Fixed-time AI protocols replacing eCG with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows

The aim of the present study was to evaluate the effects of a single treatment with FSH on diameter of the largest follicle and on conception rates of suckled Bos indicus beef cows submitted to timed artificial insemination (TAI). Four hundred fifty-six suckled anestrous Nelore beef cows at 30-60 days postpartum were assigned to treatments. At the first day of the estrous synchronization protocol (Day 0), all cows received a progesterone-releasing intravaginal device plus 2mg of estradiol benzoate. On Day 8, cows were assigned to blocks according to the diameter of the largest follicle and then allocated to one of three treatment groups (Control, FSH, or eCG) within each block. Simultaneously to progesterone device withdrawal on Day 8, cows in the eCG treatment group (n=150) received 300 IU of eCG and cows in FSH treatment group (n=153) received 10mg of FSH, and Control cows (n=153) did not receive any additional treatment. Additional treatments with 150 μg of cloprostenol and 1mg of estradiol cypionate (EC) were also administered concurrently to progesterone device removal in all cows on Day 8. Two days later (D10), TAI and ovarian ultrasonic examinations to evaluate follicle size were performed in all cows. On Day 12, a subset of cows (n=389) were submitted a second ultrasonic exam to confirm ovulation. Final follicular growth (mm/day) was less (P=0.006) in both Control (0.95±0.11) and in FSH-treated cows (0.90±0.10) than in eCG-treated cows (1.40±0.13). Interestingly, there was a treatment-by-BCS interaction in ovulation results (P=0.03), in which, eCG treatment increased percentage of cows having ovulations with a lesser BCS. Similarly, there was a treatment-by-BCS interaction for conception (P=0.04), where the eCG treatment increased fertility in cows with a lesser BCS. In conclusion, FSH failed to stimulate final follicular growth, ovulation, and conception rate in sucked-anestrous beef cows submitted to TAI as effectively as eCG. However, physiological effects of eCG seem to be more evident in cows with a lesser BCS.     

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.     

Comparison of different transvaginal ovum pick-up protocols to optimise oocyte retrieval and embryo production over a 10-week period in cows

The objective was to develop a simple and effective ovum pick-up (OPU) protocol for cows, optimised for oocyte harvest and subsequent in vitro embryo production (IVP). Five protocols differing in collection frequency, dominant follicle removal (DFR) and FSH stimulation were tested on groups of three cows each, over an interval of 10 consecutive weeks. Performance was evaluated on per OPU session, per week and pooled (3 cowsx10weeks) basis. Among the non-stimulated groups, on a per cow per session basis, once- or twice-weekly OPU had no effect on the mean (+/- S.E.M.) number of follicles aspirated, oocytes retrieved and blastocysts produced (0.6+/-0.8 and 0.7 +/- 0.7, respectively). However, DFR 72 h prior to OPU almost doubled blastocyst production (1.2 +/- 1.3). In stimulated groups, FSH treatment (80 mg IM and 120 mg SC) was given once weekly prior to OPU. Treatment with FSH, followed by twice-weekly OPU, failed to show any synergistic effect of FSH and increased aspiration frequency. When FSH was given 36 h after DFR, followed by OPU 48 h later, more (P < 0.05) follicles (16.0 +/- 5.0), oocytes (10.6 +/- 4.5) and embryos (2.1 +/- 1.2) were obtained during each session, but not on a weekly basis. Pooled results over 10 weeks showed an overall improved performance for the treatment groups with twice-weekly OPU sessions, due to double the number of OPU sessions performed. However, the protocol that consisted of DFR, FSH treatment and a subsequent single OPU per week, was the most productive and cost-effective, with potential commercial appeal.     

The effect of eCG or estradiol at or after norgestomet removal on follicular dynamics, estrus and ovulation in early post-partum beef cows nursing calves

In post-partum anestrous beef cows suckling calves, neither the choice of hormonal regime to ensure the presence of a healthy dominant follicle at the end of a progestagen treatment nor the optimum hormone to induce estrus and ovulation is clear. Twenty-eight beef cows, in good body condition, 25-30 days post-partum, were assigned to one of four treatments: (i) 3mg norgestomet (N) implant with 5mg estradiol valerate (EDV) and 3mg N injection at the time of insertion (Crestar) for 5 days followed by 600 IU eCG at the time of implant removal; (ii) Crestar for 5 days as in (i) followed by 0.75 mg estradiol benzoate (EDB) 24h later; (iii) Crestar for 9 days followed by 600 IU eCG at the time of implant removal; and (iv) Crestar for 9 days followed by 0.75 mg EDB 24h later. Ovarian scanning was preformed from 4 days before implant insertion until ovulation and 4 days postovulation to detect the CL. Daily blood samples were collected from day 20 post-partum until second ovulation for FSH and E(2) assay. Data were analyzed using analysis of variance. There was no effect of the stage of follicle wave at the time of implant insertion on interval to new follicle wave emergence (range 1-7 days; mean 4.7 days). FSH concentrations were decreased to 5.9+/-2.0 and 7.7+/-1.1 ng/ml for pre- and post-selection cows 1 day after start of treatment; thereafter, they increased on Day 2 to 7.9+/-2.0 and 11.0+/-1.1 ng/ml and on Day 3 to 10.3+/-2.7 and 11.4+/-1.7 ng/ml for pre- and post-selection cows, respectively, despite high-estradiol concentrations at that time. There was no effect of treatment on the interval from implant removal to ovulation (3.2-4.0 days) or on the number of cows detected in estrus (26 of 27 cows). The size of the ovulatory follicle in cows given 0.75 mg EDB 24h post implant removal was decreased in animals at the pre-selection stage (12.2+/-0.1mm) of the follicle wave compared with those at the post-selection stage (15.3+/-0.9 mm) at implant removal. Cows given 600 IU eCG at the pre-selection phase of follicular growth had multiple ovulations (4.0+/-1.1). Cows given EDV at the start of a 5-day implant period had higher estradiol concentrations before and on the day of implant removal than those given EDV at the start of a 9-day implant period. The injection of 0.75 mg EDB 1 day after implant removal tended to increase concentrations of estradiol one day later. In conclusion, 5mg EDV and 3mg N at insertion of a 3mg N implant resulted in variable new follicle wave emergence 1-7 days later in post-partum beef cows nursing calves (22 of 27); both eCG and EDB were equally effective at inducing estrus after implant removal in cows in good BCS, but eCG resulted in a significant increase in ovulation rate in cows treated before dominant follicle selection.     

Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH superstimulation

The objective in this study was to investigate the effect of repeated oocyte collection by transvaginal, ultrasound-guidance, oocyte pick-up (OPU) in nine, prepubertal (8-12 months), swamp buffaloes. Animals were treated with FSH for 3 days and received GnRH on the third day, 24 h before OPU. This session was repeated on five occasions at 2 weekly intervals. Over the five sessions of hormone treatment followed by OPU, 39/42 (92.9%) animals responded and had 6.6+/-3.6 follicles with a follicular diameter of 5.0+/-2.0 mm. The oocyte recovery rate was 5.4+/-3.7 and averaged 82.8% oocytes, except for session 4, when oocyte recovery was around 75.0%. Most oocytes were denuded (39.5%), whilst 28.8% had a substantial cumulus mass. There were no differences in the ovarian responses and the recovery rates between the collections. It was concluded that five repeat cycles of FSH and OPU did not influence the follicular response to superstimulation or the number of oocytes recovered from prepubertal, buffalo calves.     

Comparison of methods to stimulate ovarian follicular growth in cynomolgus and African green monkeys for collection of mature oocytes

The objective was to compare various gonadotropin-based methods to stimulate ovarian follicular growth in female cynomolgus (n=16) and African green monkeys (n=8) for collection of mature oocytes. On the 1st day of menstruation, the monkeys were treated with 3.75 mg leuprorelin acetate (a GnRH agonist). Starting 2-3 weeks later, ovarian follicular growth was stimulated as follows: (a) 25 IU/kg of human FSH (hFSH) in a glycerol solution given once daily for 9 d; (b) 200 IU of eCG given six times during a 9-d interval; (c) 75 IU/kg hFSH in a glycerol solution given three times (72 h intervals) during a 6-d interval. In addition, the monkeys were given 1200 or 4000 IU of hCG 36 h (Methods A and B) or 60 h (Method C) after the last gonadotropin treatment, and oocyte collection was attempted 36-38 h after hCG. Although there were no significant differences among methods in the number of oocytes collected, in cynomolgus monkeys, hFSH (Methods A and C) was better than eCG (Method B; 12 and 10 versus 7 mature oocytes, respectively), whereas in African green monkeys, eCG (Method B) was more effective than hFSH (Method A; 12 versus 7 mature oocytes). Furthermore, in cynomolgus monkeys, Method C was nearly as effective as Method A; using a glycerol solution as a solvent decreased the frequency of hFSH administration from nine to three times. In conclusion, in cynomolgus and African green monkeys, ovarian response depended on the species and on the individual, and in cynomolgus monkeys, hFSH in a glycerol solvent was effective.     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius)

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 μg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal).Overall an average of 12.12 ± 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 ± 4.1) and 26-27 h (82.1 ± 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 ± 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 ± 2.1 vs. 60.9 ± 6.6) and developed to blastocyst stages (52.4 ± 4.1 vs. 30.5 ± 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation.In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro.     

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.     

Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

The objective of this study was to determine the best combined hormonal treatment to utilize in order to obtain a high number of good quality in vivo and in vitro matured oocytes from prepuberal Mediterranean Italian buffalo calves (Bubalus bubalis). Transvaginal ultrasound follicular aspiration was employed to recover oocytes from antral follicles. Fifteen barn housed buffalo calves, between 5 and 9 months of age were used in this study and randomly divided into control (Group A) and treated groups. A commercially available preparation of 2000 IU eCG was administered to animals in the treatment groups, followed by 2000 IU of hCG given either 12 h (Group B), or 24 h (Group C) before ovum pick up (OPU). From the time of administration of eCG treatments, the best timing for hCG administration before OPU was determined and integrated with the administration of 500 IU of FSH-LH in a decreasing dosage protocol over 4 days (Group D). Expanded cumulus oocyte complexes (COCs) recovered from all groups were immediately fixed for later aceto-orcein staining. All other COCs were processed for in vitro maturation using standard procedures and then fixed and stained for assessment of nuclear maturation. Collectively, hormonal stimulation did not increase the number of ovarian antral follicles available compared to the control group (P > 0.05), but did result in higher output of medium (Group B: 9.8 +/- 7.1; Group C: 3.4 +/- 6.7; Group D: 15.6 +/- 4.9 versus Group A: 1.6 +/- 2.2) and large follicles (Group B: 44.8 +/- 22.9; Group C: 8.7 +/- 6.1; Group D: 70.2 +/- 10 versus Group A: 6.1 +/- 6.3). Administration of hCG 12 h before follicle aspiration proved to be the best strategy to obtain high numbers of immature and mature oocytes from antral follicles (P < 0.05; Group B: 70.8 +/- 12 and Group D: 82 +/- 12.6 versus Group A: 43.6 +/- 13.9 and Group C: 27.2 +/- 13.9). A significantly higher number of expanded COCs was obtained from hormonally stimulated groups compared to the control group (P < 0.05; Group B: 28.7 +/- 16.8, Group C: 16.3 +/- 5.9 and Group D: 27.1 +/- 16.9 versus Group A: 6.2 +/- 6). A higher oocyte maturational competence (P < 0.05) was found in Groups A, B and D (80.8 +/- 7.9, 87.5 +/- 8.2, and 86.5 +/- 4.3, respectively) compared to Group C (60 +/- 26.2). In conclusion, in prepuberal buffalo calves combined gonadotrophin stimulation protocols yielded higher numbers of medium to large size follicles compared to a control group. A high number of good quality oocytes were recovered by transvaginal ultrasound follicle aspiration, and a high rate of metaphase II progression was reached after in vivo and in vitro maturation.     

Effect of eCG on early resumption of ovarian activity in postpartum dairy cows

The purpose of the present study was to hasten the resumption of ovarian activity early postpartum in lactating dairy cows, using equine chorionic gonadotropin (eCG), to enhance follicular growth, followed by hCG, to induce ovulation. Primiparous Holstein dairy cows (n=21) were assigned equally into eCG, eCG-hCG and Control groups. Cows in the eCG and eCG-hCG groups received an i.m. injection of eCG (500 IU Folligon?) on Day 6 postpartum. Cows in the eCG-hCG group were also given an i.m. injection of hCG (500 IU Chorulon?), once dominant follicle reached the diameter of 13-16 mm following eCG injection. Cows in Control group did not receive any treatment. Daily blood sampling and ultrasound examination were conducted, starting at Day 6 postpartum until confirming the third ovulation. Follicles ≥10 mm in diameter were detected on Day 11.5±1.48, 10.1±0.52 and 11.1±1.36 after calving in Control, eCG and eCG-hCG groups, respectively (P>0.05). The first wave dominant follicle ovulated in 71.4% of cows treated with eCG and eCG-hCG. In contrast, none of the first wave dominant follicles ovulated in Control cows. By Day 20 postpartum, all cows in eCG group, 6/7 cows in eCG-hCG group and none of the cows in Control group ovulated (P<0.05). Short estrous cycles (≤16 days) were detected in 2/7, 1/7 and 6/7 cows in eCG, eCG-hCG and control groups, respectively (P<0.05). In conclusion, injection of eCG on Day 6 postpartum could assist the early resumption of ovarian activity by enhancing ovarian follicle growth and early ovulation in postpartum cows. In this context, subsequent hCG injection may not provide any more beneficial effect.     

Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery

Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50. mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF(2)alpha (prostaglandin F(2)alpha) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P>0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; P<0.01) and with the number of ova and/or embryos recovered (r=0.88; P<0.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%.     

In vitro and in vivo maturation of llama oocytes

Cumulus-oocyte complexes (COC) were collected from abbatoir-derived llama ovaries and cultured in vitro for 28, 30, or 36 h at 39 degrees C in 5% CO2 to determine the time required for maturation. The majority of COC (n=298, 87%) were classified as categories 1 and 2 (COC with > or =5 layers or 2-4 compact layers of cumulus cells, respectively) and homogeneous ooplasm, and the proportion that underwent nuclear maturation (MII) was 78, 81 and 80%, after 28, 30 and 36 h, respectively (P=0.65). To compare the effectiveness of FSH versus eCG for inducing in vivo maturation, in experiment 2, llamas (n=20 per group) were treated with: (1) 25 mg FSH, twice-daily for 4 day, plus 5 mg armour of LH at the end of FSH treatment; or (2) 1000 IU of eCG, plus 5 mg armour of LH 4 day after eCG treatment. The FSH- and eCG-treated groups did not differ (P=0.85) with respect to the number of follicles > or =6mm at the time of COC collection (17.9+/-2.2 versus 17.7+/-2.2), the number of COC collected (10.7+/-2.1 versus 11.2+/-2.3 per llama), or the collection rate per follicle aspirated (71 versus 74%). As well, no difference (P=0.49) was detected between the FSH and eCG groups in the number of expanded COC collected (8.3+/-2.1 versus 10.6+/-2.2) or the number of COC at the MII stage (6.9+/-1.8 versus 8.9+/-1.9). In conclusion, llama oocytes reached MII as early as 28 h after in vitro culture and both FSH and eCG were equally effective in inducing ovarian superstimulation. Treatment with LH after either FSH or eCG superstimulation permitted the recovery of a preponderance of expanded COC in metaphase II that may be suitable for in vitro fertilization without in vitro maturation.     

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).     

Manipulation of follicular development to produce developmentally competent bovine oocytes.

Superstimulation in donor cows increases the number of cumulus-oocyte complexes (COC), but when compared to in vivo maturation, in vitro maturation results in only half as many blastocysts after prolonged in vitro culture. The objective of this study was to establish a superstimulation protocol that would produce a maximal number of competent COC for standard in vitro embryo production. During experiment 1, eight cyclic Holstein heifers were superstimulated with four doses of FSH. Half the heifers received an injection of LH 6 h before ovum pick-up (OPU). The COC were collected following OPU either 33 or 48 h following the last FSH injection (coasting period). During experiment 2, six cyclic Holstein heifers were superstimulated with six doses of FSH, and in half the heifers, LH was administered 6 h before OPU. The COC were collected following ultrasound-guided transvaginal aspiration of both ovaries 48 h after the last FSH injection (coasting period). The COC originating from follicles with a diameter of 5 mm or more (n = 180 for experiment 1 and 57 for experiment 2) were subjected to standard in vitro maturation, fertilization, and development. When animals were administered four doses of FSH, 48 h of coasting resulted in significantly more 5- to 10-mm follicles (P < 0.01) than 33 h of coasting. If a 33-h coasting period was used, administration of LH 6 h before OPU resulted in a significant increase in both percentage of blastocysts and embryo production rate at Days 7 and 8 (P < or = 0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P < 0.08), were obtained when animals received LH 6 h before OPU, with 80% +/- 9% (mean +/- SEM) blastocysts and 0.8 +/- 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.     

Effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows

Two experiments were conducted to test the effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows. The objective of the first experiment was to determine if aspiration of all visible follicles would influence the estrous cycle. All visible follicles > or = 5 mm in diameter were punctured and the oocytes aspirated once during the second and fourth cycle, with no aspirations occurring during Cycles 1, 3 and 5 in 4 Holstein cows. Between aspirations the follicles were scanned ultrasonically every 2 to 3 d to monitor follicular waves. Estrus was monitored twice daily and plasma progesterone concentrations were measured daily. The inter-estrus periods were significantly longer when aspiration was performed than in the 3 cycles in which aspiration did not occur (25.0 vs 21.1 d; P < 0.01). To determine the maximum number of follicles harvestable on a continuing basis, a second experiment was conducted to contrast unstimulated cows subjected to ultrasonically-guided oocyte collection twice-weekly with animals from which oocytes were collected once a week after gonadotrophin stimulation. All visible follicles of multiparous Holstein cows were aspirated every 3 or 4 d (unstimulated group, n = 4), and the total number of follicles was compared with the number of follicles in cows that were aspirated weekly after 400 mg FSH were administered over 2 d (stimulated, n = 6). The study continued for 8 consecutive weeks. The weekly mean number of follicles available for aspiration in the unstimulated cows was 15.7 +/- 3.3 (mean +/-SEM), which was not different from the 14.2 +/- 1.9 follicles available for aspiration from the FSH-stimulated cows. Within the unstimulated cows, there was an increased number of follicles available for aspiration over time during the period of study (P < 0.001). The number of follicles available for aspiration from the stimulated cows did not change, but plasma concentrations of progesterone increased over time as persistent luteal tissue developed on the ovaries.     

Factors affecting oocyte quality and quantity in commercial application of embryo technologies in the cattle breeding industry

With the introduction of multiple ovulation, embryo recovery and transfer techniques (MOET) plus embryo freeze-thaw methods in the early 1980s, the breeding industry has the tools in hand to increase the number of calves from donors of high genetic merit. In the early 1990s, the introduction of ovum pick-up followed by in vitro embryo production (OPU-IVP) opened up even greater possibilities. Using these technologies, we challenge biological mechanisms in reproduction. Where normally one oocyte per estrous cycle will develop to ovulation, now numerous other oocytes that otherwise would have degenerated are expected to develop into an embryo. Completion of oocyte growth and pre-maturation in vivo before final maturation both appear to be essential phases in order to obtain competence to develop into an embryo and finally a healthy offspring. In order to increase oocyte quality and quantity in embryo production technologies, current procedures focus primarily on improving the homogeneity of the population of oocytes with regard to growth and state of pre-maturation at the start of a treatment. In the case of MOET, dominant follicle removal (DFR) before superovulation treatment improves the number of viable embryos per session from 3.9 to 5.4 in cows but not in heifers and a prolonged period of follicle development obtained by preventing release of the endogenous LH surge increases the number of ova but not the number of viable embryos per session. In the case of OPU-IVP, the frequency of OPU clearly affects quantity and quality of the collected oocytes and FSH stimulation prior to OPU every 2 weeks resulted in 3.3 embryos per session. Analysis of 7,800 OPU sessions demonstrated that the oocyte yield is dependent on the team, in particular, the technician manipulating the ovaries. It is concluded that an increased understanding of the processes of oocyte growth, pre- and final maturation will help to improve the efficiency of embryo technologies. However, somewhere we will meet the limits dictated by nature.     

Using hormone-treated pregnant cows as a potential source of oocytes for in vitro fertilization

In vivo collection of oocytes during pregnancy may be alternative method of obtaining gametes for in vitro fertilization (IVF) from genetically superior gestating cattle. The objectives of this experiment were to induce follicular growth in mature beef cows during each trimester of pregnancy, and then to collect oocytes and verify oocyte competency by IVF and subsequent embryo culture in vitro. Cyclic beef cows in Treatment A and pregnant cows in Treatment B were administered a total dose of 40 mg of FSH in descending dose levels (6, 5, 4, 3 and 2 mg) twice daily for 5 consecutive days. Cows in Treatment A were administered 25 mg of PGF(2)alpha and in Treatment B an equal volume of 0.9% saline at the seventh FSH injection. Pregnant cows in Treatment C were administered neither FSH nor PGF(2)alpha and served as a control group. Following a gonadotropin treatment, the ovaries of each female were evaluated for follicular development by ultrasonography. Oocytes were collected by follicle aspiration from cows in the first trimester. Following IVF procedures, the embryos were co-cultured on caprine oviductal cells, or in the chicken embryo co-culture system, or were placed in goat oviducts in vivo. The mean number of follicles per ovary 12 hours after FSH treatment was not different for cows in Treatments A and B, (8.1 vs 7.7) and both numbers were greater (P<0.05) than the 1.1 follicles per ovary for the control cows in Treatment C. Oocytes collected in vivo and exposed to IVF, resulted in 20% cleaving, and of these embryos 50% developed to the morula stage in culture. In summary, stimulating supplemental follicular development with FSH treatment during pregnancy and collecting the oocytes for IVF may be an alternative method for obtaining supplemental gametes from valuable donor cattle.     

Methods for collecting and maturing equine oocytes in vitro

The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbecco's PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultured in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in oocyte recovery when follicles were flushed with PBS, with or without hyaluronidase. There was also a difference (P<0.05) in the percentage of maturation of oocytes between 30 and 24 h (86 vs 48%), but no further increase was seen by 36 h (84%). In Experiment 3, follicles were aspirated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocytes were cultured for 30 h in TCM-199 either with or without 100 IU/ml eCG. There was no effect of eCG or time from slaughter on oocyte maturation or cumulus expansion (P>0.05).