Superovulation and embryo quality in beef cows using PMSG and a monoclonal anti-PMSG

The long half-life of pregnant mare serum gonadotrophin (PMSG) reduces its application in the superovulation of cattle; thus, a monoclonal antibody to PMSG (anti-PMSG) was administered at the onset of estrus to increase the number of transferable embryos. Angus, Hereford and Angus x Hereford cows (n = 149) 3 to 9 yr old were assigned randomly to one of three dosages of PMSG (1500, 3000 or 6000 IU) with or without an equivalent dosage of anti-PMSG. Embryos were collected nonsurgically on Day 8 (estrus = Day 0), and all cows were ovariectomized on Day 9. The percentage of cows exhibiting estrus and ovulating decreased (P<0.05) with an increasing dosage of PMSG (82, 76 and 44% for 1500, 3000 and 6000 IU, respectively). Ovarian and total corpora lutea (CL) weight increased (P<0.001) linearly as PMSG dosage increased, but were reduced (P<0.001) curvilinearly by anti-PMSG, resulting in a PMSG by anti-PMSG interaction (P<0.001); the interaction was also significant (P<0.05) for ovulation rate (14.0 vs 14.3, 21.5 vs 24.4 and 29.2 vs 6.6 CL for 1500, 3000 and 6000 IU PMSG, without vs with anti-PMSG, respectively). Anti-PMSG increased (P<0.001) the number of small ovarian follicles (1 to 3 mm diameter) and decreased (P<0.001) the number of large follicles (>10 mm) at ovariectomy; the number of large follicles increased (P<0.001) with PMSG dosage. The number of total and transferable embryos recovered did not differ among PMSG and anti-PMSG dosages; however, the percentage of transferable embryos decreased (P<0.01) with increasing PMSG dosage. In general, neither PMSG dosage nor anti-PMSG influenced embryo quality.     

Induction of superovulation in cyclic heifers: The inhibitory effect of large doses of PMSG

In two different experiments, superovulation was attempted with a PMSG-PG treatment; a bovine anti-PMSG serum was injected at estrus. After 2500, 5000 and 7500 IU of PMSG injected during the luteal phase, the mean ovulation rates were respectively 16.2 +/- 7.7, 3.2 +/- 2.1, and 1.4 +/- 0.6 in the first experiment (17 heifers) and 18.3 +/- 12.6, 8.5 +/- 8.2, and 2.2 +/- 2.3 in the second (19 heifers). The estradiol-17beta and progesterone patterns and the observations of the ovaries on the day of estrus (Day 0) by ultrasonic echography and on Day 8 by endoscopy show that the ovaries were highly stimulated and suggest that the inhibition observed with the largest doses reflects the absence of the preovulatory LH discharge or its effect.     

Multiple ovulation resulting from low level administration of PMSG to cattle at different stages of the oestrous cycle

Two experiments were carried out to determine whether differences in sensitivity to exogenous gonadotrophin which exist during the oestrous cycle can be used effectively in the induction of multiple pregnancy in cattle. In Experiment I, Hereford heifers and cows were injected with 800 IU pregnant mare serum gonadotrophin (PMSG) on approximately day 10 of the oestrous cycle, followed 48 h later by 30 mg prostaglandin F_2α (PGF_2α). Controls were treated with PGF_2α alone. Mean ovulation rates based on rectal palpation were 1.33 ± 0.10 (range: 1–2) and 3.05 ± 0.68 (range: 1–13) for 21 control and 21 treated animals, respectively (P < 0.02). In Experiment II, Hereford cows were treated with 800 IU PMSG on either day 5 (14 cows) or day 10 (12 cows) of the oestrous cycle, followed 48 h later by PGF_2α. Mean numbers of ovulations for animals that became pregnant were 1.50 ± 0.26 (range 1–3) and 3.80 ± 0.74 (range 1–7), respectively (P < 0.02). A high incidence of embryonic wastage occurred by 120 days of gestation in animals treated on day 10. The results of this study indicate that taking advantage of an animal's reduced responsiveness to exogenous gonadotrophin during the early portion of the oestrous cycle may be useful in developing methods for inducing multiple births with exogenous gonadotrophins.     

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.     

Superovulation in cattle: Effect of goat anti-PMSG serum

Sixteen heifers were superovulated using 5 000 i.u. PMSG on days 9 to 11 of the oestrous cycle (day 1 of the experiment) followed by two injections of 500 mug Estrumate 48 and 54 hours later. Eight of them were injected with goat anti-PMSG serum 5 hours after the first signs of oestrus were observed. Compared with the control group, the treatment with anti-PMSG serum resulted in a shorter heat period (25.8 vs. 51.3 hours), a higher mean number of ovulations (22.1 vs. 18.0) and a lower number of follicles over 10 mm in diameter (4.1 vs. 22.3). The mean numbers of eggs recovered in the experimental and control groups were 17.8 and 6.9, of which 70.2 and 42.0 per cent, respectively, were viable embryos. The concentrations of progesterone and 17-beta oestradiol in the blood plasma showed no significant differences between the experimental and control animals. A higher oestradiol in the control group on day 9 of the experiment was in keeping with the histological picture of the target organs and with a significantly higher number of follicles at slaughter on days 12 to 14 of the experiment.     

Embryo transfer in cattle: Factors affecting superovulatory response,number of transferable embryos,and length of post-treatment estrous cycles

The effect of multiple ampules of frozen semen on conception rate in superovulated Holstein heifers was studied using 3 breeding regimens (n=25): 1 ampule at 12 hr (0 hr = beginning of estrus), 3 ampules at 12 hr, and 1 ampule at each of 0, 12, and 24 hrs. There was no significant effect of breeding regimen on recovered number of ova or percentage of fertilized ova. In another project, months during which heifers were superovulated with PMSG (5–8 heifers/month) did not significantly affect rate of superovulation (number of CL). Clinical records for 173 superovulatory treatments in 150 Holstein heifers were studied to obtain preliminary information on efficacy of treatment regimens, repeatability of response within heifers, and relationships between superovulation and length of estrous cycle; where indicated, contemporary, nontreated heifers were used for comparisons. Efficacy of PMSG vs FSH treatments did not differ in number of CL or number of ova recovered, but percentage of recovered ova that were transferable was greater (P<.05) for FSH (58.3) than for PMSG (42.9). There was an intraclass correlation coefficient of 0.33 (n=37) indicating repeatability within heifers in the magnitude of response to superovulatory treatment. Mean length (days) and coefficient of variation were significantly greater for superovulatory estrous cycles (cycles during which multiple CL were present; n, 141; mean, 31.2; SE, ± 1.2; CV, 46.2%) than for contemporary cycles in nontreated heifers (n, 63; mean, 20.8; SE, ± 0.4; CV, 13.9%). Treated heifers with short cycles (<15 days) had fewer CL (6.8 ± 1.4; mean ± S.E.) than heifers with intermediate cycles (15 to 27 days; 9.4 ± 0.6) or prolonged cycles (>27 days; 11.5 ± 0.7). Collection of an ovum from nontreated heifers resulted in shortening (P<.05) of the estrous cycle (n, 16; mean, 18.1 days) when compared to cycles from contemporary heifers in which collections were not done (n, 16; mean, 20.4 days).     

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.     

Ovarian response to hCG treatment during the oestrous cycle in heifers

The aims of this study were to investigate whether treatment with a single ovulatory dose of hCG, between the day of oestrus and the end of the luteal phase, could induce extra ovulations in heifers and whether the presence of an existing corpus luteum (CL) affected the response. Heifers (N = 32) were injected with 1500 i.u. hCG or saline on a given day of the oestrous cycle. Treatments were repeated during subsequent cycles to provide a total of 71 observations, 57 of which followed an injection of hCG, given between Day 0 (oestrus) and Day 16, and 14 of which followed saline injections as controls. Ovulatory responses were noted by laparoscopy 2 days after hCG treatment. No heifers injected with saline produced additional CL. Of the hCG-treated cycles, 23 resulted in the formation of an additional CL, and this was significantly affected by the stage of the oestrous cycle when hCG was given; a greater response was observed during the early (Days 4-7) and late (Days 14-16) stages of the luteal phase than at the mid-luteal phase of the oestrous cycle. Two heifers were also treated with hCG on Days 17 or 18 of the oestrous cycle, but before oestrus; both had induced CL. There were no significant differences between the left-right orientation of the existing CL or the hCG-induced CL. These results demonstrate that the large, luteal-phase follicle of the cow is capable of ovulating in response to hCG and that the induced CL is not affected by the presence of an existing CL.     

Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle

Based on the premise that superovulation in cattle is optimal when superstimulation is initiated at the time of follicular wave emergence, the present study was done in beef heifers to determine if the superovulatory response following a single bolus of gonadotrophin treatment after follicle ablation (induced wave) at random stages of the oestrous cycle is comparable to the same gonadotrophin treatment at mid-dioestrus (spontaneous wave). In Experiment 1, heifers were assigned to nonablation (n = 18) and ablation (n = 20) groups. In nonablated heifers, superstimulatory treatment was given as a single subcutaneous injection (Folltropin-V, 400 mg) at mid-dioestrus to coincide with emergence of the spontaneous follicular wave 8 to 12 days after oestrus. In ablated heifers, the same superstimulatory treatment was given 1 day after ablation of all follicles ≥ 5 mm at random stages of the oestrous cycle to coincide with emergence of the ablation-induced wave. In both the nonablation and ablation groups, PGF_2α (Estrumate, 500 μg) was given 48 h after the superstimulatory treatment and artificial insemination was done 60 and 72 h later. Reproductive tracts were collected at the time of slaughter 6 or 7 days after insemination. Observations made in Experiment 1, indicated that some ablated heifers had only partial luteal regression at the time of insemination, while some others exhibited behavioral oestrus as early as 24 h after PGF_2α treatment. The design was amended in Experiment 2 to address these problems. Heifers were assigned to nonablation (n = 17), ablation-alone (n = 20) or ablation plus progestogen (n = 20) groups. Follicle ablation, superstimulatory treatment, artificial insemination and collection of reproductive tracts were done as in Experiment 1. However, all heifers were given two doses of PGF_2α (500 μg/dose) 48 and 60 h after superstimulatory treatment to ensure complete luteal regression, and heifers in the ablation plus progestogen group received a norgestomet ear implant at the time of follicle ablation to prevent early ovulations. The implant was removed at the time of the second PGF_2α treatment. In Experiments 1 and 2, the means for the ovarian and superovulatory responses were not significantly different between groups. Averaged over the nonablation and all ablation groups for Experiments 1 and 2, the mean number of corpora lutea, fertilized ova and transferable embryos were 22.9 vs 18.6, 7.3 vs 7.8 and 5.4 vs 5.6, respectively. In summary, follicle ablation at random stages of the oestrous cycle followed by a single bolus of gonadotrophin treatment 1 day later resulted in a superovulatory response that was comparable to the same superstimulatory treatment administered around the time of spontaneous wave emergence at mid-dioestrus. The ablation/superstimulation method described herein offers the advantage of initiating superstimulatory treatment forthwith and assuring that treatment is concomitant with wave emergence to achieve an optimal superovulatory response. Moreover, the full extent of the oestrous cycle is available for superstimulation and the need for detecting oestrus or ovulation and waiting 8 to 12 days to initiate treatment is eliminated.     

SUPEROVULATION AND SUPERPREGNANCY IN THE GOLDEN HAMSTER*

Pregnant mare serum gonadotropin (PMSG) treatment given in the morning or afternoon on any day of the four-day estrous cycle and human chorionic gonado tropin (HCG) given two days later successfully induced superovulation in the golden hamster. The minimum interval between PMSG and HCG necessary to obtain consistent superovulation was approximately 44 hr. The lowest ovulation rate was obtained following PMSG treatment on the afternoon of day 4 despite the fact that this time coincides with the maximum endogenous FSH level, necessary for the maturation of the next crop of follicles destined to ovulate. Thirty-eight to one-hundred percent of superovulated females in four different treatment groups became superpregnant after natural mating. Some treated females exhibited two consecutive nights of estrus with ovulation apparently occurring during the second night. Superpregnant females delivered “super” size litters, up to 27 live-born pups. The ultimate litter size appeared to be established after day 3 and prior to day 8 of superpregnancy. A one-day extension of the normal 16-day gestation period was observed in 31% of superpregnancies. Unilateral pregnancies were observed at autopsy in 44% of treated females which received the high dose of PMSG (30 IU). The progeny of superovulated females reproduced normally at maturity. The results indicate that ova from superovulated female hamsters are capable of full normal development.     

Ultrasonic study of follicular dynamics following superovulation in German Merino ewes

Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be convenient and less expensive to use a single dose of FSH, but results of various trials have been controversial. We wished to investigate ovarian dynamics using ultrasonography after superovulation with a single dose of pFSH and hMG as compared with a single dose of eCG. Estrus was synchronized during the breeding season with fluorogestone acetate-containing intravaginal sponges in adult German Merino ewes (n = 38). They were superovulated with single doses of pFSH (17 mg; n = 10), hMG (600 IU FSH and 600 IU LH; n = 9) or eCG (1250 IU; n = 10) given at the time of sponge removal, or pFSH (17 mg; n = 9) given 36h before sponge removal. Follicular and luteal development were observed by ultrasonic scanning every 8 h from the gonadotrophin injection until the end of estrus, and then once daily until Day 6 after estrus. Jugular venous blood was collected starting immediately before and 1 h after superovulation treatment, then twice daily until the end of estrus and once daily for the following 7 days. Concentrations of estradiol-17beta (E2) and progesterone (P4) were measured in plasma. Differences in the follicular dynamics of the 4 superovulation groups were obvious. The functional duration of the pFSH action was estimated to last approximately 48 h, whereas eCG and hMG were active for up to 72 h. The diameter of the ovulatory follicles proved to be smaller than it was described for unstimulated ewes. Single applications of pFSH or hMG can induce a superovulatory response, although the post-estrus progesterone profile revealed a high premature luteal regression rate in the different superovulation groups. Premature corpus luteum regression could not be seen by ultrasonography at this early stage of the luteal phase, indicating that the technique may fail to detect these corpora lutea in an embryo transfer program. However, ultrasonography represents a suitable method to observe follicular dynamics following different superovulation regimens in sheep.     

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.     

The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

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.     

Premature regression of the corpora lutea in superovulated cows

Morphological and functional regression of the CL was shown by $\text{15}\text{55}$ (27%) superovulated dairy cows at slaughter on D 14. Neither the routine injection of a PMSG-antiserum at the induced superovulatory oestrus, nor intrauterine infections proved to be responsible. Animals with regressed CL however had significantly lower progesterone levels in the peripheral blood at the time PMSG (D 10, P = 0,05) and prostaglandin (D 12, P = 0,02) were injected, as compared to superovulated animals with normal CL. At the induced superovulatory heat, the differences between the 2 groups disappeared. From D 4 after superovulation the differences in progesterone concentration between the 2 groups became significant again (P = 0,02 – 0,05). It is not yet clear whether the observed luteal dysfunction is due to an inadequate luteotrophic stimulus or to a premature luteolysis. The luteal regression could be caused either by a genuine luteal insufficiency of the midcyle CL or by poor heat detection.     

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.     

Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

Mature nonlactating Altamurana ewes (n = 168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for 12 d. In Experiment 1, 48 ewes were divided into a 3 x 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, 1, 2, 3 microl/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divided into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at -24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without anti-PMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that 1 microl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes. Estradiol-17 beta levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P < 0.01) and increased the rate of ovulation (P < 0.05). Moreover, embryo production showed a more than two-fold increase (P < 0.01) of viable embryos following AP injection at 12 or 24 h after the onset of estrus (3.2 to 3.3 vs 1.3, with vs without anti-PMSG). It is concluded that superovulatory treatment with 1000 IU PMSG plus AP administered at a fixed time after the onset of estrus may improve ovarian response and the yield of viable embryos in ewes.     

Effect of injection time of anti-PMSG antiserum on ovulation rate and quality of embryos in superovulated cows

Eighteen cows were superovulated by injecting 3000 IU of PMSG during the luteal phase, followed 48h later with an injection of Estrumate. They were then placed in a control group or were given anti-PMSG antiserum at either 12h or 24h after the onset of oestrus. Sixteen of these animals were used for the same experiment five months later. The results were pooled because they were not significantly different between the two treatment periods. The timing of the injection of anti-PMSG antiserum, either 12h (11 cows) or 24h (12 cows) after the onset of oestrus, did not significantly affect the ovulation rate, the number of embryos collected or the number of good embryos. The antiserum significantly increased the number of good embryos but did not affect the ovulation rate or embryo recovery. It is concluded that even with a moderate dose of PMSG, the use of anti-PMSG at 12h or 24h after the beginning of oestrus improves the quality of embryos. The mean number of embryos to be transferred (5.5) is in the range of those obtained after the FSH treatments, but the procedure required only three injections compared with nine for the FSH treatment.     

The effect of PMSG-priming on subsequent superovulatory response in dairy cows

Superovulation was induced in 56 dairy cows to evaluate the effect of two different regimens using pregnant mare serum gonadotropin (PMSG). Thirty-two cows (controls) were superovulated between Days 9 and 12 of the estrous cycle with a single dose of PMSG (2 800 IU), while remaining 24 cows (PMSG-primed) received 200 IU of PMSG on Day 4 of the estrous cycle and subsequently a single dose of PMSG (2 800 IU) between Days 8 and 12. The cows in both treatments were each given 0,5 mg of cloprostenol at 48 h after the superovulatory PMSG treatment. They were then artifically inseminated twice, 48 h and 72 h later. Embryos were recovered at sloughter between Days 2 and 5 of the cycle and morphologically evaluated. The number of corpora lutea (CL) in the ovaries of the cows was recorded. The mean number of CL (7.2 vs 17.8) was significantly higher (P 0.01) for PMSG-primed cows. The percentage of recovered ova (60.5 vs 70.2 %) and good embryos (79.3 vs 70.7%) were not significantly different between groups. The percentage of fertilized ova (91.4 vs 83.8%) was significantly (P 0.025) greater for the controls. Results of the study indicate that PMSG-priming increased the ovulation rate in the cows superovulated with PMSG.     

Effects of monoclonal antibody against PMSG administered shortly after the preovulatory LH surge on time and number of ovulations in PMSG/PG-treated cows

Normally cyclic heifers received 2500 i.u. PMSG i.m. at Day 10 of the oestrous cycle and 15 mg prostaglandin (PG) i.m. 48 h later. From 30 h after PG the LH concentration in the peripheral blood was estimated every hour using a rapid RIA method which allowed the LH concentration to be known within 4 h. Monoclonal antibody against PMSG was injected in the jugular vein of 29 heifers at 4.8 h after the maximum of the preovulatory LH peak; 28 heifers were not treated with anti-PMSG (controls). Peripheral blood concentrations of PMSG, LH, progesterone and oestradiol were compared. Ovaries were collected by ovariectomy at fixed times, 22-30 h after the LH peak, and numbers were counted of small (2-10 mm), large (greater than 10 mm) and ovulated follicles, and of follicles with a stigma. In anti-PMSG-treated cows, the PMSG concentration fell sharply to non-detectable levels within 2 h of the treatment, indicating that PMSG was neutralized in these cows at the onset of final follicular maturation. In all cows, the concentration of oestradiol showed a significant decrease at about 8 h after the LH peak. After anti-PMSG treatment ovulations took place from 24 until 30 h after the LH peak, whereas in control cows follicles had already ovulated at or before 22 h and ovulations continued until 30 h. At 30 h 90% of the follicles had ovulated in anti-PMSG-treated cows vs 72% in the controls, resulting in 15 and 8 ovulations per cow respectively (P less than 0.05). Also, administration of monoclonal antibody against PMSG synchronized final follicular maturation and shortened the period of multiple ovulations. In conclusion, neutralization of PMSG shortly after the preovulatory LH peak suppresses adverse effects of PMSG on final follicular maturation, leading to an almost 2-fold increase of the ovulation rate.