The presence of estradiol benzoate in the cervical relaxation treatment for non-surgical embryo collection does not impair embryonic morphological quality, cryosurvival, and gene expression profile.

Non-surgical embryo recovery (NSER) is usually preceded by a cervical relaxation in ovine donors, based on estradiol benzoate (EB), prostaglandin (PGF), and oxytocin (OT). However, it is hypothesized that, due to poorly understood mechanisms, EB can result in embryotoxic actions. To evaluate this, 20 min before NSER superovulated sheep were induced to cervical relaxation with 0.0 (G0.0), 0.5 (G0.5), or 1.0 mg (G1.0) of EB associated with 37.5 µg of PGF 16 h before NSER and 50 IU of OT. In doing so, the efficiency and duration of the NSER procedure showed no compromise (P > 0.05). Additionally, the presence of EB did not affect (P > 0.05) the embryo's morphological quality, the development dynamics, or the abundance of transcripts associated with embryonic quality (OCT4 and NANOG), cellular stress (HSP90 and PRDX1), and apoptosis (BCL2 and BAX). A similar result (P > 0.05) was also observed when comparing embryonic cryosurvival at 24 (52.0, 52.0, and 54.0) and 48 h (60.0, 54.0, and 58.0) of in vitro culture (G0.0, G0.5, and G1.0, respectively). Thus, we can conclude that EB use does not compromise embryonic quality and cryoresistance.

Intravaginal progesterone device reinsertion during the early luteal phase affects luteal function and embryo yield in superovulated ewes.

This study checked the efficacy of progesterone (P4) device reinsertion during the early luteal phase on luteal function and embryo yield in superovulated crossbred ewes. Twenty multiparous ewes received an intravaginal P4 device for nine days (D0 to D9) followed by six decreasing doses (25, 25, 15, 15, 10, 10%) of 133 mg pFSH i.m. at 12 h intervals, starting 60 h before P4 device removal. Ewes were naturally mated at 12 h intervals while in estrus. On D13, ewes with viable corpora lutea (CL; n = 19) were equally allocated for receiving their P4 device reinsertion (G-P4; n = 10) or not (G-Control; n = 9). On D17, the P4 device was removed, and all females received the cervical relaxation protocol 16 h to 20 min before non-surgical embryo recovery. CL count and their functionality classification were performed on D13 and D17 by transrectal B-mode and color Doppler ultrasonography (US). Plasma P4 concentrations (ng/mL) of G-P4 ewes increased (P < 0.05) over the days, being greater on D17 (9.2 ± 2.8) than on D9 (1.9 ± 0.7) and D13 (1.6 ± 0.4). The overall CL count per ewe tended to be greater (P = 0.09) in G-P4 compared with G-Control. The occurrence of premature regression of corpora lutea did not differ (P > 0.05) between G-P4 (30.0%) and G-Control (44.4%). The number of ova/embryos recovered was greater (P < 0.05) in G-P4 (11.6 ± 2.9) compared with G-Control (3.7 ± 2.0), respectively. Altogether, the reinsertion of the P4 device for four days after superovulation in ewes promotes greater P4 concentrations, resulting in greater ova/embryos recovered.

Effects of different doses of estradiol benzoate used in a cervical relaxation protocol on the success of non-surgical embryo recovery and luteal function in superovulated ewes.

This study investigated the effectiveness of different doses of estradiol benzoate (EB) to promote cervical relaxation and their effects on luteal function and outcomes of non-surgical embryo recovery (NSER) in sheep. Multiparous (MULT) and nulliparous (NULL) crossbred Lacaune X Santa Inês ewes were superovulated and naturally bred. Seven days after progesterone withdrawal, females were randomly assigned to one of three distinct cervical relaxation protocols, consisting of i.m. treatment with 37.5 µg d-cloprostenol and different doses of EB: 0.0 mg (0.0EB group; n = 3 NULL and 14 MULT); 0.5 mg (0.5EB group; n = 4 NULL and 12 MULT) or 1.0 mg (1.0EB group, n = 6 NULL and 11 MULT) 16 h before NSER. All ewes received 50 IU of oxytocin 20 min before NSER (D17). Blood samples were collected and ultrasound exams (B-mode and color Doppler) were performed at two timepoints: immediately before d-cloprostenol and EB treatments and prior to NSER. Estrous behavior, corpora lutea count and NSER success outcomes were not affected by EB treatments nor parity (P > 0.05). Embryo recovery rate was greater for ewes in the 0.5EB group and in the NULL ewes (P < 0.05). Ovarian biometrics differed between the two evaluation timepoints in all groups (P < 0.05). Plasma estradiol increased over time, reaching a significant greater level in 1.0EB ewes compared to controls on D17 (P < 0.05), whereas progesterone concentrations decreased over time in all groups (P > 0.05). In conclusion, treatments did not affect NSER success but they did affect luteal function by altering P4 and E2 concentrations. Therefore, the NSER technique can be successfully performed in ewes with or without prior treatment with EB.

Single dose of 300 IU hCG in the early luteal phase in superovulated ewes: Effects on corpora lutea, progesterone profile, and embryo recovery.

This study aimed to evaluate the effects of hCG treatment during the early luteal phase on ovarian function, progesterone profile, and embryo yield in superovulated ewes. Superovulated sheep were randomly assigned to receive 300 IU hCG i.m. (GhCG, n = 24) or not (GControl, n = 25) at 96 h after the removal of the progesterone (P4) device (D13). Non-surgical embryo recovery (NSER) was performed eight days after P4 withdrawal. Ultrasound evaluations were performed on D13, D14, D16, and D17. Blood samples were collected on D14, D16, and D17. Superovulation scores were recorded based on the number of corpora lutea (CL) as follows: 1 (≤ 2), 2 (3-5), 3 (6-8), and 4 (≥ 9). NSER efficiency, superovulation response, and luteal tissue area were similar in both groups (P > 0.05). Structural luteolysis tended to be higher in GControl (P = 0.07; 47.0 %) while functional luteolysis was similar in both groups (P > 0.05; 0.0 % and 5.9 %). The recovery rate was greater (P < 0.05) in GhCG (89.8 %) compared with GControl (71.0 %), with similar overall ova/embryo numbers observed for both groups (P > 0.05). GhCG showed a higher concentration of animals with a superovulatory response score of 4 (54.5 %; P < 0.05) compared with the lowest scores. Plasma progesterone on D16 was higher (P < 0.05) in GhCG ewes (11.1 ± 1.5 vs 6.9 ± 1.5 ng/mL). In conclusion, the hCG treatment improved circulating P4 and embryo recovery rate, tended to maintain luteal functionality, and thus constitutes an additional tool for improving embryo yield in superovulated ewes.

Transcervical Versus Laparotomy Embryo Recovery: What Strategy Is Best for Embryo Bank Formation in the Canindé Goat Conservation Program?

By allowing for the creation of embryo banks, reproductive biotechnologies play an essential role in the preservation of endangered goat breeds' genetic diversity. This study focused on comparing both available embryo collection methods [laparotomy (LAP) vs. nonsurgical embryo recovery (NSER)] in Canindé goats to create an embryo bank for later use in a breed conservation program. Twelve females were superovulated and subjected to either the LAP or NSER technique for embryo recovery. The recovery rate was similar (p > 0.05) between NSER (86.8% ± 5.6%) and LAP (92.8% ± 4.0%). Moreover, there were no differences (p > 0.05) in the number of structures recovered, the viable embryos, and the freezable embryos per goat, respectively, for NSER (11.7 ± 1.3, 11.2 ± 1.5, and 10.2 ± 1.1) and LAP (10.3 ± 1.0, 8.7 ± 0.7, and 8.0 ± 0.8). Overall, 132 structures were collected out of 151 ovulations (∼12.6 ± 1.2 corpora lutea per goat). Finally, the procedure duration time was also similar (p > 0.05) for NSER versus LAP, respectively: 32.3 ± 3.3 versus 30.8 ± 3.9 minutes. In conclusion, the NSER method results proved to be similar to the LAP technique in small-sized Canindé goats. It was noticeable, however, that the NSER technique is simpler and provides the possibility for successive procedures with few health risks and sequels for females. This study may hopefully boost in vivo embryo production programs in the Canindé breed, facilitating the formation of embryo banks and so assuring the availability of genetic diversity before any decline becomes irreversible.

Study of the factors affecting embryo yields and quality in superovulated Morada Nova ewes that underwent non-surgical uterine flushing.

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9SOV , n = 21) or 12-day (G-12SOV , n = 21) progesterone (P4 )-based estruses synchronization protocol coupled with superovulatory treatment with decreasing doses of porcine follicle-stimulating hormone (133 mg of pFSH given over 3 days). Non-surgical embryo recovery (NSER) was performed 6-7 days after the onset of oestrus. Total antral follicle count doubled from the first to the sixth pFSH dose in both groups (p < .05). Oestrus responses did not vary between the two groups of animals (95.2%). Corpora lutea (CL) were detected in 85.0% and 60.0% of ewes that previously manifested oestrus behaviour in G-9SOV and G-12SOV respectively. NSER was successfully completed in 86.2% of ewes that had CL (p > .05). The mean number of CL per ewe/successfully flushed donor ewe was greater (p < .05) in G-12SOV (12.3 ± 1.7/12.1 ± 1.9) than in G-9SOV (7.9 ± 1.4/8.2 ± 1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p > .05) in G-12SOV (5.8 ± 1.9 and 3.7 ± 1.7) than G-9SOV (3.5 ± 1.1 and 0.8 ± 0.3 respectively). The total follicle count (all follicles ≥2 mm in diameter) at the sixth pFSH dose (at P4 -device removal) was positively correlated (p < .05) with the number of CL (r = .95) and viable embryos (r = .91) in G-12SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12SOV group had better superovulatory responses compared with G-9SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P4 for 12 days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes.

NonSurgical Embryo Recovery from Estrus-Synchronized or Superovulated Morada Nova Ewes: A Feasible Strategy for Sheep Embryo Banking.

This study assessed the feasibility of in vivo embryo production and nonsurgical embryo recovery (NSER) in Morada Nova ewes (an endangered native Brazilian breed of sheep) subjected to different estrus synchronization and/or superovulation protocols. Ewes received intravaginal sponges soaked with 60 mg medroxyprogesterone acetate (MAP), which were kept in place for six (G6; n = 12), nine (G9; n = 12), or 12 (G12; n = 12) days. Half of the ewes in each group remained estrus synchronized only (SYNCH) and the other half was superovulated (SOV) with 133 mg porcine follicle-stimulating hormone (pFSH). There were no differences (p > 0.05) in antral follicle counts determined with ultrasonography 60 hours before MAP sponge removal (or at the time of the first pFSH dose) among G6 (6.4 ± 0.9), G9 (6.2 ± 0.7), and G12 (5.5 ± 0.6). Estrus responses and NSER success rates did not vary (p > 0.05) among the three progestin-treatment groups of ewes for either estrus-induced or superovulated animals. The onset of estrus occurred 10-12 hours later (p < 0.01) in G9SYNCH ewes compared with G6SYNCH and G12SYNCH, and the duration of estrus was ∼19 hours greater (p < 0.01) in G9SOV than in G6SOV. The average duration of the NSER procedure was 32.6 ± 1.3 minutes. At least one structure was recovered in 85.7% of synchronized and in 87.5% of superovulated ewes. Viable embryo recovery rates were also similar (p > 0.05) for G6 (1.0 ± 0.3 and 2.5 ± 1.5), G9 (1.3 ± 0.5 and 4.8 ± 2.0), and G12 groups (1.0 ± 0.3 and 4.8 ± 2.3; estrus-synchronized and superovulated ewes, respectively). In conclusion, progestogen pretreatment of different durations and NSER can be employed in Morada Nova ewes, resulting in reasonable viable embryo recovery rates in both estrus-synchronized and superovulated animals. Therefore, both techniques are suitable for use in commercial settings as well as small ruminant conservation programs.

Successful transcervical uterine flushing can be performed without or reduced dose of oestradiol benzoate in cervical relaxation protocol in Dorper ewes.

This study assessed the efficiency of cervical relaxation protocol using none, half or full dose (1.0 mg) of oestradiol benzoate in Dorper ewes subjected to non-surgical embryo recovery (NSER). Thirty-six pluriparous ewes received progestogen sponge (60 mg) for 9 days plus eCG administration (300 IU i.m.) 24 hr before sponge removal. Ewes were not mated and were randomly assigned to receive at 16 hr before NSER 37.5 µg d-cloprostenol i.m. and different doses of oestradiol benzoate: 0.0 mg (0EB group; n = 12); 0.5 mg (0.5EB group; n = 12) or 1.0 mg of oestradiol (1.0EB group, n = 12). All ewes received oxytocin (50 IU) i.v. 20 min before NSER, which was performed 8 days after sponge removal. Corpora lutea were counted by transrectal ultrasonography 24 hr before NSER. After procedure, the ewes were kept in natural breeding period to check their post-NSER fertility. NSER was performed in 91.7% (33/36) of the animals with overall fluid recovery efficiency over 97% (p > .05). The cervical transposing with Hegar dilator was longer (p < .05) in 0EB (4.2 ± 0.3 min) compared to 0.5EB (1.7 ± 0.3 min) and 1.0EB group (1.5 ± 0.3 min). The cervical transposing with mandrel/catheter was longer (p < .05) in 0EB (2.4 ± 0.5 min) than 1.0EB group (1.3 ± 0.5 min). Overall duration of uterine flushing was 25.4 min with structure recovery rate of 43.5%, with no difference among groups (p > .05). The post-NSER fertility was higher (p < .05) in 0.0EB (90%) than 0.5EB group (36.4%). In conclusion, NSER can be successfully performed in Dorper ewes by using a cervical relaxation protocol without oestradiol benzoate.