Human menopausal and pregnant mare serum gonadotrophins in murine superovulation regimens for transgenic applications

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F(1) hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Efficacy of exogenous gonadotrophic hormones to induce estrus in anestrous gilts

The objective of this study was to examine the response of anestrous gilts to injections of pregnant mare's serum gonadotrophin (PMSG) alone or in combination with human chorionic gonadotrophin (hCG). One hundred and eighty gilts which had failed to exhibit estrus by about 33 wk of age were given one of the following treatments: no injection, 500 IU PMSG, 1000 IU PMSG or 400 IU PMSG + 200 IU hCG. A greater number of gilts injected with 1000 IU PMSG exhibited estrus within nine days of treatment than control gilts (21/37 vs 13/41, X(2) = 5.0, P<0.05). In addition, gilts injected with 1000 IU PMSG exhibited oestrus significantly earlier than gilts receiving the other treatments. In comparisons of the proportion of gilts ovulating within 9 d of treatment and the treatment-to-ovulation interval, there were no significant differences between the three exogenous hormone treatments. There was also no significant effect of treatment on farrowing rate or subsequent litter size. The results of our study indicate that treatment of anestrous gilts with 1000 IU PMSG effectively induces ovulation and fertile estrus. Inadequate expression of estrus often accompanied the ovulation induced by the lower dosages of PMSG used with and without hCG in this experiment.     

Biochemical status of ovaries after induction of superovulation on different days of estrus cycle in mice

To investigate the role of ovarian status and to find out a suitable hormonal dose for induction of superovulation and its effect on biochemical status of the ovaries, the mice were injected with PMSG in doses of 5, 7.5, and 10 IU on different days of the estrous cycle i.e. proestrus, estrus, metestrus and diestrus followed by hCG injection 48 hr later. All these treatments increased the mean ovarian weight and ovulation rate when compared with that of control animals. Maximum response was observed by treatment with 7.5 IU PMSG on the day of estrus. This treatment resulted in a non-significant decrease in total proteins but a significant increase in the lipid concentrations while no change in cholesterol content of the ovaries of superovulated mice. The activity of acid phosphatase and lactate dehydrogenase significantly increased and alanine aminotranseferase significantly decreased in the ovaries of mice after superovulatory treatment when compared with that of control animals. This reveals that treatment with PMSG and hCG results in metabolic alterations in the ovaries which may perhaps be inducing biosynthetic deficiencies in oocytes as indicated by increased prenatal mortality in superovulated pregnant mice when compared with that of controls in the present studies.     

Superovulation and embryo recovery in rabbits (Oryctolagus cuniculus) using different doses of pregnant mare serum gonadotrophin (PMSG)

Present investigation was conducted to study the ovarian response and embryo recovery using different PMSG dose levels. Six rabbits each were assigned randomly to treatment 1 (PMSG 50 IU + hCG 100 IU), treatment 2 (PMSG 75 IU + hCG 100 IU) and the control group (no hormone administered). PMSG injection (im) was followed 68 hr later by natural mating to a fertile rabbit buck and human chorionic gonadotrophin (hCG) injection iv post coitum. Embryos were recovered 96 hr post coitum by a modified surgical method. Mean number of ovulations in the control group differed significantly from treatment 1 and 2, but no significant difference was observed between treatments 1 and 2. Mean embryo recovery percentage was lowest in treatment 2 and highest in the control group. The higher dose PMSG (treatment 2) was observed to be more disturbing in terms of recovery of embryos as well as their morphology.     

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.     

FSH-induced ovulation in intact and hypophysectomized mice

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.     

Production of transgenic rats using young Sprague-Dawley females treated with PMSG and hCG.

The aim of this study was to examine the effects of gonadotrophin treatments on estrus synchronization and superovulation in young Sprague-Dawley (SD) rats that had not yet exhibited defined estrus cycles (5 to 7 weeks old), and to produce transgenic rats using these females as embryo donors and recipients. In Experiment 1, female rats were injected with PMSG and hCG (12.5, 25, 50 and 100 IU/kg each) and were mated with stud males. The reproductive performance of young rats were highest when PMSG and hCG at doses of 25 IU/kg each were injected (delivery rate 87.5%, nursing rate 92.9%). In Experiment 2, female rats were injected with PMSG and hCG (100, 150 and 300 IU/kg each) to induce superovulation. More eggs were recovered from the rats injected with PMSG and hCG at 150 and 300 IU/kg than from those treated with 100 IU/kg (33.4 and 41.3 vs. 13.3 eggs per female, respectively; p < 0.05). In Experiment 3, pronuclear-stage zygotes from 150 IU/kg PMSG/hCG-treated rats were used for microinjection of the fusion gene of bovine alpha S1-casein gene promoter and human growth hormone gene (2.8 kb), and the microinjected zygotes were transferred into the oviduct ampullae of the 25 IU/kg PMSG/hCG-treated rats. Seventeen transgenic rats were obtained from the 334 DNA-injected zygotes (5.1%). These results indicate that recipients and embryo donors for the production of transgenic rats can be prepared by the appropriate PMSG and hCG treatments of young SD rats, regardless of their estrus stages.     

Fertilizability of Superovulated Eggs by Estrous Stage-independent PMSG/hCG Treatment in Adult Wistar-Imamichi Rats

We investigated the fertilization and developmental ability of superovulated eggs obtained from adult Wistar-Imamichi (WI) rats, by using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) treatment. Female WI rats, 11–13 weeks of age, were divided into four groups by estrous stage (metestrus [ME], diestrus [DE], proestrus [PE], or estrus [E]). PMSG (150 IU/kg) and hCG (75 IU/kg) were injected at an interval of 48 or 55 h and the female rats were mated with mature male rats. The ovulated eggs were collected 20, 24, and 27 h after hCG injection. Regardless of the estrous stage at the time of PMSG injection, the treated rats mated and ovulated similar to the untreated spontaneously ovulated rats (S group). Although the proportion of fertilized eggs in the E- and PE-treated groups was less than the S group 20 h after hCG injection, the proportion was not different among all treated and S groups 24 h after hCG injection. The proportion of fertilized eggs using in vitro fertilization and the proportion of offspring obtained from 2-cell stage embryo transfer did not differ among the treated and S groups. In comparison with PMSG/hCG-treated immature rats, mating and ovulation rate of adult rats were significantly higher. The proportion of fertilized eggs obtained from mated rats did not differ between immature and adult rats. These results demonstrate that adult WI rats are good egg donors for reproductive biotechnological studies using unfertilized or fertilized eggs.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

Effects of 4-hydroxyandrostenedione and hyperstimulation with pregnant mare serum gonadotrophin on early embryonic development in rats

A possible role of high oestradiol levels in mediating the adverse effects of hyperstimulation with pregnant mare serum gonadotrophin (PMSG) on early embryonic development in the rat was investigated using an aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), to inhibit endogenous oestradiol production. Three experiments were conducted in this study. In the first, varying doses of 4-OHA were administered either concurrently with human chorionic gonadotropin (hCG) to pro-oestrus female rats hyperstimulated at early di-oestrus stage with 20 IU PMSG or alone into nonhyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 microg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females, while low doses of 100 and 500 microg/rat were ineffective. The protective effect of 4-OHA on embryo count was optimum at 2000 microg. When administered alone, only the highest dose of 5000 microg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 microg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and 5000 microg/rat 4-OHA was able to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 microg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. Together, these results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. This may be due to its potent aromatase inhibiting properties that lead to the suppression of oestrogen production, thereby alleviating the supraphysiological level of oestradiol, which is typically present in PMSG-treated females. Interestingly, 4-OHA treatment on its own was able to positively influence embryo count when given at a high dose of 5000 microg/rat, and this may be associated with its weak androgenic properties. In conclusion, this study supports the hypothesis that excessive oestradiol is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Cytogenetic analysis of Day-4 embryos from PMSG/hCG-treated prepuberal gilts

Prepuberal gilts were treated with pregnant mare serum gonadotropin (PMSG) to study the effects of its dosage on ovulation rate, fertilization rate after artificial insemination, embryo viability, and rate of development and incidence of chromosome abnormalities in Day-4 embryos. Gilts received 750 IU, 1250 IU or 1500 IU of PMSG, followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Gilts were inseminated 28 to 30 h following the hCG injection, and resulting embryos were collected on Day 4 post ovulation. Ovulation rate was higher in the 1250 IU group than in the 1500 IU group or the 750 IU group. The 1500 IU dose caused excessive stimulation of the ovary, resulting in the occurrence of large (>10mm diameter) unovulated follicles, reduced fertilization rate and low embryo recovery rate. There was no difference in the incidence of chromosome abnormalities among the three groups, although the 1500 IU group had higher embryonic mortality than the two lower dose groups. A dose of 1250 IU PMSG increased ovulation rate above that achieved by 750 IU and, therefore, increased the number of oocytes or embryos available for transfer or for other studies, without sacrificing embryo viability or increasing the incidence of chromosome abnormalities.     

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.     

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.     

Variables affecting the yield and developmental potential of embryos following superstimulation and in vitro fertilization in rhesus monkeys

Three follicular-stimulation protocols were compared to evaluate the yield and quality of oocytes obtained from rhesus monkeys. Five animals received a high-dose regimen of PMSG (protocol I), three received a lower-dose regimen (protocol II), and two received Clomid-Pergonal (protocol III). Oocytes were recovered at laparoscopy after HCG injection, fertilized in vitro, and cultured up to the blastocyst stage. Yields of mature oocytes were 17.2 ± 13.0 (80% of total recovered), 6.7 ± 6.6 (41%), and 4.5 ± 2.5 (90%) per stimulated cycle for protocols I, II, and III, respectively. Of mature oocytes, 72%, 45%, and 89% were fertilized for protocols I, II and III, respectively. Protocol I produced the most fertilized oocytes per stimulated cycle (11.6 ± 11.6) and the greatest E₂ production (approximately fourfold that maximally expected for an unstimulated cycle). For the combined protocol I and II results, there was a significant correlation (P ? 0.05) between mean embryo development score and E₂ production. Fertilized oocytes from protocol I yielded 7.8 ± 8.0 morulae and 6.8 ± 7.2 early zonal blastocysts per cycle. After transfer of nine singleton embryos to surrogate recipients, one live birth resulted. We conclude that our high-dose PMSG regimen offers the best means at present for obtaining susbstantial numbers of developmentally competent oocytes in rhesus monkeys and that more extensive use of rapid serum E₂ assays for monitoring both stimulated cycles and those of potential surrogate recipients could help to predict the success of in vitro fertilization and embryonic development following embryo transfer in rhesus monkeys.     

The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus—including deer mice—are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.     

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.