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.

Vaginal cytology and cervical mucus as tools to predict ovulation time in small ruminants.

The possibility of using cervical mucus and vaginal cytology as tools to predict ovulation time was assessed in 11 ewes and 11 does raised under tropical conditions. Every 12 h from progesterone removal to ovulation, estrus behavior, cervical mucus, vaginal cytology, and ovarian ultrasound exams were performed. In goats, vaginal cytology had 88% of accuracy on detecting the ovulation time. However, in sheep, there was no cell pattern in the vaginal cytology and cervical mucus varied at ovulation. In conclusion, both vaginal cytology and mucus evaluation may be useful tools to determine the ovulation time in goats; however, both strategies are less accurate in sheep.

In vivo embryo production and recovery in lacaune ewes after imposing a superovulation treatment regimen is related to pFSH dose.

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 ±â€¯0.7) than G-200 (91.7 % and 11.6 ±â€¯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 ±â€¯1.1 and 67.8 %) than G-100 (1.0 ±â€¯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.

Ultrasonographic cervical evaluation: A tool to select ewes for non-surgical embryo recovery.

This study assessed the cervical ultrasonography mapping as a tool to select donor ewes for non-surgical embryo recovery (NSER). Lacaune ewes had their cervix evaluated by ultrasonography 12 hr after induced oestrus onset (Trial 1, n = 24) or 30 min before NSER (Trial 2, n = 17). Cervical rings were longitudinally evaluated and classified by their degree of misalignment on ultrasonography (DMUS) into: DMUS-1-cervix rectilinear, DMUS-2-intermediate and DMUS-3-highly asymmetrical. For predicting cervical transposing, only DMUS-1 and DMUS-2 were considered suitable. Similar ranking was attributed to degree of misalignment on the cervical map (DMCM 1-3), established immediately before NSER, which was performed at days 6 to 7 after oestrus. In Trial 1, cervical retraction for NSER was not possible only in three ewes classified as DMUS-3 (3/14, 21.4%). No difference (p > .05) was observed in the cervical transposing rates between ewes with different DMUS (ranged from 80% to 100%). In Trial 2, DMUS-1 and DMUS-2 reached 100% of transposing, and the only DMUS-3 ewe has not been transposed. In Trial 1, the prediction performance for successful cervical transposing showed low sensitivity (45%) and no specificity due to a high incidence of false negatives (52%). However, in Trial 2, sensitivity and specificity were both 100%. The DMCM and DMUS were uncorrelated, probably due to cervical stretching required to perform NSER. In conclusion, cervical ultrasound assessment immediately before NSER was more efficient to predict the cervical transposing than at induced oestrus, allowing the classification and selection of ewes eligible for NSER.

Embryo yield and quality are associated with progestogen treatment during superovulation protocol in lactating Lacaune ewes.

This study assessed the effect of progestogen treatment length on ovarian parameters and embryo yield in superovulated Lacaune ewes collected by nonsurgical embryo recovery. Twenty-three lactating ewes were superovulated 30 d apart using a cross-over design. All ewes received 60 mg of MAP intravaginal sponges for 6 (G-6 group) or 9 (G-9 group) d. A total dose of 133 mg pFSH was given in six decreasing doses (twice a day) starting at 60 h before device removal. Ultrasound examination of the ovaries was performed at the first pFSH injection and one day before embryo recovery, which was performed 6-7 d after the onset of estrus. Embryo recovery was conducted only in ewes that expressed estrus and were mated. There was no difference (P > 0.05) in the total number of follicles between G-6 (15.7 ± 1.0) and G-9 (15.6 ± 0.8) at the time of the first pFSH treatment. The percentage of responding donors with ≥3 corpora lutea (CL; 78.2% [18/23] vs 69.5% [16/23]), mean (±SEM) CL number (7.0 ± 1.2 vs 8.1 ± 1.6), transcervical passage rate (94.4% [17/18] vs 83.3% [15/18], and ova/embryo recovery rate (54.5% [60/110] vs 68.0% [83/122]) were not different (P > 0.05) between the G-6 and G-9 groups. However, the mean number of viable embryos was lower (P < 0.05) in the G-6 group (1.8 ± 0.7) than in the G-9 group. (3.5 ± 1.1). In conclusion, treatment with an intravaginal MAP sponge for 9 d during a superovulation protocol is beneficial for viable embryo yield in Lacaune ewes out of the breeding season.

Preovulatory follicular dynamics, ovulatory response and embryo yield in Lacaune ewes subjected to synchronous estrus induction protocols and non-surgical embryo recovery.

The objective of this study was to assess the effect of the duration of progesterone-based estrus induction protocols on preovulatory follicular dynamics, ovulatory response, and embryo yield after non-surgical embryo recovery (NSER) in Lacaune ewes. Females received acetate medroxyprogesterone intravaginal sponges for six (G-6; n = 14) or nine (G-9; n = 14) days plus d-cloprostenol and eCG 24 h before sponge removal (Day 0). Preovulatory follicular dynamics and the luteal characteristics are evaluated by B-mode and Color-Doppler ultrasonography. NSER was performed five to six days after ovulation. The estrous behavior rate was 85.7% for both groups, and the percentage of ewes that ovulated was 92.9% in G-6 and 100% in G-9. The day of wave emergence (relative to Day 0) did not differ (P > 0.05) between G-6 (-3.0 ±â€¯0.5) and G-9 (-4.2 ±â€¯0.5). The number of follicles of size 4.1-5.0 mm was higher (P < 0.05) in G-9 (1.4 ±â€¯0.2) compared to G-6 (0.8 ±â€¯0.2) during the Days -4 to 0. At NSER, the transcervical penetration rate was 95.2% (20/21) and its duration time was lower (P < 0.05) in G-9 (3.4 ±â€¯0.6 min) than in G-6 (7.2 ±â€¯1.3 min). The number of ovulations and viable embryos was higher (P < 0.05) in G-9 (2.9 ±â€¯0.3 and 1.3 ±â€¯0.4, respectively) than in G-6 (1.9 ±â€¯0.3 and 0.4 ±â€¯0.2, respectively). In conclusion, the 9-day protocol promoted higher ovulation rate and embryo yield; moreover, the cervical dilation treatment allowed NSER in a high percentage of Lacaune ewes.

Pregnancy rate after fixed-time transfer of cryopreserved embryos collected by non-surgical route in Lacaune sheep.

This study investigated the feasibility of applying fixed-time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non-surgically recovered from superovulated donors (n = 39) on day 6-7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed-time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non-surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.