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.     

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.     

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.     

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.     

Repeated ovum pick-up in Italian Mediterranean buffalo cows

The potential of the ovum pick-up technique, used over a long period, was evaluated in 6 Italian Mediterranean buffalo cows that had more than 500 d open. The cows were submitted to ovum pick-up twice weekly for 2 mo. An additional 2-mo cycle of ovum pick-up was performed in 3 of the buffalo. The ovum pick-up sampling did not affect the resumption of reproductive activity of these animals. In fact, all the buffalo conceived, on average, 47.5 +/- 27.5 d after the last ovum pick-up. An average of 5.48 follicles was punctured, and 2.71 oocytes were collected per session. However, only 53.5% of these oocytes were suitable for in vitro embryo production. The number of punctured follicles differed between individual cows. There were no differences in the number of collected oocytes or in the recovery rates. The number of punctured follicles, the number of collected oocytes and the recovery rate were similar in the first and second months; the quality of the oocytes was, however, better in the second than in the first month (P < 0.05). The increasing interval between 2 consecutive ovum pick-up sampling (intersession interval) caused an increase of the percentage of large follicles. Moreover, the increase of the intersession interval from 4 to 5 d decreased the quality of the collected oocytes (P < 0.05). The efficiency of in vitro production of embryos to expanded blastocysts was 16.7%.     

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.     

Superstimulation prior to the ovum pick-up to improve in vitro embryo production in lactating and non-lactating Holstein cows

The present study evaluated the efficacy of superstimulation with p-FSH (Folltropin) before the ovum pick-up (OPU) on IVP in lactating and nonlactating Holstein donors. A total of 30 Holstein cows (15 lactating and 15 nonlactating) were blocked by lactation status to one of two groups (control or p-FSH), in a cross-over design. On a random day of the estrous cycle, all cows received an intravaginal progesterone device and 2.0 mg IM of estradiol benzoate (Day 0). Cows in the control group received no further treatment, whereas cows in the p-FSH group received a total dosage of 200 mg of p-FSH on Days 4 and 5 in four decreasing doses 12 hours apart (57, 57, 43, and 43 mg). On Day 7, the progesterone device was removed, and OPU was conducted in both groups (40 hours after the last p-FSH injection in the p-FSH–treated group). There was no difference between groups (P = 0.92) in the numbers of follicles that were aspirated per OPU session (17.2 ± 1.3 vs. 17.1 ± 1.1 in control and p-FSH-treated cows, respectively); however, p-FSH-treated cows had a higher (P < 0.001) percentage of medium-sized follicles (6–10 mm) at the time of the OPU (55.1%; 285/517) than control cows (20.8%; 107/514). Although recovery rate was lower (60.0%, 310/517 vs. 69.8%, 359/514; P = 0.002), p-FSH-treated cows had a higher blastocyst production rate (34.5%, 89/258 vs. 19.8%, 55/278; P < 0.001) and more transferable embryos per OPU session were produced in the p-FSH group (3.0 ± 0.5 vs. 1.8 ± 0.4; P = 0.02). Regardless of treatment, non-lactating cows had a higher blastocyst rate (41.9%, 106/253 vs. 13.4%, 38/283; P = 0.001) and produced more transferable embryos per OPU session (3.5 ± 0.5 vs. 1.3 ± 0.3; P = 0.003) than lactating cows. Thus, superstimulation of Holstein donors with p-FSH before OPU increased the efficiency of IVP. In addition, non-lactating donors had higher percentage of in vitro blastocyst development and produced more embryos per OPU session than lactating cows.     

Embryo technologies and animal health - consequences for the animal following ovum pick-up, in vitro embryo production and somatic cell nuclear transfer

Mammalian reproductive technologies that aim either to complement or to transcend conventional livestock breeding options have contributed to some of the most remarkable achievements in the field of reproductive biology in recent decades. In so doing they have extended our horizons in two distinct dimensions, the first concerning what it is technically possible to achieve and the second relating to the time-frame within which an individual's life-long developmental capability is initially established and ultimately realized or undermined. Our impressions of the benefits and values, or otherwise, of technologies such as in vitro embryo production and nuclear transfer are rightly influenced by the extent to which they impinge on the health of animals either subjected to or derived from them. Here, we consider some of the health implications of oocyte/embryo-centric technologies applied to farm livestock.     

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).     

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.     

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.     

Genetic parameters for oocyte number and embryo production within a bovine ovum pick-up-in vitro production embryo-production program

Genetic factors influencing the outcome of bovine ovum pick-up-in vitro production (OPU-IVP) and its relation to female fertility were investigated. For the first time, genetic parameters were estimated for the number of cumulus-oocyte complexes (Ncoc), quality of cumulus-oocyte complexes (Qcoc), number and proportion of cleaved embryos at Day 4 (Ncleav_D4, Pcleav_D4), and number and proportion of total and transferable embryos at Day 7 of culture (Nemb_D7, Pemb_D7 and NTemb_D7, PTemb_D7, respectively). Data were recorded by CRV (formally Holland Genetics) from the OPU-IVP program from January 1995 to March 2006. Data were collected from 1508 Holstein female donors, both cows and pregnant virgin heifers, with a total of 18,702 OPU sessions. Data were analyzed with repeated-measure sire models with permanent environment effect using ASREML (Holstein Friesian). Estimates of heritability were 0.25 for Ncoc, 0.09 for Qcoc, 0.19 for Ncleav_D4, 0.21 for Nemb_D7, 0.16 for NTemb_D7, 0.07 for Pcleav_D4, 0.12 for Pemb_D7, and 0.10 for PTemb_D7. Genetic correlation between Ncoc and Qcoc was close to zero, whereas genetic correlations between Ncoc and the number of embryos were positive and moderate to high for Nemb_D7 (0.47), NTemb_D7 (0.52), and Ncleav_D4 (0.85). Genetic correlations between Ncoc and percentages of embryos (Pcleav_D4, Pemb_D7, and PTemb_D7) were all close to zero. Phenotypic correlations were in line with genetic correlations. Genetic and phenotypic correlations between Qcoc and all other traits were not significant except for the phenotypic correlations between Qcoc and number of embryos, which were negative and low to moderate for Nemb_D7 (-0.20), NTemb_D7 (-0.24), and Ncleav_D4 (-0.43). Results suggest that cumulus-oocyte complex (COC) quality, based on cumulus investment, is independent from the total number of COCs collected via OPU and that in general, a higher number of COCs will lead to a higher number of embryos produced. The correlation between the estimated breeding values for Ncoc and PTemb_D7 of sires in this study and the sires breeding index for female-fertility based on the Dutch cattle population was close to zero. This study revealed OPU-IVP traits (Nemb_D7, NTemb_D7, and Ncoc) that could be of potential value for selection. Introduction of such traits in breeding programs would enhance the number of offspring from superior donors as well as improve the cost efficiency of OPU-IVP programs.     

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.     

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.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

Ovum pick-up and in vitro embryo production (OPU-IVEP) in Mediterranean Italian buffalo performed in different seasons

This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%, respectively, in spring-summer and in the transitional period; P < 0.01). The results of this trial demonstrated that the morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes.     

Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.     

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.     

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.