the oestrous period. All ewes received 37.5 μg of d-cloprostenol (latero-vulvar) and 1 mg of oestradiol benzoate by either intramuscular (EBim group; n = 12) or intravaginal (EBivg group; n = 11) route 16 hr before embryo flushing. Twenty minutes before the flushing, 50 IU of oxytocin were administered intravenously. The oestrous response (i.e., the percentage of ewes that showed signs of oestrous behaviour after the second d-cloprostenol injection) was 91.3% (21/23). The proportion of successfully penetrated ewes (81.8% compared with 80.0%), the mean duration of embryo flushing (24.7 ± 2.0 min compared 26.2 ± 1.9 min), the flushing fluid recovery rate (94.8 ± 1.3% compared with 91.0 ± 2.9%) and the average number of structures recovered per ewe (0.5 ± 0.4 compared with 0.8 ± 0.4) did not vary (p > 0.05) between the EBim and EBivg groups. Viable embryos were recovered from 41.2% (7/17) of successfully penetrated ewes. It can be concluded that nonsurgical (i.e., transcervical) embryo collection can be performed in oestrous-synchronized Santa Inês ewes pretreated with d-cloprostenol, oxytocin and oestradiol benzoate, with the latter hormone administered by either the intramuscular or intravaginal route.

Comparison of the intravenous and intravaginal route of oxytocin administration for cervical dilation protocol and non‐surgical embryo recovery in oestrous‐induced Santa Inês ewes

“…In fact, the synchronous oestrous induction protocol used in this study was efficient to support the proposed methodology, not compromising the data investigated. Perhaps reduced dose of eCG used in this study resulted in less ovulation and embryo yield when compared to average embryos recovered (1.0 ± 1.1 embryos) by transcervical route in Santa Inês ewes receiving 300 IU eCG (Fonseca, Zambrini, Guimarães, Silva, Oliveira, Bartlewski, et al, ), but similar to those not receiving gonadotropin stimuli (Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão et al, ).…”

“…Therefore, the grading system was proposed by Fonseca, Zambrini, Guimarães, Silva, Oliveira, Bartlewski, et al (, Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão, et al ( and devised to establish the difficulty with which the uterine cervix can be traversed using the Hegar dilator appears to be critical for proper selection of individual animals that are suitable to undergo NSER; in the present experiment, most animals were classified as Grade four and all Grade five animals (cervical penetration times from 7 to 10 min or impenetrable cervices, respectively) could not be used for NSER. This is similar to the earlier results by Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão, et al (, where 75% of ewes that received Grade four during the traversing attempt at oestrus could not undergo NSER 6‐7 days later. A proper selection method of donor ewes for NSER procedures including vaginoscopy and a screening system including an attempt to clamp/retract the uterine cervix and traverse the cervical rings with the Hegar dilator may substantially reduce the waste of time and resources on inapt animals.…”

“…Since a similar degree of cervical dilation can be obtained with OT given by a generally more convenient and less time‐consuming i.v. route (Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão et al, ), the latter approach may be preferred in practical settings.…”

“…The cervical os was gently retracted to facilitate the passage of the Hegar dilator, and during this procedure, the cervix was classified as normal, long (i.e., >10 cm) or extremely tortuous (Fonseca, Oliveira, et al, ). Each animal received a score from 1 to 5 according to the degree of difficulty in traversing the uterine cervix with the Hegar dilator: Grade 1 (very easy; cervical penetration achieved in <1 min); Grade 2 (easy; between 1 and 3 min); Grade 3 (moderate difficulty; between 3 and 7 min); Grade 4 (difficult; between 7 and 10 min); and Grade 5 (impossible to penetrate the cervix or a lack of complete cervical passage) (Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão et al, ). Anatomical barriers such as the vestibule‐vaginal stenosis (hymen constriction did not allow cervical clipping) or poor cervical distension (when cervix was clipped but could not be retracted) were also recorded at this stage.…”

“…Immediately after successfully traversing the cervical canal with the Hegar dilator, a catheter (no. 08; Sonda Embrapa ® for goat/sheep embryo recovery; Embrapa) with a metal mandrel was used to traverse the cervical rings, following the method previously described by Fonseca, Zambrini, Guimarães, Silva, Oliveira, Brandão, et al (. If the cervix was successfully traversed with the catheter, the uterine flushing technique, described by the same authors, was performed using a custom‐designed circuit (Circuito Embrapa ® for goat/sheep embryo recovery; Embrapa) connected to the catheter positioned firstly in the right uterine horn and afterwards left (Fonseca et al, ).…”