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.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus–oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.     

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.     

家猫的胚胎工程

家猫是惟一一种没有被列为珍稀或濒危的猫科动物。通过家猫的胚胎工程研究,对保护其它濒危猫科物种有重要的借鉴意义。本文描述了家猫的一般生殖特点,着床前的胚胎在体内的发育概况;综述了近年来对家猫的超数排卵,卵母细胞的体外成熟,体外受精,胚胎的体外培养,胚胎移植,冷冻保存和胚胎克隆等方面的研究进展。     

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

Background  

The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI).     

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.     

PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).     

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.     

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.     

The effects of pokeweed mitogen (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation and embryo development in vitro

The effects of two commonly used cell culture mitogens, pokeweed (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation in vitro (IVM) and preimplantation embryo development in vitro were evaluated by randomized complete block experimental design with three treatments. Effects were measured by quantifying subsequent embryo development. Oocyte maturation was adversely affected by PWM-containing medium as indicated by a decrease in cleavage rate and subsequent embryo development to morula and blastocyst stages. Embryo developmental competence was also adversely affected by PWM. Development in PHA-containing medium was significantly better (P<0.05) than in the PWM treatment, although there was no difference (P>0.05) when compared to Control. We conclude that there are no beneficial effects in adding mitogenic agents to culture medium to enhance in vitro embryo production and development.     

In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation,pronucleus formation and embryo development

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.     

Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle

It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized.     

A novel approach for in vitro production of bovine embryos: use of the Oxoid atmosphere generating system

The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO2 Heraeus incubator and the Oxoid CO2Gen atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO2Gen gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar, an atmosphere of 6% CO2 in 15% O2 is created, which is comparable to the 5% CO2 and 20% O2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar. During experiments the AnaeroJar was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8 degrees C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.