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Cervical relaxation (CR) was performed in ewes during diestrus, to prospect a feasible protocol for non-surgical embryo transfer (NSET). In Trial 1, naturally mated ewes (n=13) received CR protocols with estradiol benzoate (EB, 1 mg on D6) and oxytocin (OT, 50 IU on D7) only (G-EB+OT) or associated with human chorionic gonadotropin (hCG, 300 IU on D7, G-EB+OT+hCG) and were compared to non-hormonally treated (G-control) ewes. Estradiol concentration increased (P<0.05) from D6 to D7 in G-EB+OT and G-EB+OT+hCG, then decreased (P<0.05) on D8. The G-EB+OT had reduced (P<0.05) progesterone on D8, and 0% (0/5) pregnancy, while G-control had 100% (3/3) conception rate. The treatment with hCG mitigated the progesterone drop on D8 and resulted in 50% (2/4) conception rate. In Trial 2, cervical transposing success after treatment with EB, OT, and hCG on D6 and D7 was assessed in nulliparous (n=25) and multiparous (n=30) ewes. The EB+OT+hCG protocol allowed efficient cervical transposition (~78% of cervical transposition success into ~5 min) in nulliparous and multiparous females and did not alter total and blood perfusion areas of corpora lutea (CL). The use of EB+OT+hCG was efficient to ensure effective cervical transposing in nulliparous and multiparous ewes, with no apparent deleterious effect on CL function, and may contribute to animal welfare, allowing a less invasive NSET procedure.

This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 μM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the "Computer-assisted Sperm Analysis" system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 μM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 μM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 μM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: Declaration of Competing Interest All authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations., (Copyright © 2022. Published by Elsevier Inc.)

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., Competing Interests: Declaration of Competing Interest Authors declare that they do not have any actual or potential conflict of interest including any financial, personal, or other relationships with other people or organizations., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9 SOV , n = 21) or 12-day (G-12 SOV , n = 21) progesterone (P 4 )-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-9 SOV and G-12 SOV 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-12 SOV (12.3 ± 1.7/12.1 ± 1.9) than in G-9 SOV (7.9 ± 1.4/8.2 ± 1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p > .05) in G-12 SOV (5.8 ± 1.9 and 3.7 ± 1.7) than G-9 SOV (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 P 4 -device removal) was positively correlated (p < .05) with the number of CL (r = .95) and viable embryos (r = .91) in G-12 SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12 SOV group had better superovulatory responses compared with G-9 SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P 4 for 12 days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes., (© 2021 Wiley-VCH GmbH.)

Two experiments were conducted in acyclic Alpine (A) and Saanen (S) goats that received intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 6 days, as well as 200 IU of eCG and 30 μg d-cloprostenol i.m. 24 h before sponge removal. On day 7 (day 0 = onset of synchronized estrus), all goats were randomly divided into two groups: animals treated with 300 IU of hCG i.m. (hCG; Exp.1: n = 8A; Exp.2: n = 75A + S) and untreated controls (Control; Exp.1: n = 8A; Exp. 2: n = 70A + S). In Exp.2, all goats were artificially inseminated. Transrectal ovarian ultrasonography and blood collection were done on days 7, 10, 13, 17, and 21 (Exp.1), and pregnancy detection on day 60 (Exp.2). Estrus and ovulations occurred in five hCG and seven Control animals. Accessory CL (aCL) were detected in all hCG does. The total luteal area of ovulatory corpora lutea (oCL) increased (P < 0.05) on day 10 in hCG does and remained greater (P < 0.05) than in Control until day 21. Total and high-velocity color Doppler area were greater (P < 0.05) for oCL of hCG does on days 13 and 17. Progesterone concentrations were greater (P < 0.05) in hCG does from days 13 to 21 and related directly to the total luteal and oCL area for the duration of the study in all does. The pregnancy rate was higher (P < 0.05) in hCG than in Control by 22.5 %. Human chorionic gonadotropin given on day 7 of the synchronized estrous cycle positively affected CL function and pregnancy rates in seasonally anovular dairy goats., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

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 G9 SYNCH ewes compared with G6 SYNCH and G12 SYNCH, and the duration of estrus was ∼19 hours greater ( p  < 0.01) in G9 SOV than in G6 SOV . 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.

This study was conducted to assess effects of different doses of pFSH on follicular recruitment, superovulatory response, ova/embryo recovery, and embryo yield in lactating ewes. Ewes (n = 24) had a superovulation treatment regimen imposed. All ewes were implanted with a progesterone intravaginal device for 9 d, and administered either 100 (G-100) or 200 (G-200) mg pFSH, proportioned into six doses administered at 12-h intervals, starting 60 h before device removal. At 7 days subsequent to progesterone device removal, there were non-surgical embryo recoveries (NSER) from ewes having three or more corpora lutea. At the time of the first pFSH injection, number of antral follicles were similar (P < 0.05) between ewes in the G-100 and G-200 group, however, there were more 3.1-4.0 mm follicles in ewes of the G-200 than G-100 group at the time of the second pFSH administration. Estrous response and CL number were less (P < 0.05) in ewes of the G-100 (66.7 % and 2.6 ± 0.7) than G-200 (91.7 % and 11.6 ± 1.2) group. There were embryo collections from 100 % and 90.9 % of ewes in the G-100 and G-200 groups, respectively (P > 0.05). Viable embryo numbers and ova/embryo recovery rate were greater (P < 0.05) in ewes of the G-200 (6.9 ± 1.1 and 67.8 %) than G-100 (1.0 ± 0.5 and 27.6 %) group. A dose of 200 mg pFSH was more effective in inducing a superovulatory response and embryo yield after NSER in ewes, however, the 100 mg dose was insufficient for these purposes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Reproductive efficiency after hydrometra (HD) treatment is usually unsatisfactory., Methods: To identify mechanisms involved in low reproductive efficiency of HD-treated goats, pluriparous dairy goats treated for HD (n=10, HD) or with no reproductive disorders (n=11, control: CONT) were induced to oestrus and superovulated. Goats were mated with fertile bucks and seven days after oestrus, non-surgical embryo recovery was performed. Embryos were evaluated and gene expression was performed., Results: There were no differences (P>0.05) in sexual behaviour parameters, superovulation response, mean number of retrieved structures and viable embryos between groups; although embryo recovery rate was higher (P=0.01) in CONT group. Structures in delayed stage (8-16 cells) were more frequent (P<0.05) in HD (29 vs 1 per cent) goats, as well as the percentage of advanced embryos was greater (P<0.05) for CONT (59.3 vs 33.3 per cent) goats. However, the expression of genes related to apoptosis ( BAX and Bcl-2 ), trophectoderm differentiation ( CDX2 ) and pluripotency maintenance ( NANOG ) was not affected (P>0.05) in embryos that reached the morulae and blastocyst stages., Conclusion: Although the HD embryos that developed to morula and blastocyst stages showed no change in the expression of genes related to their quality and implantation capacity, overall, embryo development was impaired in HD-treated goats., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

This study was conducted to evaluate effects of two administrations of d-cloprostenol at different intervals to synchronize the time of estrus and ovulation among estrous cyclic goats. In Experiment 1, 32 does were treated with 30 μg d-cloprostenol at 7.5 (T7.5, n = 16) or 11.5-day (T11.5, n = 16) intervals. In Experiment 2, the same treatments were administered and there was AI of the does (T7.5, n = 40 and T11.5, n = 38). In Experiment 1, ultrasonic assessments of ovaries were conducted at the time of the second administration of d-cloprostenol, every 12 h until detection of ovulation, and 7 days after estrous onset to detect the corpora lutea, as well as for pregnancy diagnosis 40 days after AI. In Experiment 1, the estrous response (90.6%, 29/32) was similar (P > 0.05) in both groups. Diameter of the largest follicle at the time of administration of the second dose was larger (P = 0.01) in the T7.5 than T11.5 group (7.0 compared with 5.7 mm), while the values for ovarian variables were similar (P > 0.05). In Experiment 2, the greatest (P < 0.001) synchrony in timing of initiation of estrus in does (T7.5 = 83.3% and T11.5 = 50.0%) occurred after the second day (36-48 h). The pregnancy rate tended (P = 0.0836) to be greater for does in the T7.5 (71.4%, 40/56) than T11.5 (55.6%, 30/54) group. With use of both protocols, there were acceptable estrous synchronization and pregnancy rates in estrous cyclic dairy goats., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The objective of this study is to compare the cyclic fatigue resistance of nine types of endodontic instruments of nickel-titanium., Materials and Methods: Five files of 25 mm of length of each group: Reciproc (RC) R25; WaveOne (WO) Primary; Unicone (UC) L25 25/0.06; K3XF 25/0.06; ProTaper Universal F2 (PTF2); ProTaper Next X2 (PTX2); Mtwo 25/0.06; BioRaCe 25/0.06; One Shape L25 25/0.06 were subjected to a cyclic fatigue resistance test on a mechanical apparatus. The mean fracture time was analyzed statistically by one-way analysis of variance and Tukey's honest significant difference post hoc test, with significance set at P< 0.05., Conclusion: It was observed that the groups PTX2, RC, R25, UC L25 25/0.06, and WO Primary presented greater cyclic fracture resistance than the other groups (P< 0.001).