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.     

Superovulation in immature and mature Chinese hamsters

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.     

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.     

不同激素和注射方式对家猫超排效果的比较

比较了PMSG hCG和FSH hCG两种方案以及PMSG的不同剂量和注射方式对家猫的超排效果的影响。用 1 0 0IU的PMSG超排家猫所得到的排卵点数及平均每只猫获得的卵数显著低于 2 0 0IU处理组或 30 0IU处理组 (P <0 0 5 ) ,但 2 0 0IU处理组与 30 0IU处理组之间的超排效果也无显著差异 (P >0 0 5 ) ;用皮下注射 2 0 0IU的PMSG或用肌肉注射 2 0 0IU的PMSG对超排效果无差异 (P >0 0 5 ) ;用 2 0 0IUPMSG 2 0 0IUhCG和 1 5mgFSH 2 0 0IUhCG两种方案对家猫超排 ,发现不论是每只猫的排卵点数、卵子获得数 ,还是卵子的第一极体排放率都没有显著差异 (P >0 0 5 )。实验说明 ,PMSG的注射方式不影响对家猫的超排效果 ,用 2 0 0IU的PMSG超排家猫是较适合的剂量 ,FSH和PMSG都可用于家猫的超排 ,但PMSG使用更为方便。     

Strain Differences in Superovulatory Response, Embryo Development and Efficiency of Transgenic Rat Production

The differences between rat strains in superovulation response, in vitro and in vivo development of preimplantation embryos and overall transgenic efficiency was studied. The protocols for induction of superovulation using single injections of pregnant mare’s serum gonadotropin (PMSG) or minipumps with follicle stimulating hormone (FSH) were compared in Lewis (LEW), Wistar-Kyoto (WKY), and stroke-prone spontaneously hypertensive rats (SHRSP) or Sprague–Dawley (SD) and Wistar rats as representative inbred or outbred strains, respectively. The percentage of mated animals with positive superovulatory response was similar in all strains (60.0–100%). The mean number of ova per donor was not dependent on the kind of hormonal treatment used within each rat strain. In general, females from outbred SD and Wistar rats were more responsive to hormonal treatments than animals from inbred rat strains. In addition, SD female rats produced a significantly higher number of embryos per female in response to PMSG-treatment compared to all other strains. Between the inbred strains, SHRSP was the most effective for superovulation. In vitro development of intact zygotes to the blastocyst stage was not different between SD, Wistar and SHRSP rats. In contrast, in vitro development of WKY zygotes was significantly less efficient than in other strains. However, 2-cell stage embryos in vivo produced from SD, SD × Wistar and WKY animals showed no difference in competence to develop to blastocyst stage in vitro. The proportion of offspring developing after oviduct transfer of intact zygotes was similar in all strains (44.0–56.4%) with the exception of WKY rats (35.9%). We also compared the survival rate after injection, ability of manipulated zygotes to develop to term and overall transgenic efficiency in various rat strains. SD and SHRSP zygotes survived after microinjection better than the WKY and Lewis zygotes. No differences were found in the efficiency of transgene integration per newborn in different strains ranging from 5.7 to 16.7%. The results of this study demonstrate that different rat strains have varying responses to superovulation, sensitivity to microinjection, capability to develop in vitro until blastocyst stage or in vivo to term after transfer to foster mothers. Despite these differences all studied strains can be used for efficient transgenic rat production.     

Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle-stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (401U), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continu ous s.c. infusion over a 72-h period with a purified FSH preparation containing an opti mum ratio of luteinizing hormone (LH): FSH (FSH-hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1–4 of preg-nancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH-hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH-hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovu lation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and andro-gens on days 1–3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH-hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considera bly higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rate (FSH-hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of emyryos and low levels of circulating andorgens and estradiol-17β during the preimplantation period.     

Estrous cycle stage-independent treatment of PMSG and hCG can induce superovulation in adult Wistar-Imamichi rats.

The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results demonstrate that the PMSG/hCG treatment can induce ovulation at any stage of estrous cycle in WI rats and efficient superovulation at 12 weeks of age.     

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.     

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.     

In vitro fertilization in the rabbit after delayed ovum recovery

Rabbit ovum donors were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ova were recovered 16-17 h post-hCG from oviducts immediately after killing and from excised oviducts held in saline 30 min at 33 degrees or 38 degrees C prior to ovum recovery. In vivo-capacitated spermatozoa were used to inseminate both groups of ova. Data revealed a decrease in fertilization rates following a 30-min delay at 38 degrees C in ovum recovery. Thus, 64% (44/69 ova) were fertilized with rapid recovery, whereas 43% (39/90 ova) were fertilized following a 30-min delay. The decrease in fertilization imposed by delay in ovum recovery was apparently overcome when oviduct storage was at 33 degrees C. Under these conditions, 69% of inseminated ova were fertilized. Ova inseminated with in vitro-capacitated sperm showed a similar response to delayed ovum recovery. Embryonic development in culture of ova obtained from mated does was not affected by delay in recovery at 33 degrees or 38 degrees C provided mated does had been injected only with hCG. Ova from mated does receiving both PMSG and hCG were adversely affected by a 38 degrees C delay. The data emphasize the importance of rapid ovum recovery from oviducts and suggest the possibility of altering conditions to overcome damaging effects of delayed recovery.     

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.     

The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes

The influence of estrous cow serum (ECS) or fetal calf serum (FCS) and their interaction with gonadotropins on in vitro bovine maturation and fertilization was evaluated. The addition of ECS or FCS to the medium significantly increased the percentage of oocyte maturation over that of Ham's F-10 medium alone (P<0.05). The addition of follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) or of FSH plus hCG to the medium provided no advantage in maturation over serum alone. However, FSH plus HCG added to the maturation medium significantly improved the frequency of pronucleus formation in both groups compared with a medium to which no gonadotropins were added (P<0.05). Both ECS and FCS (as medium supplements) promoted oocyte maturation. Although the addition of a combination of FSH plus hCG to the medium did not increase the rate of oocyte maturation, it did contribute to the high incidence of subsequent pronucleus formation.     

Effects of an antiandrogen, flutamide, on oocyte quality and embryo development in rats superovulated with pregnant mare's serum gonadotropin

Increased oocyte degeneration in rats is associated with enhanced ovarian androgen secretion following superovulation with pregnant mare's serum gonadotropin (PMSG). To determine whether androgens may be causally related to oocyte degeneration, we examined the effects of an antiandrogen, flutamide, on oocyte quality and embryo development after induction of superovulation with PMSG. Immature female Sprague-Dawley rats were used for two experiments. In the first experiment, the females received either 5 mg flutamide or vehicle alone 30 and 36 h after 40 IU PMSG and were killed at 48, 60, and 72 h. Although total number of oocytes was not significantly different between the two groups, flutamide significantly reduced the percentage of degenerate oocytes ovulated at all the time intervals examined (p less than 0.01). In the second experiment, the females were given 4 IU PMSG (control) or 40 IU PMSG with either 5 mg flutamide or vehicle. The rats were mated and then killed on Days 2, 3, 4, and 5 of pregnancy. Compared to control, flutamide did not effectively prevent the early loss of preimplantation embryos nor the developmental retardation which took place in the vehicle-treated rats after Day 2. However, flutamide treatment was associated with a significant decrease in embryo degeneration and a significant increase in the percentage of cleaved embryos on Day 2 (p less than 0.01). Compared to levels associated with the vehicle regimen, ovarian and/or serum androgen levels in the flutamide-treated rats significantly decreased at 60 h (p less than 0.01) and on Day 2 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the Barred Frog Mixophyes fasciolatus (Myobatrachidae)

ABSTRACT: BACKGROUND: Protocols for the hormonal induction of ovulation and oviposition are essential tools for managing threatened amphibians with assisted reproduction, but responses vary greatly between species and even broad taxon groups. Consequently, it is necessary to assess effectiveness of such protocols in representative species when new taxa become targets for induction. The threatened genus Mixophyes (family Myobatrachidae) has amongst the highest proportion of endangered species of all the Australian amphibians. This study developed and optimised the induction of oviposition in a non-threatened member of this taxon, the great barred frog (Mixophyes fasciolatus). METHODS: Gravid female M. fasciolatus were induced to oviposit on one or more occasions by administration of human chorionic gonadotropin (hCG) with or without priming with pregnant mare serum gonadotropin (PMSG). Treatments involved variations in hormone doses and combinations (administered via injection into the dorsal lymph sacs), and timing of administration. Pituitary homogenates from an unrelated bufonid species (Rhinella marina) were also examined with hCG. RESULTS: When injected alone, hCG (900 to 1400 IU) induced oviposition. However, priming with two time dependent doses of PMSG (50 IU, 25 IU) increased responses, with lower doses of hCG (200 IU). Priming increased response rates in females from around 30% (hCG alone) to more than 50% (p = 0.035), and up to 67%. Increasing the interval between the first PMSG dose and first hCG dose from 3 to 6 days also produced significant improvement (p<0.001). Heterologous pituitary extracts administered with hCG were no more effective than hCG alone (p = 0.628). CONCLUSIONS: This study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.     

Efficient collection and cryopreservation of embryos in F344 strain inbred rats

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources.     

Selective release of follicle-stimulating hormone and luteinizing hormone by 5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydroprogesterone in pregnant mare's serum gonadotropin-primed immature rats exposed to constant light

The effects of 5 alpha-dihydroprogesterone (5 alpha-DHP) and 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release were examined in the pregnant mare's serum gonadotropin (PMSG)-primed immature female rat (8 IU PMSG at 28 days of age) maintained in constant light. Control rats kept in 14L:10D conditions exhibited proestrous-like surges of LH and FSH release with peak levels attained at 1800 h on the second day after PMSG treatment. In rats exposed to constant light, the PMSG-induced surges of LH and FSH were not only delayed until 1000 h on the third day after PMSG, resulting in a delay in ovulation, but were also significantly attenuated when compared to the gonadotropin surges that occurred on Day 2 in rats kept under normal light-dark conditions. The administration of 5 alpha-DHP significantly enhanced the release of FSH at 1000 h on Day 3 when compared to constant light-exposed controls, but had no effect on LH. Treatment with 3 alpha, 5 alpha-THP selectively potentiated the release of LH at 1000 h on Day 3 and had an attenuating effect on FSH release on Days 2 and 3. These observations confirm earlier findings in the immature ovariectomized estrogen-primed rat and suggest that 5 alpha-DHP and 3 alpha, 5 alpha-THP may have significant roles in the regulation of FSH and LH secretion.     

Estrous stage- and animal age-independent superovulation in the BrlHan:WIST@Jcl(GALAS) rat.

In most strains of rats, the effects of treatment for the induction of superovulation show major strain differences and are strongly influenced by the stage of the estrous cycle. This study demonstrated, however, that superovulation was easily induced in Wistar strain Brl Han:WIST@Jcl(GALAS) rats by PMSG and hCG administration. To confirm the effects of such treatment, we studied age differences in egg collection efficiency. After superovulation was induced by intraperitoneal administration of 150 IU/kg PMSG and 75 IU/kg hCG given 48 h apart, the mean numbers of oocytes obtained from rats at 4, 8, 12, 20 and 28 weeks of age were 38.9, 33.5, 46.1, 26.9 and 21.3, respectively. No differences caused by the estrous stage at the PMSG administration were observed. In an embryo transfer experiment, fertilized eggs obtained from superovulated rats at each week of age showed equivalent viability until full-term to those from untreated rats. These results suggest that estrous stage-independent superovulation is effective not only in the pre-pubertal stage but also in adult rats.     

Improved survival of zygotes in superovulated rats following surgical or pharmacological block of ovarian steroid secretion

The purpose of this study was to test the hypothesis that the preimplantation losses of zygotes in pregnant superovulated juvenile rats was due to an imbalance of ovarian hormones. Twenty-seven or twenty-eight-day-old rats were injected with 20 iu of PMSG, 25 iu of hCG 50 hr later and mated overnight. From a mean ovulation rate of 52 ± 2 only 16 ± 4 zygotes survived after 4 days. After ligation of the cervical ends of the uteri on the day following fertilization the mean yield of zygotes was 22 ± 6. Ovariectomy on the day of fertilization increased the yield of zygotes to 39 ± 6, but the recovery of the zygotes was seriously complicated by postoperative adhesions and deformations of the adnexa. Inhibition of steroidogenesis with aminogluthehimide phosphate also increased the yield of zygotes. The optimal dose was 45 mg in six divided doses over 3 days, which gave a mean recovery of 57 ± 3 zygotes (of which 75% were blastocysts), that is 100% salvage. A lower dose (30 mg) reduced the recovery to the level of untreated animals, while increasing it to 60 mg resulted in maternal mortality and morbidity, as well as in developmental retardation of zygotes.