Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per asp?ration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.     

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.     

Transgenic production from in vivo-derived embryos: effect on calf birth weight and sex ratio

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Use of F1 progeny of HolsteinxZebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein x Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bos taurus (Holstein), Bos indicus (Zebu) and F1 crossbred Bos taurus x Bos indicus (Holstein x Zebu) genotypes were inseminated with Bos taurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein x Holstein:85%, Zebu x Holstein:84% and Zebu x Holstein x Holstein:88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P<0.05) from Zebu x Holstein and Zebu x Holstein x Holstein F1 by 5.1 and 5.8, respectively. However, the Holstein breed also yielded the lowest percentage of cleavage (45.1 vs 71.9% for Zebu x Holstein and 65.1% for Zebu x Holstein x Holstein). Of the 3 genotypes, the hybrid F1 breed was the most efficient source of oocytes for the production of embryos capable of reaching morulae and blastocyst stages (76 250 ; P< 0.001). In the second part of the study, 599 oocytes from the F1 breed were fertilized in vitro, 1 group of 150 oocytes was used for the determination of the optimal pronuclear visualization period. The highest number of oocytes with 2 pronuclei was observed between 24 to 28 h after IVF (27 to 42%). The remaining 399 oocytes were microinjected with a gene construct bearing the bacterial lacZ gene as the reporter for gene expression. Survival of embryos to microinjection was 73.8%, and 45.5% of them (50 110 ) cleaved in culture. Of the microinjected embryos, 1 out of 50 showed beta-galactosidase activity. These findings indicate that a tropical crossbreed of cattle (Zebu x Holstein x Holstein) can be used as a source of oocytes for IVF programs and gene microinjection studies.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

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

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

Development and viability of bovine preplacentation embryos treated with swainsonine in vitro.

This study investigated the effects of swainsonine (a locoweed toxin) on bovine preplacentation embryo development using in vitro procedures. We examined and confirmed the viability and developmental potential of swainsonine-treated embryos by transfer to synchronized recipient heifers. Oocytes (n = 6338) were aspirated from ovaries collected from the abattoir and subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Swainsonine was added to IVM, IVF, IVC media spatially and IVM/IVF/IVC continuously, at 0 ng/ml (TRTI, control), 200 ng/ml (TRT2), 400 ng/ml (TRT3), and 800 ng/ml (TRT4). Embryo development was evaluated with respect to oocyte cleavage rate and the rates of morula and blastocyst formation. There was no difference (P > 0.05) among treatments. The average number of nuclei per blastocyst at Day 7.5 of culture (Day 0 = IVF) was 85.9 +/- 4.3 (n = 47) and 89.3 +/- 4.4 (n = 44) for swainsonine-treated embryos (800 ng/ml) and control embryos, respectively. Pregnancy rate as determined by ultrasonography on day 35 to 40 post embryo transfer was 43.8% and 38.3% for swainsonine-treated (800 ng/ml) and control embryos, respectively. Nine (9.4%) healthy calves were delivered from heifers receiving swainsonine-exposed and nine (9.6%) from control embryos. No difference (P > 0.05) was detected in number of calves developing from TRT and control embryos. We conclude that swainsonine does not have an adverse effect on the development and viability of preplacentation bovine embryos.     

High oxygen tension during in vitro oocyte maturation improves in vitro development of porcine oocytes after fertilization

This study was conducted to evaluate the effect of oxygen tension during IVM and/or IVC on developmental competence of porcine follicular oocytes. Prospective, randomized experiments were designed, and oocytes were matured, inseminated and cultured in vitro in the designated condition. In experiment 1, either high (20%) or low (7%) oxygen tension was used for IVM. The high oxygen significantly improved blastocyst formation (23% versus 13%; P<0.01) after IVF than the low oxygen. Such treatment, however, did not significantly (P>0.05) improve the rates of nuclear maturation (89% in each treatment), sperm penetration (62-72%), monospermic fertilization (56-67%), pronuclear formation (90-96%), cleavage (49-53%) and blastocyst cell number (31-32 cells). In experiment 2, the combined effect of oxygen tension during IVM and IVC of embryos was evaluated by a 2 x 2 factorial arrangement. Again, the high oxygen tension during IVM supported blastocyst formation more efficiently (P<0.01) than the low oxygen, and this was independent of oxygen tension during IVC (26-28% versus 15-16%). In oocytes matured under the high oxygen, a tendency to increase blastomere number (P=0.0630) was found, when the low oxygen was used for IVC after insemination (39-45 cells/blastocyst). In conclusion, the use of high oxygen tension (20% maintained by exposure to 5% CO2 in air) for IVM of porcine oocytes promoted blastocyst formation in vitro.     

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.     

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.     

Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.     

Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon)

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus)

Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.