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.     

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.     

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.     

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.     

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.     

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 production of bovine embryos using individual oocytes

The development of a bovine in vitro embryo production system where individual oocytes could be followed through to the morula or blastocyst stage would be of interest to several fields of study and would allow us to characterise developmentally competent oocytes and their corresponding follicular environment. Several studies have, however, reported significantly reduced embryo development when oocytes or embryos were cultured individually compared to in groups. The aim of this study was to establish such an embryo production system, with embryo development rates similar to that observed under control (grouped) conditions. This study showed that conservation of the oocyte/embryo medium densities generally employed for grouped culture does not facilitate embryo development if oocytes/embryos are cultured individually. However, individual oocytes could effectively undergo IVM/IVF/IVC to the expanded blastocyst stage with some small modifications to the standard protocol. Individual IVF was effective if carried out in either 100 μl of medium in wells or in 50 μl droplets. Individual IVC, if carried out in 10 or 20 μl droplets of SOF with FCS added at either 0 or 24 hr, was effective in terms of blastocyst yields but 20 μl droplets did yield significantly fewer hatched blastocysts compared to grouped controls (P < 0.05). An entirely individual embryo production system was effective when it included individual IVM in 10 μl droplets of M199 + 10 ng/ml EGF resulting in day 8 blastocyst yields not significantly different from controls (38% vs. 35% respectively). The use of 10% FCS during individual IVM appeared, at least under our experimental conditions, to be detrimental to subsequent development. The uses of an individual system for embryo production are many and varied. The results of this study show clearly that a large proportion of bovine oocytes can develop to the blastocyst stage when matured, fertilized, and cultured individually. This opens the way for studies regarding the quality of specific oocytes in such a way as will greatly improve our understanding of the events of late folliculogenesis. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Evaluation of novel SexedULTRA-4M technology for in vitro bovine embryo production

SexedULTRA-4M™ is made using an improved method of sex-sorting sperm in a less damaging environment for better retaining sperm integrity throughout the sorting process. The objective of this research was to compare conventional (CONV) and SexedULTRA-4M™ (ULTRA-4M) semen for bovine IVP using four Angus bulls. Matured slaughterhouse oocytes (n = 4000) were divided into the CONV group and the ULTRA-4M group (2000 COCs for each semen type). The IVF process was implemented with CONV and ULTRA-4M semen from the same bull. The cleavage rates, eight cell embryos and blastocysts on day 7 of culture were evaluated for each semen type and each bull. The statistical analysis was carried out with the ANOVA procedure SAS software. The results were 54.45% ± 1.03 and 58.10% ± 1.07; 35% ± 1.57 and 39.15% ± 1.62; 22.8% ± 1.09 and 27.15% ± 1.12 for CONV and ULTRA-4M, respectively, for cleavage rate, eight cell embryos and blastocysts on day 7 for the average of all bulls, comparing only the semen type. Concerning only the semen type, ULTRA-4M was significantly superior to CONV for cleavage rates (P = 0.01) and blastocysts on day 7 (P = 0.009). There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analyzed for Bull 1 and Bull 4, however, for Bull 2 ULTRA-4M was significantly superior to CONV for cleavage rates and blastocysts on day 7 (P< 0.05). In Bull 3, ULTRA-4M was significantly higher (P< 0.05) for blastocysts on day 7 compared to CONV. In conclusion, under the conditions of this research the ULTRA-4M and CONV semen produced similar bovine IVP results overall.     

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the survivability of bovine oocytes vitrified in droplets

Vitrification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine oocytes subjected to vitrification according to the minimum drop size approach. In total, 748 bovine, in vitro matured oocytes were vitrified using VS14 vitrification solution, containing 5.5-M ethylene glycol and 1.0-M sucrose after different pre-equilibration and equilibration protocols performed at 35 degrees to 37 degrees C. Experiment 1 showed no significant toxic effect during pre-equilibration treatments of oocytes in 2%, 4% or 6% ethylene glycol solutions, except the lower cleavage rate of oocytes exposed to 6% ethylene glycol (77.2% vs. 93.9% in control, P< 0.05). In Experiment 2, 12 to 15 min of pre-equilibration treatments in 0%, 1% or 2% ethylene glycol solutions were tested, followed by 30 or 45 sec of equilibration in VS 14 solution and vitrification in droplets of medium dropped directly into liquid nitrogen. The development rate of vitrified oocytes to the blastocyst stage tended to be higher after 30-sec equilibration treatment (9.5%, 13.9% and 13.8% in groups of oocytes pre-equilibrated in 0%, 1% or 2% ethylene glycol solutions, respectively). Experiment 3 tested pre-equilibration treatments in 0%, 1%, 2%, 3%, 4%, 5% or 6% ethylene glycol solutions, followed by 30-sec equilibration and vitrification in droplets. The highest cleavage, blastocyst and hatched blastocyst rates, which were not significantly different from control, were achieved in a group of oocytes pre-equilibrated in 3% ethylene glycol solution (76%, 30% and 15% vs. 89%, 42% and 21% in control, respectively). A healthy calf was born on Feb 22 1999, after transfer of 4 morula/blastocyst stage embryos developed from oocytes vitrified in droplets after pre-equilibration in 3% ethylene glycol solution. We conclude that gentle pre-equilibration of bovine oocytes in diluted, 3% ethylene glycol solution is an important factor improving the effectiveness of vitrification in droplets of bovine 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.     

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.     

Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro

Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.     

Production and breeding of transgenic cattle using in vitro embryo production technology

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.