In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.     

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.     

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.     

In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.     

In vivo and in vitro embryo production in goats

Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a greater production of valuable goats. Also, IVP of goat embryos will provide an excellent source of embryos for basic research on development biology and for commercial applications of transgenic and cloning technologies. Different protocols of IVP of embryos have been used in goats. However oocyte quality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes. One of the principal determinant factors in the results of blastocyst development is the age of the oocyte donor females. In goats, oocytes from prepubertal and adult females do not show differences in in vitro maturation and in vitro fertilization; however the percentage of oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertal and adult goats, respectively.     

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.     

Meiotic and developmental competence of prepubertal and adult swine oocytes

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.     

Refrigeration of rainbow trout gametes and embryos

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and embryos resulted in an average embryo survival rate of 71% in optimal source variants after 17 days of refrigeration (7 days oocytes+10 days embryos). The study shows that both gamete and embryo refrigeration can be successfully used as an efficient tool for prolonging availability of rainbow trout embryos in early developmental stages.     

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 leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.     

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.     

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined. Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and subjected to in vitro fertilization followed by embryo culture. After fertilization, ova from unstimulated prepubertal monkeys displayed lower development to morula (4%) than those from unstimulated adult females (18% in breeding season and 22% in nonbreeding season). No developmental difference was found between ova from unstimulated adult monkeys in breeding and nonbreeding seasons. However, ova from FSH-primed prepubertal monkeys displayed greater development to blastocyst stage (54%) than those from adult monkeys in the breeding season (16%) and nonbreeding season (0%); and ova from FSH-primed adult females in the breeding season had significantly (P < 0.05) greater developmental competence than those obtained in the nonbreeding season (> or = morula stage, 54% vs. 3%; blastocyst stage, 16% vs. 0%). These data indicate that 1) rhesus monkey oocytes acquire developmental competence in a donor age-dependent manner, and 2) animal age and breeding season modulate the effect of FSH on oocyte developmental competence in the rhesus monkey.     

Selection of prepubertal goat oocytes using the brilliant cresyl blue test

Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB-). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6+/-6.3 microm) was higher (P < 0.001) than that of BCB- oocytes (125.5+/-10.2 microm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher (P < 0.05) than that of BCB- (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB- (8.2%; P < 0.0001) and control oocytes (11.9%; P < 0.05). The percentages of total embryos undergoing development to >8-cell and the morula plus blastocyst stages were higher (P < 0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB- oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.     

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.     

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.     

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.     

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.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.