Impact of semen cryopreservation season on in vitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. In vitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo in vitro capacitation.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Luteal function in cyclic goats treated with human chorionic gonadotropin administered by intramuscular or intravaginal routes at the time of artificial insemination.

Human chorionic gonadotropin (hCG) has been used to improve goats reproductive efficiency. This study aimed to (i) evaluate if hCG administered by the intramuscular (i.m.) or intravaginal (i.vag.) route can be detected by a rapid ß-hCG test in blood plasma samples and (ii) document ovarian effects of hCG administered by both routes at the time of artificial insemination (AI) performed 60 h after oestrus synchronization in goats. Twenty-two Alpine goats received two i.m. injections of 30 µg of d-cloprostenol (Prolise®, Tecnopec, São Paulo, Brazil) 7.5 days apart. One day after the onset of oestrus (at the time of AI), the goats were randomly allocated to one of the three groups that received: control (n = 7): 0.3 ml of saline solution intravaginally; hCGi.m. (n = 7): 300 IU of hCG (Vetecor®; Hertape-Calier, São Paulo, Brazil) i.m. and hCGi.vag. (n = 8): 300 IU of hCG deposited intravaginally. Blood samples were drawn at -1, 3, 6, 9 and 24 h after as well as on days 3, 7, 10, 13, 17 and 21 after hCG treatment/AI. All animals tested negative for hCG (ECO Diagnóstica, Corinto, Brazil) at -1 h, and all control animals tested negative throughout the entire blood collection period. All hCGi.m. animals tested positive from 3 h until D3 post-AI but only 50% of hCGi.vag. goats tested positive during the present study. In all animals studied, mean circulating P4 concentrations increased (p < .05) from D3 to D7 after AI and then declined (p < .05) from D10 to D17 in control and hCGi.m. groups and from D17 to D21 in the hCGi.vag. group. Total cross-sectional luteal area (CLA), mean colour Doppler area (DA), DA/CLA, mean high-velocity Doppler area and HVDA/CLA all declined (p < .05) by D17-D21 in all animals studied. In summary: (i) human chorionic gonadotropin could consistently be detected in blood samples using the rapid ß-hCG test only in the hCGi.m. group; and (ii) there were no significant differences in the mean pregnancy rate, circulating P4 concentrations and various luteal parameters studied among Control, hCGi.m. and hCGi.vag. dose.

Climatic seasons and time of the day influence thermoregulation and testicular hemodynamics in Santa Inês rams raised under humid tropical conditions.

This study evaluated the possible association between the diurnal variations of climatic factors during the rainy (RS) or less rainy (LS) seasons on the testicular hemodynamics and thermoregulatory responses of hair sheep rams raised in a humid tropical climate. Santa Inês rams (n = 6) underwent evaluation of general and testicular physiological parameters (heart and respiratory rates, internal and scrotal temperatures, internal-scrotal temperature gradient, scrotal distention, and color Doppler ultrasound evaluation of the spermatic cords and spectral analyses of testicular arteries) over six consecutive weeks per season at three separate times daily (morning = 8:00 a.m., noon = 12:00 p.m., and afternoon = 5:00 p.m.) during the RS and LS. Climatic air temperature and relative humidity data were recorded, and the temperature and humidity index (THI) was calculated. Higher thermal challenge was observed in LS relative to RS (air temperature = 28.0 vs. 30.9 °C; relative humidity = 84.1 vs. 69.9%; THI = 80.0 vs. 82.5; P < 0.05). In both seasons, respiratory rate and internal temperature were normal, demonstrating the animals' adaptability. In RS, however, a higher scrotal temperature was recorded in relation to LS (35.0 vs. 34.7 °C; P < 0.05), with a gradual increase from morning to afternoon. Lower resistivity (0.40 vs. 0.64; P < 0.05) and pulsatility (0.55 vs. 1.14; P < 0.05) indices, and a higher rate of high-velocity blood flow of testicular arteries (71.1 vs. 60.6%; P < 0.05) were observed in RS compared to LS. The lowest correlations between testicular hemodynamic, physiological variables, and environmental parameters (P < 0.05) were observed in the morning. In conclusion, testicular thermoregulation and testicular hemodynamics were influenced by the climatic seasons and time of the day, being more efficient in the LS season and with less interference from environmental factors in the morning.

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.

Effect of eCG in a short-term synchronization treatment on ovarian status, estrus synchrony, and ovulation in dairy goats managed under tropical conditions.

The aim of this study was to assess the need of using eCG on short-term estrus synchronization protocol in nulliparous (NUL) and multiparous (MULT) dairy goats during the breeding season. Alpine (n = 20), Nubian (n = 20), and Saanen (n = 16) goats received 60 mg medroxyprogesterone acetate intravaginal sponges for 6 days plus 30 µg d-cloprostenol and 200 IU eCG (G-eCG, n = 28) or saline (G-Control, n = 28) 24 h before sponge removal. The NUL and MULT goats of each breed were equally assigned into the two treatments. Transrectal ultrasonography was used to evaluate ovulatory parameters, and teaser goats were used for estrus detection every 12 h from sponge removal to ovulation. eCG did not affect (P > 0.05) estrus response (~86%), diameter of ovulatory follicles (~6.8 mm), and number of ovulations (~1.6). Nevertheless, eCG led to earlier (P < 0.05) ovulation (G-eCG = 65.1 and G-Control = 73.2 h) and increased (P < 0.05) the ovulation rate (G-eCG = 96.4% and G-Control = 67.9%). In the absence of eCG, no differences regarding reproductive parameters (P > 0.05) were found between parity orders. Alpine MULT goats underwent a superior (P < 0.05) number of ovulations (2.2) in comparison to NUL goats (1.3). In conclusion, the exclusion of eCG from short-term estrus synchronization protocol did not interfere with estrus response but decreased the ovulation rate.

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.

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.

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.

Changes in testicular size, echotexture, and arterial blood flow associated with the attainment of puberty in Dorper rams raised in a subtropical climate.

There is a paucity of information on the relationships of testicular morphology, echotextural attributes, and blood flow dynamics with pubertal development of rams raised in a subtropical climate. Forty-five Dorper rams (24 rams aged 8-11 months and 21 rams aged 12-24 months) were examined using a portable ultrasound scanner connected to a 7.5-MHz transducer. Computer-assisted analyses of testicular ultrasonograms utilized commercially available Image ProPlus® analytical software. Spectral Doppler scans of testicular arteries were performed immediately after scrotal (B-mode) ultrasonography to determine peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI = [PSV-EDV]/PSV), and pulsatility index (PI = [SPV-EDV]/mean velocity) of the blood vessels. The length of the testes (9.7 ± 0.3 compared with 9.0 ± 0.2 cm) and scrotal circumference (33.3 ± 0.5 compared with 31.8 ± 0.4 cm) were greater (p < 0.05) but testicular depth (4.5 ± 0.1  compared with 4.9 ± 0.08 cm) was less (p < 0.05) in sexually mature compared with peripubertal rams. [Corrections added on 9 Jan 2019 after initial online publication: The testicular size values in the sentence were corrected.] There were no differences (p > 0.05) between the two age groups of Dorper rams in blood flow indices of testicular arteries. Mean numerical pixel values (100.5 ± 4.1 compared with 89.2 ± 4.8) and pixel heterogeneity (25.6 ± 0.6 compared with 23.6 ± 0.5) of testicular parenchyma were greater (p < 0.05) in peripubertal than in postpubertal rams. Semen volume was negatively correlated with PI of testicular arteries (r = -0.57, p = 0.04). In summary, the attainment of sexual maturity in the rams of the present study was associated with significant changes in testicular length and depth, scrotal circumference, and parenchymal echogenicity/hetrogeneity but not in testicular volume and blood perfusion rates. Testicular artery PI can be used to predict the volume of ejaculate in rams.

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

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.

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.

Early luteal development in Santa Inês ewes superovulated with reduced doses of porcine follicle-stimulating hormone.

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty-nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B-mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11-15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose.

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.

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous-induced Santa Inês ewes (6-day treatment with progestin-releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6-7 days after oestrous onset and mating. All ewes received 37.5 µg of d-cloprostenol via latero-vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non-surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17-40 min) and ~96% of flushing fluid could be recovered (range: 85%-100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep.

Non-surgical embryo transfer in goats and sheep: the Brazilian experience.

Brazil has presented tremendous progress in non-surgical embryo transfer (NSET) in sheep and goats. New instruments and techniques for non-surgical embryo recovery (NSER) and NSET in small ruminants were implemented. Recent improvements include refinement of the protocols for cervical relaxation combining oestradiol-oxytocin-cloprostenol treatment at specific times before NSER in sheep; recipient goats do not require any hormonal drugs to induce cervical dilation and direct embryo transfer by the cervical route yields excellent results. Transrectal ovarian ultrasonography (B-mode but especially colour Doppler) have proven to be accurate methods to localise and enumerate corpora lutea and luteinised unovulated follicles in recipient and donor does and ewes. An array of new criteria for selecting superior animals for NSER and NSET (e.g. cervical mapping) have been developed by Brazilian researchers. Extensive studies on both technologies were initially conducted in commercial breeds of goats and sheep but have been gradually extended to some native breeds of sheep (germplasm conservation) and dairy goat operations. It is speculated that, in future, NSER and NSET may become methods of choice for caprine and ovine embryo recovery and transfer in Brazil, and then globally. Due primarily to the efficiency of NSET in goats, a novel interspecies (e.g. bovine) IVP method may soon be developed on a large scale. The Brazilian experience is an invaluable source of information and know-how promoting the replacement of conventional surgical assisted reproductive technologies with non-surgical procedures and hence supporting the rapid development of the embryo transfer industry in small ruminants.

Reproductive and productive performances of Santa Inês ewes submitted to breeding in different periods of the Amazonian humid tropical climate.

The objective of this study was to evaluate the reproductive and productive performance of Santa Inês ewes bred at different times of the year in humid tropical climate. One hundred and forty-eight Santa Inês ewes were grouped according to the time of the year of their breeding season (i.e., mating period) (dry/wet, wet, wet/dry, and dry season). The service type was natural mating and the ewes and rams were kept together every night for 45 days. Reproductive efficiency was assessed by service, pregnancy, lambing, prolificacy, twinning, pregnancy loss, weaning, and lamb mortality rates. Ewes were weighed at the beginning and at the end of the breeding season and before and after parturition, and sequential weighing of the lambs was performed (at birth, 15, 30, 60, and 90 days). Reproductive efficiency index (number of lambs weaned/total of served ewes) and productive efficiency (kg of weaned lamb/kg of served or lambed ewes) were calculated. All ewes expressed estrus early in the breeding season; however, a higher percentage (53.5 and 7.1 % at 30 and 45 days, respectively) of ewes returned to estrus during the wet/dry period. The lower rates (13.9 %) of return to estrus at 30 days were during the wet season (P < 0.05). There were no (P > 0.05) effects of breeding seasons on the remaining reproductive rates. Ewes that lambed during the wet/dry transition period weighted less, before (40.5 ± 2.5 kg) and after (38.6 ± 1.6 kg) parturition, than those of other groups (P < 0.05). Lamb weight at birth did not vary between groups, however, weight at weaning was higher (15.6 ± 2.1 kg) in lambs born during the wet season (P < 0.05). The reproductive efficiency index was lower (0.66) when the breeding season took place during the dry/wet period (P < 0.05). Productive rates were significantly higher (0.29 and 0.33 for kg of weaned lamb/kg of served and lambed ewes, respectively; P < 0.05) in ewes served in the dry season. The reproductive performance of Santa Inês ewes was not significantly influenced by the period of the year in which the breeding seasons took place, allowing for four breeding seasons a year in the Amazon region. Variations between periods in return to estrus rates, weight of ewes close to parturition and lamb weight at weaning indicate that climate changes can also affect reproductive rates.

Transcervical uterine flushing and embryo transfer in sheep: Morphophysiological basis for approaches currently used, major challenges, potential improvements, and new directions (alas, including some old ideas).

At present, the success of non-surgical embryo recovery (NSER) and transfer (NSET) hinges upon the cervical passage of catheters, but penetration of the uterine cervix in ewes is problematic due to its anatomical structure (i.e., long and narrow cervical lumen with misaligned folds and rings). It is a major obstacle limiting the widespread application of NSER and NSET in sheep. While initial attempts to traverse the uterine cervix focused on adapting or re-designing insemination catheters, more recent studies demonstrated that cervical relaxation protocols were instrumental for transcervical penetration in the ewe. An application of such protocols more than tripled cervical penetration rates (currently at 90-95 %) in sheep of different breeds (e.g., Dorper, Lacaune, Santa Inês, crossbred, and indigenous Brazilian breeds) and ages/parity. There is now sufficient evidence to suggest that even repeatedly performed cervical passages do not adversely affect overall health and reproductive function of ewes. Despite these improvements, appropriate selection of donors and recipients remains one of the most important requirements for maintaining high success rates of NSER and NSET, respectively. Non-surgical ovine embryo recovery has gradually become a commercially viable method as even though the procedure still cannot be performed by untrained individuals, it is inexpensive, yields satisfactory results, and complies with current public expectations of animal welfare standards. This article reviews critical morphophysiological aspects of transcervical embryo flushing and transfer, and the prospect of both techniques to replace surgical methods for multiple ovulation and embryo transfer (MOET) programs in sheep. We have also discussed some potential pharmacological and technical developments in the field of non-invasive embryo recovery and deposition.

Luteal tissue characteristics of Morada Nova ewes with hCG application 7.5 days after the end of estrus synchronization protocol in the breeding season.

Results with the use of hCG after synchronization protocol are still inconsistent, which may vary according to breed, season, day of application and dose of the drug used. The aim of the present study was to evaluate the functionality of luteal tissue and ovarian perfusion after hCG treatment during early luteal phase. Estrus-synchronized ewes were randomly assigned to receive i.m. injection of 300 IU of hCG (G-hCG; n = 40) or 1 mL of saline (G-Control; n = 32) on Day 7.5 after progesterone withdrawal. Ultrasonographic evaluations of the ovaries and ovarian and iliac arteries were performed on Days 7.5, 10.5, 13.5, and 21.5. The accessory corpus luteum (aCL) formation rate was 52.5% for G-hCG. There was interaction (p > 0.05) for treatment (G-hCG and G-Control), days (7.5, 10.5, 13.5 and 21.5) and PD (Pregnant and Non-pregnant) for the variables of biometric characteristics of the corpus luteum B-Mode and Color Doppler on days 7.5, 10.5, 13.5 and 21.5. There was no difference (p > 0.05) for pregnancy rates and mean fetuses per ewe between the treatment groups. It is concluded that the application of hCG 7.5 days after the hormonal protocol in Morada Nova ewes in a breeding season is efficient in inducing aCL formation and increasing luteal tissue biometry. However, there was no effect on pregnancy rate.

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.

The effect of flunixin meglumine on the premature regression of corpus luteum, recovery rate, and embryo production in superovulated Dorper ewes.

This study evaluated the use of flunixin meglumine to prevent the occurrence of premature corpus luteum (CL) regression in superovulated ewes, improving embryo recovery and viability. Ewes (n=23) submitted to conventional superovulatory protocol and laparoscopic artificial insemination were treated with 2.2 mg/kg/day of flunixin meglumine (FLU, n=12) or 1.5 mL saline solution (CONT, n=11) on Days 2, 3, and 4 (Day 0 = 48 h after device removal). Serum progesterone (P4) concentrations were measured (Day 1-6). Ultrasound (US, Days 3 and 6) and laparoscopic evaluation (Day 6) were performed to identify luteinized structures. In the US, laparoscopy, and P4 assessments, the percentage of ewes with premature CL regression differed (P<0.05) between CONT (54.5; 63.6; and 54.5 %) and FLU (0.0; 0.0; and 0.0 %), respectively. The US exams revealed the effect (P<0.05) of treatment on the number of regressing CL between CONT (1.4 ± 0.6) and FLU (0.0 ± 0.0). Greater (P<0.05) number of normal CLs (10.5 ± 1.8 vs. 4.4 ± 1.5), ova/embryos (9.1 ± 2.1 vs. 3.7 ± 1.3), viable embryos (5.1 ± 1.1 vs. 2.6 ± 1.2), and recovery rate (79.5 ± 9.6 vs. 41.3 ± 15.0 %) were observed in FLU compared to CONT, respectively. The embryo viability rate did not differ (P>0.05) between FLU (60.7 ± 10.5 %) and CONT (45.5 ± 16.1 %). In conclusion, the flunixin meglumine protocol was able to prevent the occurrence of premature CL regression in superovulated ewes, increasing the recovery rate and embryo production.