The pFSH dose affects the efficiency of in vivo embryo production in Santa Inês ewes.

This study compared the follicular growth, superovulatory response, and in vivo embryo production after administering two doses of porcine follicle-stimulating hormone (pFSH) in Santa Inês ewes. The estrous cycle of 36 multiparous ewes was synchronized with the Day 0 protocol and superovulated with 133 mg (G133, n=18) or 200 mg (G200, n=18) of pFSH. Ultrasonographic evaluations of the ovaries were performed, ewes were mated and submitted to non-surgical embryo recovery. Viable blastocysts were stained with Nile Red and Hoechst. The G200 had a greater number of medium and large follicles, as well as a larger size of the third largest follicle. A total of 97.2% (35/36) of the ewes came into estrus and it was possible to transpose cervix in 80.6% (29/36). There were no effects of treatments in the response to superovulation, the proportion of ewes in which was possible to transpose the cervix, the number of corpora lutea, the number of anovulatory follicles, the proportion of ewes flushed with at least one recovered structure, number of recovered structures, number of viable embryos, viability rate, and recovery rate. The G200 ewes were in estrus for a longer period of time than the G133 ewes (54.0 ± 4.5 h vs. 40.3 ± 3.6 h) and produced more freezable embryos (6.5 ± 1.6 vs. 2.3 ± 0.7) than G133. Both doses promoted an efficient superovulatory response and did not affect embryonic lipid accumulation. The dose of 200 mg of pFSH showed greater potential to increase the superovulatory response, as it increased follicular recruitment and the recovery of freezable embryos.

Transcervical artificial insemination in the brown brocket deer (Subulo gouazoubira): a promising method for assisted reproduction in deer.

The present study aimed to test the efficiency of transcervical artificial insemination techniques with cervical immobilization (TCAI-CI) or cervical traction (TCAI-CT), associated or not with the use of oxytocin (OT) as a protocol for cervical dilation, in the brown brocket deer (Subulo gouazoubira). The study was carried out in a crossover design using four adult females in two replicates with an interval of 60 days. Estrus was synchronized with oral melengestrol acetate (MGA) associated with estradiol benzoate and sodium cloprostenol. TCAI techniques were performed from 18 to 24 h after estrus onset. All females received either an i.v. application of 50 IU of OT (G-OT, n = 4) or 1 mL of saline solution (G-Control, n = 4) 20 min before the TCAI procedure. The TCAIs were performed using frozen-thawed semen motility 40%, vigor 3, acrosome integrity 87%, membrane integrity of 95% and 13% of total post-thaw defects from the same batch. Behavioral estrus was observed in 100% of the females, in both replicates. It was achieved a 50% (4/8) success of cervical transposition with semen deposition in the uterine. Regarding inseminations, most of them (87.5%) were performed using the TCAI-CT technique, and the overall conception rate was 50%. Cervical transposition times (< 1 min) and TCAI procedures (~ 17 min) were considered satisfactory. Thus, the performance of the TCAI-CI and TCAI-CT techniques was successful, regardless of using OT as a cervical dilation protocol. This procedure is proposed as a method of choice for artificial insemination with greater applicability in different conservation centers, compared to more advanced reproductive biotechniques, and with a favorable impact on the conservation of deer species.

Use of meloxicam with or without dipyrone in non-surgical embryo recovery in hair sheep: Effects on animal welfare.

The aim of this study was to determine the effectiveness of meloxicam with or without dipyrone on the welfare of ewes subjected to non-surgical embryo recovery (NSER). Two studies were carried out using 51 multiparous Santa Inês ewes. All animals received a standard oestrous synchronization treatment and a superovulatory protocol. In Study 1, 12 ewes received meloxicam (GM) before cervical transposition (1 mg kg-1 , i.v.), repeated 24 h after (1 mg kg-1 , i.m.), while the other 10 received a saline solution, remaining as a control group (GC1). In Study 2, ewes were allocated into a group of 15 ewes treated as GM of Study 1 associated with dipyrone (GMD; 50 mg kg-1 , i.m.) before cervical transposition, 12 h, and 24 h after, or a control group (GC2) of 14 ewes treated with saline solution. In both studies, heart and respiratory rates (RR), cortisol, glucose, total proteins, albumin and globulins blood concentration were recorded before sedation (BS), after sedation (AS), after cervical transposition, immediately after collection (IAC), and 0.5, 1.5, 3, 6, 12, 24 and 48 h after embryo collection (hAC). In Study 1, RR tended to be greater in GC1 (p = .08), serum total proteins and globulins values were lower and serum albumin values were greater in this group than GM (p = .003, p < .0001, and p < .0001, respectively). In Study 2, treatment of GMD tended to reduce the glycaemia at AS (p = .052) and reduced it at 3hAC (p < .0001), and 6hAC (p = .03). It also tended to reduce cortisol concentrations (p = .10). The other variables varied with NSER without interaction with the experimental treatments. In conclusion, in this study condition, NSER in sheep induced transient changes indicative of stress and possibly pain, therefore, affecting animal welfare. The administration of meloxicam was ineffective to reduce those responses, and the association of dipyrone had only slight effects without modifying the main welfare indicative responses in ewes subjected to NSER.

Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

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.

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.

Follicular dynamics and in vivo embryo production in Santa Inês ewes treated with smaller doses of pFSH.

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P <  0.05), ovulation time later (P <  0.05), and PFS rate greater (P <  0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P <  0.05) than in the G133 group (P <  0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P <  0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.

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.

Anti-Müllerian hormone and antral follicle count are more effective for selecting ewes with good potential for in vivo embryo production than the presence of FecGE mutation or eCG pre-selection tests.

This study aims to compare four different methods for selecting high responding sheep donors for in vivo embryo production. These methods include a pre-selection eCG test (eCG), antral follicle count (AFC), plasma anti-Müllerian hormone measurement (AMH) and genotyping for the presence of the FecGE mutation (a polymorphism in the GDF9 gene associated with increased ovulation rate). Santa Ines ewe lambs (n = 25) underwent superovulation (SOV) with 800 IU equine chorionic gonadotropin (eCG) and the corpus luteum (CL) count was recorded by laparoscopy after eight days. At the D0eCG, blood samples for AMH and genotyping analysis were collected. Twenty-one days after the end of the eCG test, the same animals underwent SOV with 200 mg of FSH, administered in six decreasing doses, and then naturally mated. Immediately before the beginning of the FSH protocol (D0FSH), and at the moment of the first FSH dose (D9FSH), the AFC was assessed. Plasma AMH was again determined at the D9FSH. After each screening process, animals were classified as having a high (HR), or low (LR), potential of response (using specific thresholds for each method). Then, the ewes' response to SOV and embryo yield for each screening method, classified as HR or LR, were compared. Animals classified as HR by AFC (HRAFC) and by AMH concentration (HRAMH) at the D9FSH, produced more viable embryos than those classified as LRAFC and LRAMH (HRAFC 6.2 ±â€¯3.2 vs LRAFC 2.8 ±â€¯3.0 and HRAMH 6.6 ±â€¯3.6 vs LRAMH 3.0 ±â€¯2.9). Pre-selection tests with eCG and different FecGE genotypes, either heterozygous (+/E) or wild type (+/+), were unable to discriminate HR or LR animals. A tendency (P = 0.06) to have lower plasma AMH was observed in heterozygous FecGE (+/E) ewes. In conclusion, both AFC and plasma AMH can be used to select donor ewes with a higher potential of response for in vivo embryo production.

Use of two doses of cloprostenol in different intervals for estrus synchronization in hair sheep under tropical conditions.

This study evaluated the effect of two doses of prostaglandin at different intervals on reproductive parameters of crossbred ewes. In Experiment 1, 30 ewes received two doses of 120 µg cloprostenol at 7 (G 7 days), 9 (G 9 days), or 11.5 (G 11.5 days) days apart. Ultrasound assessments were performed from the first and second cloprostenol administration for 5 days or ovulation detection. Estrus signs were checked by a teaser male. Plasma progesterone concentration was measured before each cloprostenol dose. In Experiment 2, 95 ewes were allocated into the same treatments and after the second dose, ewes in estrus were mated. At 30 days after breeding, pregnancy diagnosis was conducted and prolificacy was evaluated at lambing. In Experiment 1, at the first cloprostenol administration, 50% of ewes had an active CL and all showed estrus. At the second administration, 66.7% of ewes had an active CL and one did not present estrus. There was no difference (P > 0.05) after the second dose for as follows: overall estrous response (90%), interval from cloprostenol administration to estrous onset (42.0 ± 4.9 h), estrus duration (31.5 ± 2.1 h), ovulation rate (100.0%), and number of ovulations (1.5 ± 0.3). In Experiment 2, both pregnancy and prolificacy rates were similar (P > 0.05) for G 7 days (73.3; 145%), G 9 days (75.9; 125%), or G 11.5 days (75.9; 145%), leading to an overall pregnancy rate of 75.0% (66/88) and prolificacy rate of 137%. Therefore, the three treatments proposed were able to promote high pregnancy and prolificacy rates in crossbred ewes.

Effects of hCG administration on accessory corpus luteum formation and progesterone production in estrous-induced nulliparous Santa Inês ewes.

The effect of hCG administration on accessory corpus luteum (ACL) formation, CL area, and plasma progesterone (P4) concentration (ng/mL) seven days after breeding was studied in nulliparous Santa Inês sheep. Intravaginal 60 mg MAP sponges were inserted into ewes for six days and 300 IU eCG i.m. and 30 µg d-cloprostenol latero-vulvar were administered 24 h before sponge removal. Ewes were naturally bred and, seven days after first mating (Day 0; D0), were treated with either 250 IU hCG (hCG group; n = 7) or 1 mL saline solution (control group; n = 7). Blood was collected to determine plasma P4 concentrations and sonograms were performed on Days 7, 10, 13, 16, 19, and 22. Number of CL on D7 was similar (P > 0.05) between hCG (1.3 ± 0.5) and control (1.3 ± 0.5) groups; however, on D13, it was greater (P < 0.05) in the hCG group (2.3 ± 0.5) than in the control group (1.3 ± 0.5). A greater (P < 0.05) luteal tissue area was detected in hCG-treated ewes (n = 4) on Days 16 to 22 than in the animals in the control group (n = 7). Plasma P4 concentration on D13 to D22 was higher (P < 0.05) in hCG-treated animals than in control ewes. Administration of hCG seven days after estrus onset efficiently induced accessory CL formation in ewes, increasing luteal tissue area and plasma P4 concentration.

Effect of different hormonal combinations on follicular wave emergence and superovulatory response in sheep.

The aim of the present study was to compare hormonal treatments to induce and synchronize follicular wave emergence to improve the results of superovulatory (SOV) treatments in ewes. In Experiment 1 (n = 66), ewes were treated with a progesterone intravaginal implant plus a PGF2α analogue (group GP4), or with the same treatment plus estradiol benzoate (GP4+EB), a GnRH agonist (GP4+GnRH), or both, estradiol benzoate and a GnRH agonist (GP4+EB+GnRH) in a 2 × 2 factorial arrangement. Follicular wave emergence was determined by ultrasound. Follicular wave did not emerge during the studied period in 10 females (one from GP4, six from GP4+EB and three from GP4+EB+GnRH). Follicular emergence was less synchronized (P = 0.007) when estradiol was administered (GP4+EB: 103.6 ± 22.0 h), without any interaction with GnRH treatment (GP4+EB+GnRH: 80.1 ± 21.4 h, GP4+GnRH: 52.5 ± 8.7 h, GP4: 56.6 ± 10.4 h). Estradiol administration delayed the moment of follicular emergence (P = 0.007) and the follicular wave emergence moment in which follicular dominance was achieved (P = 0.009), without interactions between estradiol and GnRH in the moment of follicular wave emergence or dominance. In Experiment 2 (n = 22), two SOV protocols were compared: the best treatment of Experiment 1 (GP4) was used to synchronize follicular wave emergence, initiating the SOV treatment 2.5 days later; in the control treatment, SOV treatment started 80 h after a short-term protocol to synchronize ovulation (Gcontrol). The number of corpora lutea (CL) and the evaluation of the collected embryos were performed six days after estrus. Blood samples were collected daily for plasma progesterone determination. Although the number of CL was similar in Gcontrol (7.1 ± 1.0) and GP4 (6.9 ± 5.1), the number of structures and viable embryos recovered were greater in Gcontrol (P < 0.05). The occurrence of luteal premature regression was significantly greater in GP4 (60%) than in Gcontrol (8.3%). The use of GnRH agonist alone did not improve synchronization of follicular wave emergence. When EB was used (alone or associated) follicular wave emergence was less synchronized. The SOV protocol proposed had a similar ovarian response; however, it resulted in less transferable embryos.

Efficiency of different hormonal treatments for estrus synchronization in tropical Santa Inês sheep.

The aim of this study was to evaluate the effect of (i) the duration of hormone treatment with progestogen sponges during the seasonal anestrus and (ii) the administration of two doses of prostaglandin at 7 days apart during the breeding season on reproductive parameters of Santa Inês ewes. In experiment 1, 32 ewes received intravaginal MAP sponges for 6 (G6 days), 9 (G9 days), or 12 (G12days) days and 75 µg D-cloprostenol i.m. and 300 IU eCG i.m. 1 day before sponge removal. In experiment 2, 23 ewes received two doses of 0.48-mg sodium cloprostenol i.m. 7 days apart. Ovarian follicular dynamic was assessed through transrectal ultrasonography. Blood samples were collected daily to determine progesterone concentrations. In experiment 1, estrus and ovulation rates did not differ (P > 0.05) among protocols and between cyclic and acyclic ewes at the beginning of the experiment. The G9 days treatment showed a lower dispersion of ovulations in relation to onset of estrus when compared to G6 days and G12 days. In experiment 2, all ewes exhibit estrus and ovulated after the second dose of prostaglandin, although ewes that were in diestrus at D0 showed subluteal concentrations of progesterone during the follicle development stage of the treatment. In conclusion, the use of progestogen device during 9 days promotes lower dispersion of ovulation when compared to its use for 6 or 12 days, and the protocol of two doses of prostaglandin 7 days apart synchronizes estrus efficiently but results in follicular development under low progesterone concentrations.

Reproductive performance, metabolic and hormonal profiles of Santa Inês ewes in winter and summer under tropical conditions.

The study aimed to evaluate the reproductive, metabolic and hormonal profiles of Santa Inês ewes during winter and summer in the tropical climate of Rio de Janeiro State, Brazil. The ewes (n = 16) were prepared for induction of synchronised oestrus with a short-term hormonal treatment, and follicular dynamics were assessed with ultrasonography. Blood samples for biochemical and hormonal analyses were collected on days zero, six and eight of the protocol at 7, 13 and 19 h. Reproductive behaviour did not differ (P > 0.05) between seasons. Based on ultrasonography data, the ovulation rate was 100% in summer and 81.3% in winter. Most of the ewes showed double ovulation in summer, and the diameter of the largest and second largest follicles differed (P < 0.05) between seasons. Among the biochemical parameters, plasma glucose, plasma urea and serum albumin differed (P < 0.05) between the study periods. Regarding the hormonal profile, higher serum triiodothyronine, thyroxine and prolactin concentrations (P < 0.05) were observed in summer. The average progesterone concentrations demonstrated low reproductive seasonality. In conclusion, the reproductive performance of the ewes was superior in summer, and higher levels of plasma glucose and serum albumin during this season may have played a role in the observed ovulation rates. The higher levels of serum prolactin and serum thyroxine in summer demonstrated that these compounds were not involved in the regulatory mechanisms underlying the onset of anoestrus in these Santa Inês ewes under tropical conditions.

Is the Santa Inês sheep a typical non-seasonal breeder in the Brazilian Southeast?

This study aimed to characterize the annual reproductive cycle of Santa Inês sheep in the Fluminense lowland region (latitude 22° 27' 45″ south, Rio de Janeiro, Brazil) between September 2011 and August 2012. Ten ewes were maintained in a semi-intensive system under natural photoperiods with access to pasture and shelter. Blood samples were collected every 2 weeks to determine plasma progesterone concentrations. The body condition score (BCS) was determined each month. There was no seasonal variation in the plasma progesterone concentration from the months of September to January, April, and May to August. In the months of February and April, the plasma hormone levels were higher than August to November. Seventy percent (7/10) of the sheep studied had short seasonal anestrus. The periods of anestrus were concentrated between the months of September and December (spring season) in 85.7 % (6/7) of the cases evaluated. In these cases, 57.1 % (4/7) also had short periods of reproductive inactivity during other months of the year. The progesterone values obtained in the spring corroborate the higher reproductive anestrus observed in this season. Higher plasma progesterone values were found in summer and autumn with reduction in the winter to lower values in the spring. No changes in the BCS during the study period were observed. Under the studied conditions, the Santa Inês sheep showed a low degree of reproductive seasonality. However, some individual ewes had seasonal anestrus during the spring. Further studies that include management techniques are needed to improve reproductive efficiency without hormone therapy in this breed under tropical conditions.