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.     

Transvaginal ovum pick-up (OPU) in the cow: a new disposable needle guidance system

A study was conducted to modify the routinely used ovum pick up (OPU) devices to permit use of disposable needles and to simplify the technique and to make it more economical and practical to use. Long nondisposable needles are commonly used in transvaginal OPU despite several disadvantages. A new OPU device was developed using 19-g disposable needles to eliminate these disadvantages and to make the technique more successful. The disposable needle was connected to silicone tubing by means of a stainless steel connector. The system was inserted into a stainless steel tube, creating a rigid structure within which to move the needle back and forth. A blunt needle can be changed simply by replacing it with a new one, even while the device is in the vagina of a cow. The needle guidance system is incorporated into a new OPU device together with the transducer of an ultrasonographic scanner with an unilateral orientated scanning field, making it possible to utilize the needle length to its maximum. This combination permits easy manipulation of the ovaries, easy positioning of follicles on the puncture line, and enables the use of shorter needles which directly enter the scanned area without loss of useful needle length. As a preliminary result we obtained an overall oocyte recovery rate of 42%. Although this is promising, additional puncture sessions are needed to establish more consistent recovery rates. When OPU is used routinely, application of short disposable needles is more practical and economical.     

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.     

Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments

Endoscopically and ultrasonographically guided ovum pick up were studied in 15 heifers once per week. Althogether 60 sessions were carried out. The number of aspirated follicles per animal (13.0 vs 10.3) did not differ significantly between both methods. Similar recovery rates (39 % vs 44 %) were achieved after repeated sessions. As a consequence the number of recovered oocytes per animal (5.2 vs 4.7) were not significantly different. Inter-animal- and intra-animal-variations were more important for the results than the applied methods. In contrast to these findings the quality of recovered cumulus-oocyte-complexes (COC) was significantly influenced by the methods. The COC were divided into 4 categories a) oocytes with compact cumulus, b) oocytes with rest of compacted cumulus, c) oocytes with expanded cumulus and d) oocytes without cumulus. There was a higher denudation rate of COC when using the endoscopic aspiration (62.0 % vs 6.6 %) because of turbulent current in this aspiration system. Advantages and disadvantages of both methods are described and discussed. Generally, the ultra sonographic method is the less traumatic procedure for the vagina, fornix and for abdominal organs. The other method is less traumatic for the ovaries.     

Ovarian synchronization following ultrasound-guided transvaginal follicle ablation in heifers

In Experiments 1 and 2, ultrasound-guided transvaginal follicle aspiration was used as a method of follicle ablation to induce and synchronize subsequent follicular wave emergence and enhance ovulation synchrony following PGF(2alpha) administration. Heifers were at unknown stages of the estrous cycle at the start of both experiments in which all follicles >/=5 mm in diameter were ablated; luteolysis was induced 4 d later with cloprostenol (500 ug/dose, im). In Experiment 1, heifers were randomly assigned to either an ablation (n=17) or a procedural control (no follicle ablation, n=17) group. Ablation-induced wave emergence was indicated by a significant increase in the total number of follicles >/=5 mm within 2 d of ablation (mean, 1.5 d), which was preceded by a significant surge in circulating FSH. Although the mean (+/-SEM) interval from PGF(2alpha) administration to ovulation did not differ between follicle-ablated heifers (5.1+/-0.5 d range, 3 to 9 d) and control heifers (5.1+/-1.0 d; range, 1 to 5 d), the variability of the interval was different (P<0.05). Inequality of variance between the 2 groups was attributed to a greater (P<0.08) degree of ovulation synchrony in the ablation group than in the control group; 13 16 (81%) versus 9 17 (53%), respectively, ovulated within 5 d of cloprostenol administration. Relative asynchrony of ovulations in control heifers was associated with the status of the follicular wave at the time of PGF(2alpha) administration and, in part, to incomplete luteolysis following a single dose of PGF(2alpha). Experiment 2 was designed to examine the efficacy of 2 doses of cloprostenol 12 h apart (n=7) versus a single dose (n=8) to induce complete luteolysis subsequent to follicle ablation-induced wave emergence. Two doses of cloprostenol potentiated ovulation synchrony; more (P<0.05) 2-dose heifers (7 7 , 100%) than single-dose heifers (4 8 , 50%) ovulated within 5 d after PGF(2alpha) administration. In summary, ultrasound-guided transvaginal follicle ablation, done at random during the estrous cycle, induced and synchronized subsequent follicular wave emergence, and resulted in a high degree of ovulation synchrony among heifers after PGF(2alpha) induced luteolysis, especially when 2 doses of PGF(2alpha) were administered 12 h apart.     

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.     

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.     

Two different schemes of twice-weekly ovum pick-up in dairy heifers: effect on oocyte recovery and ovarian function

The aim of the present study was to compare two different schemes of twice-weekly ovum pick-up (OPU), continuous (C) and discontinuous (DC), with special emphasis on differences in oocyte yield and quality, estrous cyclicity, ovarian dynamics, and progesterone production. Subsequent to characterization of their normal estrous cycles (pre-OPU period), eight dairy heifers were subjected to 4 months of twice-weekly OPU under two different schemes: the DC (OPU restricted to Days 0-12 of the cycle) and the C schemes. Effects of the two different schemes on oocyte yield, quality, and in vitro competence, together with effects on ovarian dynamics and progesterone production, were monitored. The mean numbers of punctured follicles and recovered oocytes per session were slightly higher (not significant (n.s.)) using the DC scheme, but in total, similar numbers of oocytes were obtained. The quality of the oocytes as well as cleavage rate after in vitro fertilization of the oocytes did not differ between the two OPU schemes. There was no influence of a corpus luteum (CL) producing progesterone on the oocyte yield and quality, whereas the presence of dominant follicles appeared to decrease the number of recovered ooctyes. During the pre-OPU period, all heifers showed normal cyclicity. In the DC scheme, the heifers showed regular and normal cyclic activity throughout the puncture period, with one to two complete follicular waves during the interval from the last OPU to the next estrus. In the C scheme, the heifers occasionally revealed cyclicities with irregular interestrous intervals and weaker signs of estrus. No complete follicular waves were seen during the OPU period in this scheme. The CL developed from the ovulation of the preovulatory follicles in the DC scheme showed similar characteristics to the CLs of the pre-OPU period; however, the CL-like structures from the puncture of follicles, in both the DC and the C schemes, revealed a shorter life span and inferior competence in producing progesterone (P<0.05). The present results indicate that the DC OPU scheme, which allows animals to go into natural ovulation prior to the first OPU, does not affect their ovarian function, whereas the C OPU scheme does. Our study further demonstrates that an equal number of oocytes can be obtained with both schemes, but that fewer OPUs are needed when the DC scheme is applied.     

Prepubertal propagation of transgenic cloned goats by laparoscopic ovum pick-up and in vitro embryo production

The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.     

Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies.

Laparoscopic ovum pick-up (LOPU) is a convenient methodology by which oocytes can be recovered and used either for in vitro production of zygotes or as a source of cytoplasts in nuclear transfer (NT) procedures. The pregnancy and transgenesis rates achieved with IVM/IVF of LOPU-sourced oocytes followed by subsequent DNA microinjection of zygotes are similar to the rates obtained when using in vivo-produced oocytes or zygotes. Similarly, pregnancy rates and kids born by using LOPU-sourced and in vitro matured oocytes as recipient cytoplasts in NT programs are comparable with those reported by others using in vivo matured oocytes collected by oviduct flushing. The use of LOPU allows for improved control over the stage of maturation/development of the oocytes and produced zygotes, a less invasive means of recovery, thereby allowing for repeated usage of the oocyte donor animals and the ability to source the oocytes from live animals of known health status. In addition, because of large follicular responses that can be obtained from prepubertal animals, LOPU followed by IVM/IVF has demonstrated great potential for the early propagation of valuable animals, in particular, transgenic animals.     

Potential use of ovum pick-up for embryo production and breeding in cattle

The efficacy of transvaginal ultrasound-guided puncturing of ovarian follicles for collecting immature oocytes in cattle was studied. Three experiments were conducted to examine the effects of puncturing on follicle recruitment and on the number of oocytes collected. Puncture sessions were executed twice weekly at regular intervals of 3 and 4 d respectively. The oocytes were matured, fertilized and allowed to develop in vitro and the number of transferable embryos was recorded. The health of the cows was checked daily. In Experiment 1, dairy cows (n=10) were punctured over a period of 5 mo, and the collected oocytes were fertilized with the semen of 1 bull. In Experiment 2, oocytes were collected from one 12 year old high pedigree dairy cow and an one month pregnant cow were punctured. The oocytes of the old cow were fertilized with semen of 8 different bulls. In Experiment 3, beef cows (n=6) were punctured over a 2 mo period and the semen of 2 different bulls of the same breed was used to fertilize the oocytes from 3 of these cows. In Experiment 1, 14.5 +/- 0.4 (mean +/- SEM) follicles were punctured per session, and 8.0 +/- 0.3 (mean +/- SEM) oocytes were recovered. A mean of 16% of the oocytes developed into transferable embryos with a pregnancy rate of 40%. The results did not differ between the months of the experiments, indicating that the transvaginal puncturing method can be used successfully over a 5 mo period. No detrimental effects were observed after clinical and post mortem examinations, nor did breed, age or reproductive status appear to affect the results. However, large differences were observed between individual cows and between cow/bull combinations.     

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.     

In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr)

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.     

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.     

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n = 10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9 mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2 ± 0.3 mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P < 0.0001). There was a tendency (P = 0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P < 0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P < 0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.     

Factors affecting recovery and quality of oocytes for bovine embryo production in vitro using ovum pick-up technology

In Experiment 1, different vacuum pressures (30, 50, 70 and 90 mm Hg) were used to aspirate 4156 bovine follicles in vitro, to assess their effect on flow rate and the recovery, morphology and blastocyst formation of the recovered oocytes. Cumulus oocyte complexes (COCs) were classified according to the morphology of the cumulus cells. Data were analyzed using Chi Square analysis. Overall recovery rate declined as the aspiration pressure increased above 50 mm Hg (P<0.05). The recovery rate of Grade 1 oocytes decreased significantly (P<0.05) as the vacuum pressure increased with a corresponding increase in the number of denuded oocytes recovered (P<0.05). The blastocyst yield, expressed as a percentage of recovered COCs decreased significantly as the aspiration pressure increased beyond 50 mm Hg (P<0.05). In Experiment 2, the holding media (hepes- or bicarbonate-buffered TCM 199) and holding time (1 h or 5 h) did not affect the blastocyst formation of the oocytes (P>0.05). In Experiment 3, it was found that individual culture of the oocyte during fertilization or culture had a detrimental effect on the oocytes blastocyst formation (8.8% to 16% blastocyst yield on Day 8) when compared to control (31.3%). In Experiment 4, groups of 5, 10 and 25 oocytes were compared with singly cultured oocytes. There were no significant differences (P<0.05) in the blastocyst formation rate among groups of 5, 10, or 25 oocytes, but there was a significant difference between oocytes processed in groups and those processed individually. In Experiment 5, when groups of 10 oocytes were cultured in different drop sizes, there was no significant difference in cleavage rates between oocytes cultured in 100 microL (85.0%, n = 280) and in 10 microL (86.8%, n = 280) of media, but culture in 50 microL (79.3%, n = 280) resulted in cleavage rates significantly lower (P<0.05) than culture in 10 microL drops. There was no significant difference in the blastocyst formation. However there was a significant difference (P<0.05) in cell numbers of Day 8 blastocvsts, with oocytes cultured in 100 microL drops having significantly lower cell counts than oocytes cultured in 50 or 10 microL drops.     

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus–oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus–oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus–oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.     

Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and quality

The present study was undertaken to explore the potential of the Murrah breed of buffaloes as donors of oocytes and to find out the recovery rate and oocyte quality in cyclic Murrah buffaloes subjected to oocyte recovery once a week. Murrah buffaloes (n = 5) were synchronized for estrus by a single prostaglandin injection schedule. The animals were subjected to transvaginal oocyte retrieval (TVOR) once weekly for 6 weeks, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). TVOR was performed using an ultrasound machine with a 5 MHz transvaginal transducer, single lumen 19-gauge, 60 cm long needle and a constant vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (> or = 10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes (COCs) with > or = 5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. The mean (+/- S.E.M) number of small, medium and large follicles, and the number of total follicles observed per animal per session, which was 2.2 +/- 0.3, 0.6 +/- 0.2, 0.9 +/- 0.1 and 3.7 +/- 0.3, respectively, did not differ between animals or between puncture sessions. Small follicles constituted a major proportion (59%) of the total observed follicles. A mean (+/- S.E.M) number of 3.0 +/- 0.3 follicles were punctured and 2.0 +/- 0.3 oocytes recovered per animal per session, with a recovery rate of 68%. Out of the total 61 oocytes recovered, 36 (59%) were of grades A + B whereas 25 (41%) were of grades C + D. In conclusion, this study describes the potential of cyclic Murrah buffaloes as donors of oocytes collected by repeated TVOR once a week, without any adverse effects on follicular growth and oocyte recovery. It also describes an efficient system for carrying out TVOR in buffaloes.     

Laparoscopic ovum pick-up and in vitro production of sika deer embryos: effect of season and culture conditions

Amongst the 200 deer subspecies worldwide, more than 40 are considered as endangered. In vitro embryo production may represent an efficient way to produce and disseminate offspring from sparse remaining individuals in these species. With a view to establishing a method of in vitro embryo production, we assessed the ovarian response after hormonal stimulation (oFSH), oocyte yield following laporoscopic ovum pick-up (LOPU) and oocyte developmental competence according to seasonal reproductive status in sika deer (Cervus nippon nippon). Twelve adult sika deer hinds were allocated between two groups and submitted weekly to oFSH follicular growth stimulation followed by LOPU. Hinds in Group A (n=6) were treated first during the breeding season (5 weeks), and then during the non-breeding season (3 weeks). Hinds in Group B (n=6) were submitted to similar procedures but in the reverse order (treated first during the non-breeding season). Cumulus-oocytes complexes (COC) recovered from Group B were allowed to mature in vitro for 24 h in TCM-199 medium supplemented with oFSH, goat follicular fluid and 100 microM cysteamine. In vitro fertilization was performed with frozen/thawed semen in SOFaa medium supplemented with 20% estrous sheep serum and presumptive zygotes were cultured in the presence or absence of ovine oviductal epithelial cell monolayer (oOEC) in SOFaa-BSA medium. Mean number of follicles aspirated per hind per session decreased significantly between breeding and non-breeding season in Group A (9.8+/-0.7 versus 3.2+/-0.7, mean+/-S.E.M., respectively, P<0.001) but did not change between the non-breeding and the subsequent breeding season in Group B (5.3+/-0.7 and 5.7+/-0.7, respectively, P>0.05). Irrespective of the season, good quality COC with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Whereas development to the blastocyst stage did not occur in SOF medium alone, high development rates to the blastocyst stage were observed in oOEC co-culture regardless of season (22% and 34% of total oocytes in co-culture during non-breeding and breeding season, respectively).