Follicular Wave Synchronization Research Articles

Effect of follicular wave synchronization using a progesterone-releasing intravaginal device-based protocol on in vitro embryo production in Bos taurus cows subjected to 14-day intervals ovum pick-up

Follicular wave synchronization (FWS) before ovum pick-up (OPU) is one of the strategies used to improve the efficiency of in vitro embryo production (IVP). This study aimed to evaluate the effect of FWS on the total follicular number, cumulus-oocyte complex (COC) recovery, and in vitro embryo development in Angus cows (n = 33) subjected to OPU with 14-day intersession intervals. Additionally, it was also evaluated the presence of carryover effects given the short intersession interval used. The experiment was run as a 2-treatment (FWS vs. Control) x 2-period (1 vs. 2) crossover design. Animals in the FWS group received an intravaginal progesterone implant (1gr), estradiol benzoate (2 mg), and D-cloprostenol (150 μg) on day 0 and the OPU was performed on day 5. Control group animals did not receive any hormone treatment. The FWS increased the number of 6–10 mm follicles (P = 0.05), but it decreased the COC recovery rate (P < 0.01). The FWS did not affect the total or frozen embryo numbers (P = 0.49 and P = 0.17; respectively), but it increased the total blastocyst cell number (P < 0.01). A carryover effect was found on the total and < 6 mm follicles number (P = 0.10 and P < 0.01; respectively), and on the regular, atretic, viable, and total number of COC (P = 0.01, P = 0.08, P = 0.02 and P < 0.01; respectively). We concluded that the FWS increased the quality of embryos after OPU with 14-day intersession intervals in Angus cows and that this kind of OPU/IVP scheme enabled the existence of a carryover effect, especially on the follicle number and COC morphology.

Influence of ovarian follicular wave synchronization and single-dose eCG superstimulation on oocyte collection and in vitro embryo production in bison during the ovulatory and anovulatory seasons

In an effort to develop an effective, minimum-handling protocol for the conservation of wood bison, the present study was designed to determine the effects of ovarian synchronization and superstimulation on cumulus oocyte complex (COC) collection and in vitro embryo production in wood bison during the ovulatory (Exp. 1) and anovulatory seasons (Exp. 2). We tested the hypotheses that COC collection and in vitro embryo production are 1) greater after follicular wave synchronization than at random stages of the follicular wave, 2) repeatable within individuals, 3) greater after ovarian superstimulation with a single dose of eCG than without treatment, and 4) greater during the anovulatory season than the ovulatory season. In Exp. 1, ultrasound-guided COC collection was performed on Day −1 in wood bison to induce follicular wave emergence the following day (Day = 0). Immediately after the COC collection on Day −1, bison were given a single im dose of 2500 IU eCG or saline (n = 6 per group). Subsequent COC collections were on Days 4 and 9. A similar design was used in Exp. 2, with an additional treatment group given 5000 IU eCG (n = 8 per group). In Exp. 1, compared to the saline-treated group, a single dose of 2500 IU eCG resulted in a greater number of ≥8 mm follicles at the time of the Day 4 COC collection (P = 0.03), but not at the Day 9. In Exp. 2, treatment with 5000 IU eCG resulted in a greater number of ≥8 mm follicles than 2500 IU eCG or the saline treatment (37.5 ± 6.9, 17.5 ± 2.0, 16.9 ± 2.0; P = 0.01, respectively). Although the number of embryos produced/COC submitted to IVM was not different among groups (mean = 18.6%), treatment with 5000 IU eCG produced more than twice as many embryos per bison as unstimulated bison (0.8 vs 1.9). In summary, embryo production rates were higher from COC collected subsequent to follicular wave synchronization vs random stages of the wave, and ovarian superstimulation with eCG resulted in a dose-related increase in the number of ≥8 mm follicles, COC collected, and embryos produced. Repeated COC collections after successive wave synchronization resulted in similar follicular counts and embryo production rates within individuals, and the greatest number of follicles aspirated, COC collected, and embryos produced was in the anovulatory season. We conclude that the minimum-handling COC collection protocols in the present study are effective and provide realistic options for embryo production in wild bison.

Effects of lipopolysaccharide on beta-catenin, aromatase, and estrogen production in bovine granulosa cells in vivo and in vitro

Diseases resulting from Gram-negative bacterial infection can induce an immune response by releasing a lipopolysaccharide (LPS) endotoxin that may lead to impaired fertility in cows. To evaluate the effects of LPS on follicular dynamics in a subacute inflammatory disease state, 14 Angus heifers (BW = 413 kg±14) were blocked by weight and assigned to vehicle (n = 7) or LPS treated (n = 7) groups. Heifers received subcutaneous injections of saline (CON) or 2.0 μg/kg LPS on d 2, 5, and 8 of a select synch plus controlled internal drug release device (CIDR) follicular wave synchronization protocol. Fifty hours following CIDR withdrawal, ovaries were harvested, and follicular fluid was collected for hormone and LPS analysis. Daily blood samples were collected from d 0 to d 7. Beginning on d 8 blood samples were collected at 0, 16, 24, 32, 40, and 50 h following LPS challenge. Rectal temperatures were recorded prior to treatment and at regular intervals after each LPS challenge. Heifers treated with LPS exhibited mild (+0.5 °C) hyperthermia (P < 0.05) at 3, 4, and 8 h after the initial LPS challenge (d 2) when compared to vehicle-treated controls. Follicular fluid concentrations of estradiol (E2) increased (P = 0.04) in LPS-treated heifers compared to controls (1,595 ng/mL and 808 ng/mL±240, respectively), while follicular fluid progesterone (P4) concentrations did not differ (P = 0.27) between treatment groups. Additionally, LPS concentrations tended to be increased (P = 0.59) in dominant follicles of LPS-treated heifers, but no difference was detected (P = 0.81) in small developing follicles. To further delineate the impact of LPS on ovarian signaling pathways, a granulosa cell line (KGN) was incubated in the presence or absence of LPS (10 μg/mL) for 48 h. Cells were then collected for gene expression and protein analysis. Cells in both treatment groups expressed toll-like receptor 4, myeloid differentiation factor-2 receptor, and CD-14 complex genes required for LPS signaling. Cells treated with LPS exhibited decreased mRNA expression of aromatase (P = 0.03) and beta-catenin (P = 0.02). However, no change (P > 0.10) was detected in abundance of total beta-catenin protein or beta-catenin phosphorylated isoforms at serine 552 or 675. Based on results from this in vivo experiment, these investigators concluded that low doses of LPS can alter E2 concentrations and this effect may be modulated in part through beta-catenin regulation of aromatase transcription.

Follicular wave synchronization prior to ovum pick-up

Among the reproductive biotechnologies, in vitro embryo production (IVEP) is an important tool for multiplying genetic material of superior merit. Recently, the number of embryos produced and transferred in vitro became significantly higher than that produced in vivo worldwide. In this context, the enhancement was attributable to ovum pick-up (OPU). With the advent of genomic technology, shortened breeding intervals, and increased selection accuracy, IVEP has attracted increasing attention for commercial use. The IVEP technique is well-established, but the embryo production rate has reached a plateau at 30–40%. Despite constant advances, the OPU/IVEP programs face some challenges that hinder the efficient application of the technique. Previous studies have shown that the quantity and quality of aspirated oocytes are essential factors for successful IVEP. This paper presents a brief overview of alternatives that can be employed to improve the process-seeking methods that assist in the recovery of better-quality oocytes and higher competence in OPU to improve embryo production. These strategies include using follicular wave synchronization prior to OPU, employing the influence of antral follicle populations, using the pre-OPU gonadotrophic stimulus and applying non-hormonal methods for selecting female donors.

How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro? (12 years of practical results at a California dairy farm).

Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.

107 Follicular wave synchronization and FSH stimulation prior to ovum pickup for invitro embryo production

Invitro embryo production (IVP) has become a reliable alternative for genetic improvement in beef and dairy herds. Maximizing embryo yield and pregnancy per donor are key factors. The aim of this study was to compare ovum pickup (OPU) yields, developmental competence of cumulus-oocyte complexes (COCs), and pregnancy rates from Angus donors in a commercial IVP setting. Donors (&amp;gt;4-year-old pluriparous open dry cows) were handled under the same feeding and environmental conditions. Treatment groups were organised as follows: Group 1: no synchronization (SYNCH; n=5); Group 2: SYNCH with no superstimulation (SOV; n=5); Group 3: SYNCH + SOV (n=5) and OPU 36h after last FSH injection; Group 4: SYNCH + SOV (n=5) and OPU 48h after last FSH injection; and Group 5: SYNCH + SOV (n=5) and OPU 72h after last FSH injection. Follicular waves in groups 2, 3, 4, and 5 were synched by gonadotrophin-releasing hormone (GnRH), prostaglandin F2α (PGF), and controlled internal drug release (CIDR). No pre-synch was used. Injections of FSH (pFSH=180mg, Folltropin) were performed IM twice a day, for three days. A minimum of three replicates were performed for each donor. A Mindray DP30V equipped with a micro-convex transducer 5.0-8.5MHz probe, disposable 20-gauge needle, and a flow rate of 15mL min−1 were used for OPU. All visible follicles (Foll) were punctured and retrieved into a 50-mL 36°C warmed tube with media (phosphate-buffered saline, bovine serum albumin (BSA), and heparin). Viable oocytes were classified according to IETS guidelines. The COCs were matured in 100µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL−1), epidermal growth factor (50ngmL−1), oFSH (50ngmL−1), bLH (3μgmL−1), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was carried out using highly fertile sires selected by discontinuous 40%/80% layers (PureSperm) and diluted to a final concentration of 1×106 spermmL−1. Matured oocytes were fertilized in 50µL of modified synthetic oviductal fluid (SOF) media supplemented with fructose (90µgmL−1), penicillamine (3µgmL−1), hypotaurine (11µgmL−1), and heparin (10µgmL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in 50-µL drops of SOF-BSA for 7 days. On Day 3.5, 2% of FBS was added. On Day 7, fresh transferable (grade 1 and 2, IETS standards) blastocysts were implanted into synchronized recipient cows. Around Day 30, ultrasound diagnosis was performed to determined pregnancy rate (PR). We used ANOVA for comparisons of mean values and X2 test for proportions, α=0.05 (Table 1). In conclusion, synchronization, FSH stimulation, and 48-h coasting before OPU in Angus cows increased the number of collected viable oocytes and embryo development rates. More transferrable embryos and higher rates of PR per OPU were obtained using 36- and 48-h coasting, respectively. Table 1.Follicles (Foll), viable oocytes, cleavage, blastocysts, and pregnancy rates (PR) in Angus cows after ovum pickup (OPU) or invitro embryo production (IVP) SYNCH1 FSH Coasting Foll/OPU Oocytes/OPU Viable oocytes Cleavage,% Embryos,% Embryos/OPU PR,% PR/OPU NO NO NO 10.8a 9.0a 8.9a 63a 25a 2.3a 45a 1.0a YES NO NO 10.1a 8.4a 8.1a 73b 27b 2.2a 51b 1.1a YES YES 36H 16.5b 13.8b 13.6b 70c 29b 4.0b 46a 1.8b YES YES 48H 16.1b 13.4b 13.3b 69c 28b 3.8c 52b 2.0b YES YES 72H 15.6b 13.0b 12.6b 64a 22c 2.8d 55b 1.6c a-dValues with different superscripts in the same column differ (P&amp;lt;0.05). 1SYNCH=synchronized.

Ovarian hormones and antioxidant biomarkers in dromedary camels synchronized with new and re-used controlled intravaginal drug release (CIDR)/GPG (Ovsynch) program during breeding season.

This study aimed to investigate the effect of CIDR, re-used wCIDR, and Ovsynch protocols for the synchronization of follicular waves on ovarian hormones, oxidative stress, and antioxidant biomarkers during the breeding season. Dromedary camels (N = 18) were divided into three equal groups. The first group received CIDR. The second group received previously used wCIDR after thorough cleaning and disinfection. The third group was subjected to GPG protocol. Progesterone (P4), estradiol (E2) l, total antioxidant capacity (TAC), catalase (CAT), lipid peroxide product (MDA), nitric oxide (NO), and glutathione reduced (GSH) were measured. Days during CIDR affected P4 (P =0.0001), E2 (P =0.047), TAC (P =0.01), NO (P =0.028), and GSH (P =0.005). Days during re-used wCIDR effected P4, TAC, CAT, NO, GSH, and MDA (P ≤0.001). Days during GPG effected P4, E2, TAC, GSH (P =0.0001), MDA (P =0.036), and NO (P =0.02). CIDR-treated camels had high P4 (P =0.0001), E2 (P =0.0001), TAC (P =0.012), and NO (P =0.0001), with low GSH (P =0.001), and MDA (P =0.003). Exogenous progesterone improved ovarian hormones and the antioxidant capacity and minimized the oxidative stress than the GPG treatment and is recommended for future reproductive management of camels.

Efficient follicular wave synchronization using a progesterone-releasing intravaginal device (PRIDΔ) in Camelus dromedarius

The reproductive efficiency of camels can be improved using artificial insemination and embryo transfer programs that require a dependable method for synchronization of follicular waves. This study aimed to evaluate the efficacy of a progesterone-releasing intravaginal device (PRIDΔ®) to induce synchronization for the follicular wave in Camelus dromedarius during the rutting season, by ovarian monitoring, assessing sexual receptivity and fertility, and quantifying progesterone (P4) and estradiol (E2) concentrations. Twenty animals received a new PRIDΔ with 1.55 g of P4 for 2 wk (Day 14); another 20 camels were used as the control. Ultrasound ovarian monitoring and camel sexual receptivity were evaluated each day during the course of the experiment for all camels in the two groups. The proportion of animals in the ovulatory (follicle diameter: 12 to 18 mm) and non-ovulatory phases was calculated according to the ovarian monitoring results using ultrasound. Blind natural mating occurred on Day 16, and ovulation, non-return, and fertility rates were calculated. Blood samples were collected. Serum P4 and E2 levels were measured daily during the trial period (21 d) using ELISA kits. The results revealed that the proportion of females in the ovulatory phase on Day 16 in the PRIDΔ group was significantly higher than that in the control group (85 and 50, respectively). Serum P4 levels increased significantly after PRIDΔ insertion and reached the maximum values (5.47, 5.02, 5.55, and 4.88 ng/mL) on Days 4, 5, 6, and 7, respectively. P4 levels significantly decreased after PRIDΔ removal and reached the minimal levels (1.23 and 0.67 ng/mL) on Days 15 and 16, respectively. There was no significant difference in the E2 levels between the groups. Ovulation, non-return, and fertility rates in inseminated camels were significantly higher in the PRIDΔ group (85%, 80%, and 75%, respectively) than in the control group (50%, 45%. and 45%, respectively). In the control group, the P4 level was maintained at a minimal level (≥1 ng/mL). In conclusion, the treatment of dromedary camels with PRIDΔ led to a uniform increase in serum concentrations of P4; however, it could not stop follicular growth. After PRIDΔ removal, P4 concentrations dropped and stimulated the development of a new follicular wave; most female camels were in the ovulatory phase 2 d after PRIDΔ removal. Therefore, PRIDΔ is efficient at inducing follicular wave synchronization in C. dromedarius with good fertility.

Monitoring and controlling follicular activity in camelids

This paper reviews that state of our knowledge concerning follicular wave dynamics, monitoring and manipulation. All camelids have overlapping follicular waves in absence of ovulation which is induced by a seminal plasma factor (βNGF). The interval between follicular waves varies. The size of the ovulatory follicle varies between 11 and 25 mm in camels and between in 6 and 13 mm in South American Camelids. The interval between induction of ovulation and next ovulatory follicle is 15 ± 1 day for all camelids. Follicular activity is best monitored by transrectal ultrasonography. Progesterone therapy for 7–15 days seems to suppress follicular dominance but does not completely inhibit follicular recruitment. Combination of estradiol and progesterone seems to provide better control of follicular activity. Both methods have provided variable results in the synchronization of follicular waves. Combination of induction of ovulation with GnRH and luteolysis at predetermined times shows some promise in synchronization of follicular dominance. These synchronization protocols require further investigation in order to provide practical approaches for fixed-time breeding. Ovarian superstimulation with FSH and eCG alone or in combination is somewhat successful. The best results are obtained when treatment is initiated at the emergence of a new follicular wave after induction of ovulation or following treatment with progesterone for 7–14 days. However, response remains extremely variable particularly in terms of ovulation rate and number of recovered embryos. Sources of this variability need to be studied including the effects of season, nutrition, doses and frequency of administration of gonadotropin.

Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick-up

This study evaluated the effects of the synchronization of ovarian follicular wave emergence on the efficiency of in vitro embryo production. Bos indicus cows (n = 20) were divided into two groups (control vs. synchronization) and subjected to repeated ovum pick-up (OPU) sessions (8 replicates each, with an interval of 21 days in a 2 × 2 crossover design) and subsequent in vitro embryo production. Cows in the control group (n = 10) were submitted to OPU procedures without any stimulation every 21 days. Animals in the synchronization group received a protocol-based progesterone implant, estradiol benzoate and prostaglandin on a random day of the estrus cycle (Day 0) and the OPU was performed on Day 5. After in vitro production, embryos were transferred to recipients synchronized at a fixed time and the diagnosis was performed 60 days later. An evaluation of the parameters for each OPU session revealed that donors that received the synchronization protocol pre-OPU showed a greater number of embryos (5.9 ± 0.5 vs. 4.5 ± 0.4; P = 0.037), higher rate of embryo production (45.8% vs. 38.5%; P = 0.001) and higher mean number of conceptions per group (2.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.07) in relation to the group that did not receive hormonal treatment. We concluded that synchronization of the follicular wave prior to OPU showed positive effects on in vitro embryo production as well as on pregnancy rates.

Superovulatory response following FSH, eCG-FSH and hMG and pregnancy rates following transfer of hatched blastocyst embryos with different diameter and shape in dromedary camel

The objectives of this study were to compare three superovulation protocols and to investigate the effect of embryo diameter and shape on pregnancy rates in dromedary camel. In Experiment 1, following follicular wave synchronization of donors using two injections of GnRH (25 μg, im), superovulation was initiated 2 days after the second GnRH (Day 0 of experiment). Then, donors were assigned into three groups (n = 5) and received 1) FSH alone (390 mg), 2) eCG (1000 IU; single dose on Day 0) and FSH (330 mg) and 3) hMG (17.5 ampules). FSH and hMG were administered twice daily, in decreasing doses, for 5.5 days. Thirty-six hours after the last dose of FSH or hMG, donors were mated once. On Day 4 of superovulation, the diameter and number of follicles and on Day 7.5 after mating, total number of corpora lutea and follicles ≥9 mm were recorded using ultrasound examination. On Day 8.5 after mating, embryos were recovered non-surgically. In Experiment 2, on Day 6.5 after inducing ovulation, recipients (n = 139) received a single 7.5 days old hatched blastocyst. The diameter and shape (round, oval, collapsed) of transferred embryos were recorded. Pregnancy was detected on Day 25 after embryo transfer. Data were analyzed using Proc GLM, Proc FREG, Proc CORR and Proc Genmod of SAS. The number of corpora lutea was greater in FSH (22.4 ± 2.25) than eCG-FSH (11.6 ± 2.58) and hMG (7 ± 3.19; P < 0.01). Total number of embryos was higher in FSH (16.2 ± 2.72) than hMG (1.6 ± 1.17; P < 0.01). In FSH group, there was correlation between the ratio of follicles >6 mm/follicles ≤6 mm on Day 4 of superovulation and total number of corpora lutea (r = 0.9; P < 0.05) and embryos (r = 0.91; P < 0.05). In the latter group, there was also correlation between corpora lutea and number of embryos (r = 0.92; P < 0.05). Transfer of embryos collected from different superovulation protocols did not affect pregnancy rates (P > 0.05). The diameter of hatched blastocysts had significant effect on pregnancy rates (P < 0.05). Medium size embryos (601–985 μm) produced greater pregnancy rates (50.8%) compared to small (275–600 μm; 25%) and large (986–1571 μm; 32%) embryos (P < 0.05). In conclusion, FSH protocol provided better and predictable superovulatory response. Greater pregnancy rates could be achieved by transferring medium size hatched blastocysts in dromedary camel.

Ovarian follicular growth in Morada Nova sheep supplemented with crude glycerin before mating

This study was aimed to investigate the follicular growth of Morada Nova sheep supplemented with crude glycerin before mating. Eighteen ewes were subjected to follicular wave and estrus synchronization with three injections of D-cloprostenol administered via IM, on days -14, -7 and -1 before mating. Animals were randomized into two groups, the control (n = 9), which received an oral drench of solution of 200 mL of saline solution, and the glycerin group (n = 9), which was supplied with 200 mL glycerol (n = 9), administered as an oral drench of crude glycerin in saline solution (9:1), 1h after morning feeding. Glycerin was supplied for 6 days, beginning 7 days from the mate. Growth follicular pattern was monitored daily by transrectal ultrasonography during the period of glycerin supplementation. Blood samples were taken during glycerin administration for progesterone and glucose analysis. There was a significant increase (P &lt; 0.001) in blood glucose after supplementation with glycerin (mean of 66.2 ± 1.3 vs 56.5 ± 0.8 mg/dL). The greater availability of energy near to ovulation was beneficial to Morada Nova ewes, leading to a significant increase (P &lt; 0.001) of large follicles (&gt; 3 mm). At diagnosis of pregnancy, it was observed that approximately 87% (13/15) (87.5% control group vs. 85.7% glycerin group) of the ewes were pregnant. The group treated with glycerin presented an increase of more than 40% of prolificacy (1.7 ± 0.3 vs. 1.2 ± 0.2; P=0.21). In conclusion, the monitoring of follicular growth reveals that crude glycerin increases the follicular population of Morada Nova ewes, when applied before mating.

248 SUPERSTIMULATION STRATEGIES FOR OVUM PICKUP IN HOLSTEIN DONORS

In vitro embryo production (IVP) is an important tool to enhance genetic gain in cattle. However, oocyte quality is a limiting factor for the success of IVP programs in high-producing donors. A series of studies using protocols for follicular wave synchronization and superstimulation before ovum pickup were performed to improve the efficiency of ovum pickup and in vitro production in dairy cattle. The first study evaluated superstimulation with FSH (Folltropin-V®) before ovum pickup in lactating (n = 15) and non-lactating (n = 15) Holstein donors in a crossover design. Cows underwent synchronization of follicle wave emergence (FWE) and at the expected time of FWE, the FSH group received a total dosage of 200 mg of FSH in 4 decreasing doses 12 h apart; controls received no FSH, and ovum pickup was conducted 72 h after the expected FWE in all cows. The FSH-treated cows had a higher (P &lt; 0.01) percentage of medium-sized follicles (6 to 10 mm) at the time of ovum pickup (55.1%) than control cows (20.8%) as well as lower cumulus‐oocyte complexes (COC) recovery rates (60.0 v. 69.8%, respectively; P = 0.002). However, FSH-treated cows had a higher blastocyst production rate (34.5 v. 19.8%; P &lt; 0.01) and more transferable embryos per ovum pickup session (3.0 ± 0.5 v. 1.8 ± 0.4; P = 0.02). Subsequent trials evaluated plasma FSH profiles in 23 heifers and in vitro production following ovum pickup in 90 non-lactating Holstein donors superstimulated with a single IM injection of FSH in 0.5% hyaluronan (HA; MAP-5®, 50 mg). Controls received no treatment, while the F200 group received 200 mg of FSH in 4 decreasing doses 12 h apart. The F200HA and F300HA groups received 200 or 300 mg of FSH in 5 or 7.5 mL, respectively, of 0.5% HA by a single IM injection. Circulating FSH area under curve (AUC) in FSH-treated animals was greater than in the control group (P = 0.02). Although the AUC for F200 group did not differ from HA groups (P = 0.56), the total period of time plasma FSH levels were elevated was greater than in the HA groups (P &lt; 0.01). In the IVP trial, FSH-treated cows had a greater proportion of medium-sized (6–10 mm) follicles than controls (P &lt; 0.001). Also, numbers of follicles (P = 0.01) retrieved (control: 13.1 ± 1.0; F200: 16.5 ± 1.2; F200HA: 19.5 ± 2.1; F300HA: 15.4 ± 1.4; P = 0.01) and blastocysts produced per ovum pickup session (control: 2.4 ± 0.5; F200: 3.7 ± 0.7; F200HA: 4.7 ± 0.7; F300HA: 3.1 ± 0.6; P = 0.06) were greater in cows receiving FSH, regardless of treatment. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COC cultured (P = 0.04), and blastocysts per ovum pickup session (P = 0.06) than cows in the F300HA group. In conclusion, superstimulation of Holstein donors before ovum pickup increased the efficiency of in vitro production. Additionally, a single IM dose of FSH in 0.5% HA resulted in similar plasma FSH profiles to twice-daily FSH treatment. Non-lactating donors treated with FSH produced more embryos per ovum pickup session regardless of FSH treatment. Lastly, all in vitro-produced endpoints were greater following a single dose of 200 mg of FSH in 0.5% HA than 300 mg of FSH in 0.5% HA.

134 FOLLICULAR DYNAMICS OF Gyr AND HOLSTEIN OOCYTE DONORS KEPT UNDER TROPICAL CONDITIONS

Bovine in vitro production is highly relevant for dairy systems in Brazil, and the main breeds used as oocyte donors are Gyr (G) and Holstein (H). This study aimed to evaluate the ovarian follicular dynamics of G and H oocyte donors kept under tropical conditions to detect differences that could guide improvement of follicular wave synchronization protocols for ovum pickup. Fourteen cyclic cows (6 H and 8 G), assessed twice each (after a 14-day interval), had their oestrus cycle synchronized by the use of 1.0 g of progesterone via intravaginal device (Ourofino, Brazil) and administration of 2 mg of oestradiol benzoate (EB; Day 0). Withdrawal of progesterone device was followed by 7.6 mg of cloprostenol administration (Day 7); EB (1 mg) was administered after 24 h (Day 8, 0 h), and the animals were evaluated every 12 h by ultrasound for 6 days (0–132 h). All evaluations are reported regarding EB administration (0 h). Videos from each ovary were stored and processed using the ImageJ software (http://rsb.info.nih.gov/ij), by measuring the diameter of each visualised follicle. All procedures were approved by local ethics committee. Ovulation time (G = 42.0 ± 8.3; H = 42.5 ± 6.2), ovulatory follicle (F1) diameter (G = 11.5 ± 1.8; H = 12.4 ± 2.0), and F2 diameter (G = 7.2 ± 1.9; H = 7.4 ± 2.7) did not differ (P &gt; 0.05) between breeds. Growth rate (mm day–1) after ovulation was similar (P &gt; 0.05) between breeds for each follicle (F1 = G: 0.6 ± 0.2, H: 0.8 ± 0.1; F2 = G: 0.5 ± 0.1 H: 0.4 ± 0.1, F3 = G: 0.2 ± 0.1, H: 0.3 ± 0.1). In H group, the F1 growth rate was higher (P &lt; 0.05) than F2 and F3, but there was no difference (P &gt; 0.05) in G group. Follicle deviation was identified 120 h after EB in the G group (~78 h after ovulation) and 132 h in the H group (~90 h after ovulation), and at that time F1, F2, and F3 follicle diameters were 8.0 ± 0.3, 6.6 ± 0.5, and 5.3 ± 0.3 for G (120 h), respectively; 8.8 ± 0.7, 7.2 ± 0.4, and 6.2 ± 0.3 for H (132 h), respectively. There was no difference between the size of F1, F2, and F3 between breeds at any time, except at 132 h, when H F3 was higher (P &lt; 0.05) than G F3. Regarding the follicular population, follicles smaller than 3 mm were more numerous in G animals at all evaluated moments, and differed at 0 (G = 7.1 ± 1.1; H = 2.5 ± 0.5) and 132 h (G = 5.6 ± 0.8; H = 1.5 ± 0.3). Number of follicles between 3 and 8 mm increased in H compared to G at 24 (14.4 ± 1.0), 36 (15.7 ± 1.3), and 132 h (18.3 ± 1.4). Comparing 3- and 8-mm follicles in G between times, an increase (P &lt; 0.05) in number was detected from 36 h onwards, comparedto 0 h (0 h: 9.2 ± 1.0; 36 h: 13.6 ± 1.4). This increase was not significant in H group (0 h: 13.7 ± 1.1; 132 h: 18.3 ± 1.4). The main findings of this study are that the moment of deviation and the population of follicles smaller than 3 mm and between 3 and 8 mm differs from Gyr and Holstein oocyte donors. Those observations suggest ovum pickup is better performed slightly later in Holstein donors than in Gyr, and can contribute to improvement of follicular wave synchronization protocols for each of the breeds in tropical conditions. Study supported by FAPERJ, Embrapa and CNPq.

Effect of follicular wave synchronization on superovulatory response of Girolando embryo donors

The objective of this study was to evaluate the effect of the follicular wave synchronization method on the superovulatory response of Girolando embryo donors. Seven cows and five heifers were divided into three treatments according to the method of the follicular wave synchronization prior to superovulation. The donors were superovulated with eight FSH decreasing doses, adding 133 mg per animal. The superovulatory response was evaluated using an ultrasound device to quantify the number of corpora lutea. The number and quality of recovered structures were also evaluated. Animal category (cow or heifer) and genetic group influenced the superovulatory response, in which heifers showed a greater response (100% in all treatments) compared with cows, which showed 85.7%, 57.1% and 57.1% superovulatory response for standing estrus, GnRH and P4 + BE groups, respectively. The genetic group also showed influence on superovulatory response, in which the 3/8 animals had an average of 10.3 corpora lutea per donor compared with the 3/4 animals, which showed 4.9. The follicular wave synchronization treatments did not show differences in superovulatory response, recovered structures and viable structures. The treatment of follicular wave synchronization with GnRH or with P4 + BE can be used in Girolando donors with the same efficiency as the standing estrus group, and 3/8 Girolando heifers respond better to the superovulatory treatment.

Hormonal protocols for in vitro production of Zebu and taurine embryos

The objective of this work was to evaluate the effects of hormonal synchronization protocols, associated or not with follicular development stimulation, on the recovery of oocytes and on in vitro production of Bos indicus and B. taurus embryos, in different seasons. Ultrasound-guided follicular aspirations (n=237) were performed without pre-treatment (G1, control group) and after follicular wave synchronization (G2), or after follicular wave synchronization and follicle growth induction (G3). Bos indicus produced more oocytes and embryos than B. taurus (18.7±0.9 vs. 11.9±0.6 oocytes and 4.8±0.3 vs. 2.1±0.2 embryos). On average, oocyte and embryo yields were higher in G3 than in G2, and both were greater than in G1, which lead to a higher conversion of oocytes to embryos in these treatments. The hot or the cold season did not affect the B. indicus outcomes, whereas, in B. taurus, both oocyte recovery and embryo production were higher in the cold season. Follicular wave synchronization improves ovum pick-up and in vitro production of embryos in both cattle subspecies evaluated.

Ovarian response and pregnancy rates to timed artificial insemination in beef heifers after synchronization of follicular waves and ovulation via a 14-day controlled internal drug-release insert estrus synchronization protocol

ABSTRACT The effects of extended progestin exposure, gonadotropin-releasing hormone (GnRH) versus prostaglandin (PG) F 2α at controlled internal drug-release (CIDR) insertion, and GnRH on d 7 versus 9 of a 14-d CIDR on ovarian response and timed AI (TAI) pregnancy rate were evaluated. In Exp. 1 beef heifers (n = 319) received 1) GnRH and CIDR on d 0, GnRH on d 9, and 50 mg of PG on d 14 at CIDR removal (14-d GnRH-9); 2) 14-d GnRH-9 but with PG versus GnRH on d 0 (14-d PG GnRH-9); or 3) GnRH and CIDR on d 9 and 25 mg of PG at CIDR removal on d 14 (5-d CO-Synch+CIDR). Heifers (n = 452) in Exp. 2 received 1) 14-d GnRH-9, 2) 14-d GnRH-9 with GnRH on d 7 versus 9 and 25 versus 50 mg of PG on d 14 (14-d GnRH-7), or 3) GnRH and CIDR on d 7 and 25 mg of PG at CIDR removal on d 14 (7-d CO-Synch+CIDR). All heifers received GnRH at TAI. Pregnancy rate was not different (P ≥ 0.66) among treatments in either experiment. In Exp. 1, heifers in d 6 to 10 of the estrous cycle at protocol initiation had a greater (P 0.10) from d 0 to 5. Results indicate extended progestin exposure, PG at CIDR insertion, and GnRH timing did not affect pregnancy rate to TAI.

252 EFFECT OF SUPERSTIMULATORY TREATMENT TO DONOR COWS IN OXYGEN CONSUMPTION OF MATURED OOCYTES

We previously reported that follicular wave synchronization and follicular growth treatment (FGT) before ovum pick-up (OPU) were effective in improving oocyte competence, which was associated with an increase in related embryos obtained by somatic cell nuclear transfer (Sugimura et al. 2012 Cell. Reprogram. 14, 29–37). However, oxygen consumption in oocytes remained unknown. The present study was designed to examine the differences in oxygen consumption between bovine oocytes obtained by OPU with or without FGT after in vitro maturation. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two OPU sessions were conducted in each cow to collect immature oocytes, as described by Sugimura et al. (2012). The first OPU session (OPU group) was performed in cows on arbitrary days of the oestrous cycle, using a 7.5-MHz linear transducer with the needle connected to an ultrasound scanner. Follicles larger than 8 mm in diameter were then aspirated and a controlled internal drug release device (CIDR) was inserted on Day 5 (the day of the first OPU session = Day 0). Then 30 Armour units (AU) of FSH (Antrin, Kyoritsu Seiyaku, Tokyo, Japan) was administrated to cows twice a day from Day 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 AU day–1). Cloprostenol (prostaglandin F2α; 0.75 mg) was administered in the morning of Day 9. The second OPU session (FGT-OPU group) was performed 48 h after prostaglandin F2α administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected cumulus–oocyte complexes in the OPU and FGT-OPU groups were matured in vitro as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. To collect in vivo-matured oocytes (control group), the CIDR was inserted into the cows on arbitrary days of the oestrous cycle (= Day 0), and oestradiol benzoate (0.8 mg) was administered on Day 1. The cows received the FGT treatment (as described above) from Day 6 to 10; however, the CIDR was removed in the evening of Day 8. Buserelin (gonadotropin-releasing hormone; 200 µg) was then administrated in the morning of Day 10, and OPU was performed at 24 h after gonadotropin-releasing hormone administration (Day 11). Oxygen consumption of matured oocytes was measured noninvasively with a scanning electron microscopy system (HV-405SP; Hokuto Denko Co., Tokyo, Japan). Data were analysed by ANOVA followed by a Tukey-Kramer test. There was no difference in the mean oxygen consumption between the FGT-OPU group (0.34 ± 0.02 × 10–14 mol–1, mean ± SEM) and control group (0.40 ± 0.01 × 10–14 mol–1). However, oxygen consumption in the FGT-OPU and control groups was significantly lower (P &lt; 0.01) than that in the OPU group (0.50 ± 0.02 × 10–14 mol–1). These results revealed significantly lower oxygen consumption in OPU-derived in vitro-matured bovine oocytes after FGT treatment compared with those obtained without FGT treatment. Oxygen consumption of oocytes obtained from FGT-OPU was similar to that of in vivo-matured oocytes, which may reflect their cytoplasmic maturation status with high developmental competence.

Fertility after ovarian follicular wave synchronization and fixed-time natural mating compared to random natural mating in dromedary camels (Camelus dromedarius)

The objective of the study was to compare the efficiency of two ovarian follicular wave synchronization protocols coupled with fixed-time natural mating with that of random mating in dromedary camels. Dromedaries were assigned randomly to one of the three treatment groups. Group 1 animals (RM; n=46) were mated randomly. Group 2 camels (1×GnRH-FTM; n=46) were given a GnRH analog (Buserelin, 20μg/animal, i.v.; Receptal, Intervet, Holland) at random, then were mated 14 days later. In Group 3 animals (2×GnRH-FTM; n=41), random GnRH analog was followed by repeated GnRH injection 14 days later and fixed-time natural mating on Day 28. Transrectal examination and ultrasonography were performed at weekly intervals to evaluate ovarian follicular status, diagnose ovulation and pregnancy. Blood samples were collected for progesterone determination by ELISA to confirm ovulation and pregnancy. All female dromedaries were assigned randomly to one of thirteen fertile bulls and were bred once on Days 1, 14 and 28 in Groups 1–3, respectively. Ovarian follicular status and ovulation rate was similar among groups at the start of the study. Seventy-five of the 133 dromedaries (56.4%) ovulated after random natural mating or random GnRH treatment. Mean length of mating was 386±17.8 (±SEM) seconds. There was no significant difference in mating time among groups and in pregnancy rate among dromedary bulls. In Group 3 (2×GnRH-FTM), ovarian follicular status before mating (P<0.05), ovulation rate (n=37, 90.2%, P<0.001) and pregnancy rate at 21 and 60 days (PR 21 days n=22, 53.7% and PR 60 days n=19, 46.3%, P<0.05) were greater compared to random natural mating (Group 1: OR n=25, 54.3%, PR 21 days n=13, 28.3% and PR 60 days n=12, 26.1%). In Group 2 dromedaries (1×GnRH-FTM), treatment tended to improve follicular status before mating, ovulation rate (n=34, 73.9%) and pregnancy rate at 21 and 60 days (PR 21 days n=21, 45.7% and PR 60 days n=16, 34.8%), but the effect was not significant compared to random natural mating. In conclusion, this is the first study demonstrating that favorable pregnancy rate can be achieved following ovarian follicular wave synchronization with repeated GnRH analog and fixed-time natural mating at 14 days intervals in dromedary camels.

Follicular and endocrinological turnover associated with GnRH induced follicular wave synchronization in Indian crossbred cows

The goal of this study was to record the hormonal and follicular turnover in Jersey crossbred cows when subjected for follicular wave synchronization using GnRH. Six healthy, non-lactating and regularly cycling Jersey crossbred cows (5–6 y) were used for the study. In the control group, the follicular wave pattern was ultrasonographically investigated in 18 cycles (3 cycles/cow). In the treatment group, GnRH analogue (buserelin acetate 10 μg im) was administered on Day 6 of the cycle and follicular wave pattern was studied in 12 cycles (2 cycles/animal). Follicular population was categorized based on their diameter Class I, ≤5 mm; Class II, >5–<9 mm; Class III, ≥9 mm) and the number of follicles in each category was determined on Day 6, Day 8 and Day 10. Plasma FSH and progesterone concentrations were estimated in both control and treatment groups. Out of 18 estrous cycles studied, 14 cycles (77.8%), three cycles (16.7%) and one cycle (5.6%) exhibited three-, two- and four-follicular waves per cycle, respectively. It was evident that the DF of Wave I established its dominance and was in the growing phase by Day 6 of the estrous cycle in all the normally cycling crossbred cows. The DF ovulated in all the animals (100%) in the mean interval of 27.7 ± 0.2 h after GnRH administration. A synchronized homogenous group of follicles emerged two days after GnRH injection (Day of 8.0 ± 0.0) in all the animals (100%). The combination of LH surge induced ovulation of DF (abrupt termination of Wave I) and FSH surge stimulated homogenous recruitment of Class I follicles, led to a synchronized emergence of follicular wave. All the GnRH treated cows had three follicular waves because of early emergence and short period of dominance of Wave II DF.