Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in simmental heifers

Transvaginal ovum pick-up (OPU) offers several advantages over standard embryo transfer procedures. For a systematic comparison of the ultrasound-guided (U-OPU) and the laparoscopic OPU (L-OPU) method, groups of Simmental heifers were subjected to 1 of the 2 OPU-methods for 8 wk (15 sessions) followed by a treatment-free interval of 11 wk and then another 8-wk period of OPU using the alternative method. Parameters that were evaluated included the number of follicles aspirated, number and morphology of recovered cumulus-oocyte complexes (COCs), and developmental capacity of oocytes after in vitro maturation (IVM) and fertilization (IVF). Blood samples were also taken from the donors twice a wk for progesterone measurement. To evaluate effects of long-term OPU on subsequent fertility of donors, all heifers (n = 14) were inseminated during the first natural estrus after the OPU experiment. The proportion of Class I oocytes was significantly (P < 0.001) higher after U-OPU than after L-OPU (38.7% vs 21.0%). Following IVM/IVF, this difference in oocyte quality was reflected by the cleavage rate (U-OPU: 58.1%; L-OPU: 52.1%; P < 0.05) and the rate of development to morulae and blastocysts (U-OPU: 27.1%; L-OPU: 13.9%; P < 0.001). Among other factors, the greater changes in vacuum pressure during L-OPU vs U-OPU might be responsible for the difference in oocyte quality. This problem may be overcome by a more flexible system for regulating the vacuum. Progesterone levels were higher during the L-OPU than the U-OPU periods. Seven donors (50%) were diagnosed pregnant by ultrasonography on Day 28 and by palpation per rectum on Day 42.     

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.     

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7 d, followed by the administration of 150 μg d-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment 1 (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.     

Effects of long-term treatment with bovine somatotropin on follicular dynamics and subsequent oocyte and blastocyst yield in an OPU-IVF program

Fourteen Holstein-Friesian heifers between 15 and 22 months of age with normal reproductive tracts were used in an experimental set up to investigate the effect of a long term rBST-treatment on the follicular population prior to transvaginal ovum pick-up (OPU). The estrous cycle of the animals was synchronized by means of a double injection of 2 ml cloprostenol 11 days apart. The heifers were divided in 2 equal groups (n = 7) in which the animals had the same average body weight, one group receiving a weekly subcutaneous injection of 640 mg recombinantly derived bovine somatotropin (rBST) on Mondays (rBST-treatment group) and a control group, being injected with 10 ml of saline. Heifers in both groups were submitted to OPU twice a week on Mondays and Thursdays using a 5 Mhz transducer and a disposable, 55 mm long, 20-g short bevelled needle at a vacuum pressure corresponding to approximately 13 ml water/min. The experimental period lasted for 10 weeks (April to June), each animal receiving a total of 10 injections and being submitted to OPU for 20 times. Oocytes were subsequently matured and cultured in a separate drop per cow following conventional IVF procedures. A blood sample was taken on heparin immediately after each OPU session, for determination of blood progesterone concentrations to assess the influence of treatment and OPU procedure on the cow's estrous cycle. Although results show a significant increase in the total number of follicles and medium sized follicles in the rBST-treated group, no statistically significant different number of retrieved oocytes between the rBST-treated and nontreated group could be detected. The average number of retrieved oocytes per session per cow was comparable, being 6.4 for the treated and 6.0 for the control group. Additionally, the average number of blastocysts per cow per session did not differ significantly between groups, being 1.41 in the rBST-treated group and 1.53 in the control group. The number of cultured oocytes which developed to the blastocyst stage was 22% in the rBST-treated group, which was not significantly different from 25% in the control group. Repeated OPU appeared to induce a certain degree of acyclicity in both treated and nontreated animals.     

Effect of frequency of follicle aspiration on oocyte yield and subsequent superovulatory response in cattle

The effect of frequency of transvaginal follicular aspiration on oocyte yield and subsequent superovulatory response was studied in 2 experiments. In Experiment 1, 32 primiparous Hereford x Friesian cows were assigned to 4 treatments (n = 8 per treatment). Oocyte recovery was carried out once a week for 12, 8, 4 or 0 (control) wk. Embryo recovery for all animals was 7 wk after the completion of the aspiration schedules. In Experiment 2, the effects of oocyte recovery once or twice a week (n = 8 per treatment; control n = 18) for 12 wk and response to superovulation 4 wk after the last aspiration were compared using nulliparous purebred Simmental heifers. Increasing the period of once weekly aspirations from 4 to 12 wk (Experiment 1) did not affect the number of follicles observed per session (mean +/- SEM; 10.0 +/- 0.82) or aspirated (7.8 +/- 0.71), but the recovery rate of oocytes from follicles aspirated was greater for donors aspirated for either 4 or 8 wk than for 12 wk (32.3 +/- 3.73 vs 28.4 +/- 2.61 vs 20.1 +/- 2.13 %; P < 0.05). Following the last aspiration and prior to commencing superovulatory procedures, estrus or estrous activity was observed in 7 8 , 8 8 , 7 8 and 6 8 of the animals aspirated over 12, 8, 4 or 0 wk, respectively. Subsequent superovulatory responses and in vivo embryo recoveries were similar for all aspiration treatments and for control animals. Changing the frequency of oocyte recovery from once to twice weekly (Experiment 2) did not affect the numbers of follicles observed (9.1 +/- 0.63 vs 8.3 +/- 0.85), follicles aspirated (5.9 +/- 0.56 vs 6.2 +/- 0.69), oocytes recovered (1.7 +/- 0.27 vs 1.9 +/- 2.0) per session or the oocyte recovery rate (29.4 +/- 2.4 vs 30.4 +/- 2.4 %); nor was there any effect of frequency of aspiration on subsequent superovulatory response and embryo recovery. In conclusion, increasing the period of aspiration from 4 to 12 wk and the frequency from once to twice a week over 12 wk did not reduce the number of follicles observed or aspirated, or number of oocytes recovered per donor per session. Subsequent estrous cyclicity and responses to superovulation were unaffected by the periods or frequencies of oocyte recovery examined here.     

Feeding frequency has diet-dependent effects on plasma hormone concentrations but does not affect oocyte quality in dairy heifers fed fibre- or starch-based diets

The post-fertilisation developmental capacity of bovine oocytes recovered by ultrasound guided transvaginal follicular aspiration (ovum pick-up, OPU) is influenced by diet-induced changes in hormone and metabolite concentrations. The objectives of this experiment were first to determine whether post-prandial changes in hormone concentrations, induced by changing the frequency of feeding, influenced oocyte quality and second whether changes in plasma glucagon concentration were associated with oocyte quality. Using a 2 × 2 factorial design, Holstein heifers (six per treatment) were fed either fibre- or starch-based diets containing either 189 or 478 g starch/kg dry matter. The diets were offered in either two or four equal meals per day and supplied twice the maintenance energy requirement. Blood samples were obtained both at weekly intervals (three samples per heifer, collected before feeding) during the experiment and throughout an entire 24-h period (15 or 17 samples per heifer for twice or four times daily-fed heifers, respectively). Each heifer underwent six sessions of OPU (twice weekly) beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviductal fluid following in vitro fertilisation. Mean weekly plasma insulin concentrations did not differ between diets, but plasma glucagon concentrations were greatest when heifers were fed the starch-based diet twice daily compared with the other diets. When heifers were offered four meals per day, there were no meal-related changes in hormone concentrations. However, when heifers were offered two meals per day, plasma insulin concentration increased after feeding the starch-based, but not the fibre-based diet. Plasma glucagon concentration increased after meals when heifers were fed twice daily and the increase was substantially greater when the starch-based diet was fed. Treatments did not influence (overall mean with mean ± s.e.) ovarian follicle size distribution or oocyte recovery by OPU (6.2 ± 0.4 per heifer), the proportion of oocytes that cleaved following insemination (0.57 ± 0.030) or blastocyst yield (0.27 ± 0.027 of oocytes cleaved). In conclusion, by feeding diets differing in carbohydrate source at different frequencies of feeding, meal-related changes in plasma hormone profiles were altered significantly, but oocyte quality was not affected. Therefore effects of diet on oocyte quality appear not to be mediated by meal-related fluctuations in hormone concentrations.     

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.     

Preovulatory endocrinology and oocyte maturation in superovulated cattle

Preovulatory follicular and oocyte nuclear maturation was studied in donor cattle induced to superovulate with a PMSG- or FSH-prostaglandin regimen. Plasma concentrations of progesterone (P4) and luteinizing hormone (LH) and follicular fluid levels of progesterone and estradiol-17β were measured and related to the oocyte nuclear maturation stages. The oocyte donors could be divided into two distinct groups. Group I had entirely normal periovulatory P4 and LH concentrations, and the majority of follicles and oocytes followed a characteristic pattern, clearly time-related to the LH peak. Group II had deviating levels of P4 and/or LH in the pre- and periovulatory period, and a majority of their follicles and oocytes had disturbed maturation, such as abnormal P4:E2 ratio in follicles and prematurely activated or meiotically arrested oocytes. It is concluded that a certain proportion of superovulated cows and heifers develop abnormal follicular/oocyte maturation and constitute poor oocyte, and probably also embryo, donors.     

The variability of ovum pick-up response and in vitro embryo production from monozygotic twin cows

Oocytes were recovered by ovum pick up (OPU) from nine pairs of monozygotic twin German Simmental cows. The hypothesis was that there is less variability between identical twins versus among non-related individuals in the variation in the recovery of oocytes by OPU and in the efficiency of in vitro embryo production. Estrous cycles were synchronized with two doses of cloprostenol, 11 days apart. Beginning 3-4 days after synchronized estrus, OPU was done twice weekly (every 3 or 4 days; total of 11 sessions). The influence of repeated OPU on estrous cyclicity was established by estrus detection, plasma progesterone concentrations, and ovarian ultrasonography. There were no differences among days of collection for the number and quality of cumulus oocyte-complexes (COCs), and rates of cleavage and blastocyst formation. A total of 1,661 COCs, including 657 (39.6%) good-quality COCs, were recovered. From 1,457 (87.7%) cultured COCs, 827 zygotes cleaved and 314 blastocysts were produced on Day 7. The total number of COCs and the blastocyst rates varied among pairs of monozygotic twins; within pairs, only slight differences were observed. In conclusion, recovery of COCs and production of embryos had substantially less variation within pairs of monozygotic twins than among non-related cattle.     

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 long-term CIDR-based protocols to synchronize estrus in beef heifers

Two experiments evaluated long-term controlled internal drug release (CIDR) insert-based protocols to synchronize estrus and compare differences in their potential ability to facilitate fixed-time artificial insemination (FTAI) in beef heifers. In Experiment 1 estrous cycling heifers (n = 85) were assigned to one of two treatments by age and body weight (BW). Heifers with T1 received a CIDR from days 0 to 14, gonadotropin releasing hormone (GnRH) on day 23, and prostaglandin F_2α (PG) on day 30. Heifers with T2 received a CIDR from days 2 to 16, GnRH on day 23, and PG on day 30. Ovaries were evaluated by ultrasonography on days 23 and 25 to determine ovulatory response to GnRH. In Experiment 2 heifers (n = 353) were assigned within reproductive tract scores by age and BW to one of four treatments. Heifers in T1 and T2 received the same treatments described in Experiment 1. Heifers in T3 and T4 received the same treatments as T1 and T2, respectively, minus the addition of GnRH. In Experiments 1 and 2, heifers were fitted with HeatWatch transmitters for estrous detection and AI was performed 12 h after estrus. In Experiment 1 heifers assigned to T1 had larger dominant follicles at GnRH compared to T2 (P < 0.01) but response to GnRH, estrous response after PG, mean interval to estrus, and variance for interval to estrus after PG did not differ (P > 0.10). AI conception and final pregnancy rate were similar (P > 0.50). In Experiment 2 estrous response after PG did not differ (P > 0.70). Differences in mean interval to estrus and variance for interval to estrus (P < 0.05) differed based on the three-way interaction of treatment length, GnRH, and estrous cyclicity status. AI conception and final pregnancy rates were similar (P > 0.10). In summary, the greater estrous response following PG and resulting AI conception and final pregnancy rates reported for heifers assigned to the two treatments in Experiment 1 and among the four treatments in Experiment 2 suggest that each of these long-term CIDR-based protocols was effective in synchronizing estrus in prepubertal and estrous cycling beef heifers. However, the three-way interaction involving treatment length, GnRH, and estrous cyclicity status in Experiment 2 clearly suggests that further evaluation of long-term CIDR-based protocols is required with and without the addition of GnRH and on the basis of estrous cyclicity status to determine the efficacy of these protocols for use in facilitating FTAI.     

A comparison of a mechanical sector and a linear array transducer for ultrasound-guided transvaginal oocyte retrieval (OPU) in the cow

A comparison was made between a linear array and a mechanical multiple angle sector (MAP) transducer for ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU) in the cow. The ovaries of five dairy cows were punctured, in a twice-weekly OPU program lasting for 4 weeks, using two different 5.0-MHz transducers equipped with an identical disposable needle-guidance system. Both ovaries were visualized using each transducer before puncture and the number of follicles with a diameter of less than 5 mm (small) and with a diameter equal to or greater than 5 mm (large) was recorded. Subsequently, one ovary of the pair was punctured guided by the MAP, while the other was punctured using the linear array transducer. During the next puncture session on a given animal, the two systems were switched and used on the alternate ovary in a crossover design. Parameters assessed for each system were: the total number of follicles visualized in each diameter class, and the total number of retrieved oocytes per cow. A significant difference was found for the ability to visualize smaller follicles in favor of the MAP transducer, with an average visualization of 71.6 +/- 30.3 small follicles per cow during the 4-week trial period, compared to 59.8 +/- 25.7 for the linear array transducer (t-test for paired samples, P = 0.007). No differences were found in the visualization of large follicles. A numerically greater number of oocytes were retrieved using the MAP transducer, compared to the linear array, (averages of 14.2 +/- 7.2 versus 7.4 +/- 6.1, respectively), although these differences were not statistically significant. In conclusion, both systems can be effectively used for oocyte retrieval in the cow, however, the MAP transducer demonstrated superior visualization of small follicles.     

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

Hormonal treatments for increasing the oocyte and embryo production in an OPU-IVP system

The objective was to enhance the inherent developmental ability of bovine oocytes retrieved by ultrasound-guided transvaginal aspiration. Various hormonal regimes were utilized to produce partially matured oocytes in vivo, in order to improve embryo development following IVF. In the first experiment, a two-by-two factorial design was used with FSH (multiple versus single dose) and im administration of LH (yes versus no) 6h prior to OPU. In all protocols (which lasted for nine consecutive weeks), ovarian stimulation was performed in the presence of a CIDR. One FSH administration was adequate for ovarian stimulation (9.33+/-0.7 and 10.14+/-0.7 follicles per cow per OPU session); however, multiple injections increased (P<0.05) follicular response (12.97+/-0.7 and 13.97+/-0.7). In the second experiment, a two-by-two factorial design was used to compare the effects, during ovarian stimulation, of the presence or absence of CIDR, and iv treatment with LH 6h prior to OPU (yes versus no), on oocyte competence (judged by blastocyst development rates following IVF). Presence of CIDR during superstimulation had no effect on the follicular response. Administration of LH 6h prior to OPU increased (P<0.05) the oocytes of higher morphological grades, and in the absence of a CIDR, improved (P<0.05) blastocyst development rate. Treatment with LH, 6h prior to OPU without the use of CIDR during ovarian stimulation, resulted in 2.89+/-0.4 blastocysts per cow per OPU session as compared to 1.56+/-0.4, 1.56+/-0.4 and 1.33+/-0.4 for all other groups. In conclusion, compared to single administration, multiple FSH administration increased (P<0.05) available follicles for aspiration. Moreover, when ovarian stimulation in the absence of CIDR was followed by administration of LH 6h prior to OPU, it increased (P<0.05) the number of blastocysts per OPU session.     

Effects of Calf Removal at Parturition on Postpartum Ovarian Activity in Zebu (Bos indicus) Cows in the Humid Tropics

To assess endocrine and morphological responses of ovaries to total weaning at parturition, 6 Zebu (Bos indicus) cows 5 years or older were investigated. Following parturition, blood samples were collected daily during the first month and twice weekly thereafter until day 60 to determine concentrations of progesterone (P4) and prostaglandin F2α metabolite. It took between 25 to 32 days to complete uterine involution.The prostaglandin metabolite remained elevated for a mean period of 14.2 days (range, 4-21) postpartum. Five of the animals resumed cyclicity with a short estrous cycle starting between days 7 to 34 and lasting between 7 and 14 days. No estrous behavior was recorded prior to the short estrous cycles, but subsequent normal-length estrous cycles were all preceded by signs of estrus. In the 1 animal that resumed cyclicity with an estrous cycle of normal length on day 37 (length 20 days), the cycle was preceded by estrous behavior. Progesterone concentrations reached a mean maximum of 4.8 nmol liter−1 during the short estrous cycles, and prostaglandin metabolite concentrations peaked while P4 concentrations were decreasing. P4 concentrations reached a mean maximum of 12.2 nmol liter−1 during the estrous cycles of normal length. The interval from parturition to the first estrous cycle of normal length varied between 16 and 48 days, and the length of the cycle was 18 to 22 days. Starting 2 days postpartum, ovaries from 5 of the cows were scanned by ultrasonography every second day until day 30 postpartum. Medium-sized follicles were detected between days 4 to 7 postpartum in 4 of the scanned cows that later had short estrous cycles. The time between parturition and the appearance of the first dominant follicle was 7.6 days (range 6-10 days). The interval between parturition and the appearance of the first ovulatory-sized follicle was 10.2 days (range 8-13 days). In 3 of the scanned cows this ovulatory-sized follicle ovulated. We conclude that cyclic ovarian activity in Zebu cows can start early in the postpartum period in the absence of offspring, and that short luteal phases, not preceded by estrous behavior, may play an important role in establishing normal postpartum ovarian activity.     

Reproductive characteristics of cyclic beef heifers treated with the prostaglandin analog luprostiol

One hundred and twenty crossbred Angus heifers, after exhibiting a 17- to 23-d estrous cycle, were placed into six groups of 20 heifers each and administered 2 ml i.m. propylene glycol containing either 0 (controls), 3.75, 7.5, 15.0 or 30.0 mg of luprostiol, or saline containing 0.5 mg cloprostenol (Groups 1 through 6, respectively). Heifers were observed for estrus every 6 h and all treatments were given 6.5 to 8.0 d after heifers were observed in standing estrus. Blood samples were collected after treatments from 10 heifers in each groups. Blood serum was assayed for progesterone. The synchronization period was considered to be 120 h after administration of luprostiol or cloprostenol. There were 0, 16, 17, 18, 20 and 18 heifers observed in estrus during the synchronization period in Groups 1 through 6, respectively. Progesterone concentrations in blood serum dropped below 1 ng/ml in 0, 8, 9, 10, 10 and 10 of the heifers from which blood samples had been taken in the six groups. All heifers observed in estrus were artificially inseminated. During the synchronization period, 0, 12, 14, 15, 16 and 10 heifers conceived in Groups 1 through 6, respectively. The interval from injection to estrus for the 89 heifers that exhibited estrus in the synchronization period averaged 49.0 h and was not different among the luprostiol and cloprostenol treated groups. Control heifers returned to estrus an average of 13.2 d after the treatment. The number of heifers that conceived at first insemination, regardless of when estrus occurred, was 16, 15, 16, 16, 16 and 12, and the total number that conceived at the first and second inseminations was 18, 18, 17, 19, 19 and 16 for Groups 1 through 6, respectively. Based on serum progesterone concentration and/or interval from treatment to estrus, 15 and 30 mg of luprostiol effectively regressed corpora lutea (100%) when administered between 6.5 and 8.0 d after estrus, and the estrous response and conception rate for these two groups equalled or exceeded that of the control and cloprostenol groups.     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

Influence of hormonal and nonhormonal estrus synchronization methods on follicular and oocyte quality in primiparous lactating does at early postpartum period

High-yield lactating does need effective estrus synchronization methods to improve their reproductive outcome by enhancing ovarian function. The aim of the current work was to analyze ovarian follicular and oocyte characteristics of hormonal and nonhormonal estrus synchronization regimes in primiparous lactating rabbit does (Oryctolagus cuniculus) in the early postpartum period (Day 11). Females were randomly treated with either (1) a hormonal standard treatment with 25 IU equine chorionic gonadotropin (eCG) 48 h before artificial insemination (eCG group) or (2) an alternative nonhormonal treatment consisting of doe-litter separation 24 h before artificial insemination (Bio group). No significant differences were found in serum estradiol and progesterone concentrations between experimental groups. During the histologic study, the Bio group presented a higher number of primordial (P < 0.05) and primary follicles (P = 0.07) compared with that of the eCG group, whereas secondary and antral follicular populations were similar. Rates of late atretic follicles assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique were not different between treatments, but the eCG group showed a significantly higher number of mid-atretic follicles compared with that of the Bio group. Nuclear in vitro oocyte maturation (IVM), measured as metaphase II rate, and in vitro steroidogenic response of cumulus-oocyte complexes, measured by ELISA, did not show significant differences between treatments. However, confocal study showed that cytoplasmic maturation of oocytes, in terms of cortical granule migration rate, was significantly higher in the Bio group compared with that after the eCG treatment. In conclusion, transient doe-litter separation seems to improve ovarian response in terms of follicular health and oocyte competence compared with that after the eCG treatment. Therefore, a 24-h-long transient weaning could be an alternative nonhormonal method for synchronizing estrus in primiparous lactating rabbit does inseminated in the early postpartum period.