Superovulation Group Research Articles

325 INSULIN-LIKE GROWTH FACTOR-I CONCENTRATIONS IN OVIDUCT FLUID OF SUPEROVULATED EWES DURING THE PERI-OVULAR PERIOD

This study examined the concentrations of insulin-like growth factor-1 (IGF-1) in oviduct fluid during the peri-ovular period as a reference for the establishment of optimal in vitro culture conditions for sheep embryos. Six mature ewes (4–5 years, 58–67 kg) of comparable body condition were fed a standard diet for two weeks before the start of fluid collection. Ewes were superovulated using conventional treatment involving a progestagen, FSH, and GnRH treatment. Oviducts were catheterized four days (which is sufficient time to recover from surgery) before collection of oviductal fluid, which started one day (Day 1) before the time of ovulation (Day 0) and continued until five days later (Day 5). Oviductal fluid was acidified by diluting into 0.8 M acetic acid/0.2 M trimethylamine, pH 2.8, mixed, and incubated to dissociate IGFs from IGF-binding proteins (IGFBPs). Following incubation, acidified fluid was centrifuged at 10,000g through a 0.1-mm Micro-spin centrifuge filter; the filtrate transferred to glass high-performance liquid chromatography (HPLC) vials. IGFs and IGFBPs were separated from one another by high-performance size-exclusion liquid chromatography using a Protein-Pak 125 column (Waters Corporation, Milford, MA, USA) and 0.2 M acetic acid, 0.05 M trimethylamine, pH 2.8, at a flow rate of 1 mL/min. Oviductal fluid IGF-I was collected in a single 2-mL fraction directly from the HPLC and its concentration measured by an IGF-I-specific enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Inc. Webster, TX, USA). The data were analyzed by analysis of variance. The non-superovulation group had significantly higher concentrations of oviductal IGF-I compared with the superovulation group. In the superovulated group, there was, however, a significant effect of day on the oviductal fluid IGF-I concentration (P < 0.01) such that the concentrations of IGF-I first increased for three days and then decreased for the remaining four days. In the non-superovulation group, there was no significant two-way interaction between ovulation and day. It can be concluded that the levels of IGF-I increase over time and then decrease. Authors express thanks to the help of Jenn Skye and Hemish Turretfield Research Station SA.

Effect of superovulation on the expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) in murine endometrium

Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum.Design: Laboratory research.Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos.Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups.Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium.Supported by: None. Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum. Design: Laboratory research. Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos. Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups. Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium. Supported by: None.

Ultrasonic survey of follicular development following superovulation with a single application of pFSH, eCG or hMG in goats

During the breeding season, 42 adult German Improved Fawn nanny goats were superovulated with a single administration of pFSH, hMG or eCG at the end or a single dose of pFSH 36 h before the end of estrus synchronization. Development of follicles and corpora lutea were observed by ultrasonic scanning of the ovaries every 8 h from gonadotrophin treatment, until the end of estrus and once daily for the following 6 days. Differences in follicular dynamics could be realized in the four superovulation groups, and the duration of the stimulatory action following the single gonadotrophin challenge was estimated to last for 40–72 h in the case of pFSH and to exceed 72 h in the case of hMG and eCG treatment groups. Corpora lutea could first be detected 3 days after estrus, whereas an exact count was not possible until day 6. The ovulation rates were satisfactory, suggesting that a single injection of pFSH or hMG provides an adequate stimulus to induce a superovulatory reaction. Premature regression of corpora lutea could not be identified ultrasonographically at these early stages of the luteal phase. However, ultrasonography is a suitable method to follow follicular dynamics after superovulation and to estimate the superovulatory response in goats.

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 covenient 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-17β (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 application 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.

Effect of oxytocin in addition to PGF 2a injection on ovarian response, embryo recovery and progesterone profile in superovulated goats pretreated with hCG

The effect of oxytocin in addition to an prostaglandin (PGF 2a) injection, given on day 12 of the oestrous cycle (at the time of fifth and sixth FSH injection) in goats pretreated with human chorionic gonadotrophin (hCG) on day 7 of the oestrous cycle was studied. Sixteen cyclic local nondescript goats were randomly allocated to control (without oxytocin, n=8) and treatment (oxytocin, n=8) superovulation groups. All goats in the control group received a total of 20 NIH FSH S 1 units divided into eight doses (4/4, 3/3, 2/2 and 1/1 units) over 4 days (day 10 to 13 of the cycle) and 10 mg dinoprost tromethamine (PGF 2a) on day 12. Goats in the treatment group in addition also received 500 IU hCG on day 7 of the oestrous cycle and 50 IU oxytocin intramuscularly with the fifth and sixth FSH injection on day 3 of the superovulatory treatment. Goats in both the groups were administered 500 IU hCG, 10–16 h after the onset of oestrus and naturally serviced at 12 hourly intervals till the end of the oestrus. Laparotomies were performed on all goats on day 6 for assessment of ovarian response and embryo recovery. Blood samples were taken on day 7, (before hCG or any pretreatment), day 10 (before beginning of FSH administration), day 12 (before PGF 2a administration), day 0 (day of superovulatory oestrus) and day 6 (day of flushing) for the estimation of the progesterone. There was a significant decrease in the number of corpora lutea/donor in the treatment group compared to the control group (0.8±0.3 vs 5.1±0.9; P<0.01). The number of white avascular corpora lutea were significantly more in treatment group than in control group (5.6±2.2 vs 1.5±1.5; P<0.05). Similarly, the number of unovulated large follicles were also more in the treatment group (5.5±1.2 vs 2.0±0.9). The total number of embryos recovered, significantly decreased in treatment group compared to the control group (0.7±0.3 vs 2.8±0.5; P<0.05). Plasma progesterone levels on the day of oestrus in treatment and control group was almost similar (0.6±0.1 vs 0.6±0.1 ng/ml). Plasma progesterone levels on other corresponding days in both groups did not vary significantly. The injection of oxytocin in addition to PGF 2a in goats pretreated with hCG could not increase ovarian response in terms of the number of normal functional corpora lutea and number of embryos recovered.

Ovulation Time in Superovulated Ewes during the Non-Breeding Season.

The present study was undertaken to evaluate ovulation time after superovulation treatment in ewes during the non-breeding season. Ewes (n=17) were treated with vaginal sponges impregnated with fluorogesterone acetate (FGA sponge) for 12 days. To the superovulation group (n=9), an intramuscular injection of 20 mg porcine follicle stimulating hormone (pFSH) was given at 48 h before sponge removal, and 500 IU equine chorionic gonadotropin (eCG) was given 24 h later after the pFSH injection. Ewes in the control group (n=8), were given 500 IU eCG alone 24 h before sponge removal. The time of ovulation was assessed by repeated observation of the ovaries in both groups of ewes using a laparoscope. Observations were commenced 24 h after sponge removal and were conducted every 6 h for a total of 60 h. The intervals between sponge removal and the onset of estrus, and ovulations were shorter in the superovulation group than the control group (P<0.02). There were significant differences in the ovulation rate between superovulated and control ewes. In 9 superovulated ewes, 12%, 53% and 27% of the total 99 ovulations occurred within 0-24 h, 36-42 h and 42-48 h after sponge removal, respectively. The present study indicates that a single pFSH injection combined with 500 IU eCG induces a high synchrony (80%) of ovulation at 36-48 h after sponge removal in the ewes treated during the non-breeding season.

The period of ovulation and presence of the first polar body of ova ovulated in the house musk shrew (Suncus murinus).

The period of ovulation in mature house musk shrews was examined in a natural mating group and a superovulation group treated with gonadotropin. In the natural mating group, ovulation started 14 hr after mating in 3 of the 7 house musk shrews (42.8%), and occurred in all 5 house musk shrews by 15 hr after mating. In the superovulation group, ovulation started 13 hr after the administration of hCG in 3 of the 5 house musk shrews, and was observed in all 5 shrews by 16 hr after the administration. In the natural mating group, ovulated ova were collected from the ovarian bursa of 14 house musk shrews 14-20 hr after mating (mean, 2.2 +/- 1.0 ova) and from the oviduct of 42 animals 14-24 hr after mating (mean, 3.6 +/- 1.8 ova). Among the ova ovulated 14-16 hr after mating, both mature ova with the first polar body and immature ova without the first polar body were observed. In the superovulation group, ovulated ova were collected from the ovarian bursa of 31 house musk shrews 13-22 hr after the administration of hCG (mean, 9.7 +/- 6.8 ova), and from the oviduct of 28 animals 13-24 hr after the administration of hCG (mean, 20.0 +/- 11.7 ova). There were also mature and immature ova in the ova ovulated 13-16 hr after the administration of hCG. The time when ova ceased to be recovered from the ovarian bursa roughly coincided with the time when new corpora lutea ceased to be found in the ovaries. These findings suggested that the period of ovulation of house musk shrews was 14-20 hr after mating in the natural mating group and 13-22 hr after the administration of hCG in the superovulation group. Both the natural mating group and superovulation group ovulated both mature ova with the first polar body and immature ova without the first polar body.

Use of exogenous FSH to increase the yield of oocytes collected from aged beef cows: Case reports

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted.No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.

The role for the uterine insulin-like growth factor I in early embryonic loss after superovulation in the rat

To examine possible roles of the insulin-like growth factor (IGF) system in increased early embryonic loss after superovulation. Changes in the uterine IGF system were examined in superovulated rats. Insulin-like growth factor I (IGF-I) was infused to the right uterine horns to mimic enhanced IGF-I actions after superovulation. Uterine luminal fluids were collected after IGF-I infusions and embryos were cultured with uterine luminal fluids. Steroid hormones, IGF-I, IGF binding protein (IGFBP), and IGF-I receptor levels, developmental rate, and cell numbers of embryos. Elevated IGF-I levels and suppressed IGFBP levels were found from days 1 to 3 of pregnancy after superovulation. Uterine luminal fluids of the IGF-I infusion and superovulation groups impaired embryo development in vitro. Anti-IGF-I antibody infusions after superovulation reversed detrimental effects of superovulation. Dialysis of uterine luminal fluids of the IGF-I infusion and superovulation groups before culture improved embryo development. Enhanced IGF-I actions in the uterus after superovulation may be responsible for the increase of early embryonic loss. The detrimental factor for embryo development seems a small molecule and is likely a local product of the uterus in which IGF-I actions are enhanced.

Superovulation in Rubia Gallega cows with a single subcutaneous injection of FSH

The effect of oxytocin in addition to an prostaglandin (PGF2a) injection, given on day 12 of the oestrous cycle (at the time of fifth and sixth FSH injection) in goats pretreated with human chorionic gonadotrophin (hCG) on day 7 of the oestrous cycle was studied. Sixteen cyclic local nondescript goats were randomly allocated to control (without oxytocin, n=8) and treatment (oxytocin, n=8) superovulation groups. All goats in the control group received a total of 20 NIH FSH S1 units divided into eight doses (4/4, 3/3, 2/2 and 1/1 units) over 4 days (day 10 to 13 of the cycle) and 10 mg dinoprost tromethamine (PGF2a) on day 12. Goats in the treatment group in addition also received 500 IU hCG on day 7 of the oestrous cycle and 50 IU oxytocin intramuscularly with the fifth and sixth FSH injection on day 3 of the superovulatory treatment. Goats in both the groups were administered 500 IU hCG, 10–16 h after the onset of oestrus and naturally serviced at 12 hourly intervals till the end of the oestrus. Laparotomies were performed on all goats on day 6 for assessment of ovarian response and embryo recovery. Blood samples were taken on day 7, (before hCG or any pretreatment), day 10 (before beginning of FSH administration), day 12 (before PGF2a administration), day 0 (day of superovulatory oestrus) and day 6 (day of flushing) for the estimation of the progesterone. There was a significant decrease in the number of corpora lutea/donor in the treatment group compared to the control group (0.8±0.3 vs 5.1±0.9; P<0.01). The number of white avascular corpora lutea were significantly more in treatment group than in control group (5.6±2.2 vs 1.5±1.5; P<0.05). Similarly, the number of unovulated large follicles were also more in the treatment group (5.5±1.2 vs 2.0±0.9). The total number of embryos recovered, significantly decreased in treatment group compared to the control group (0.7±0.3 vs 2.8±0.5; P<0.05). Plasma progesterone levels on the day of oestrus in treatment and control group was almost similar (0.6±0.1 vs 0.6±0.1 ng/ml). Plasma progesterone levels on other corresponding days in both groups did not vary significantly. The injection of oxytocin in addition to PGF2a in goats pretreated with hCG could not increase ovarian response in terms of the number of normal functional corpora lutea and number of embryos recovered.

Alpha-adrenergic agonists and aqueous humor dynamics

The effect of oxytocin in addition to an prostaglandin (PGF2a) injection, given on day 12 of the oestrous cycle (at the time of fifth and sixth FSH injection) in goats pretreated with human chorionic gonadotrophin (hCG) on day 7 of the oestrous cycle was studied. Sixteen cyclic local nondescript goats were randomly allocated to control (without oxytocin, n=8) and treatment (oxytocin, n=8) superovulation groups. All goats in the control group received a total of 20 NIH FSH S1 units divided into eight doses (4/4, 3/3, 2/2 and 1/1 units) over 4 days (day 10 to 13 of the cycle) and 10 mg dinoprost tromethamine (PGF2a) on day 12. Goats in the treatment group in addition also received 500 IU hCG on day 7 of the oestrous cycle and 50 IU oxytocin intramuscularly with the fifth and sixth FSH injection on day 3 of the superovulatory treatment. Goats in both the groups were administered 500 IU hCG, 10–16 h after the onset of oestrus and naturally serviced at 12 hourly intervals till the end of the oestrus. Laparotomies were performed on all goats on day 6 for assessment of ovarian response and embryo recovery. Blood samples were taken on day 7, (before hCG or any pretreatment), day 10 (before beginning of FSH administration), day 12 (before PGF2a administration), day 0 (day of superovulatory oestrus) and day 6 (day of flushing) for the estimation of the progesterone. There was a significant decrease in the number of corpora lutea/donor in the treatment group compared to the control group (0.8±0.3 vs 5.1±0.9; P<0.01). The number of white avascular corpora lutea were significantly more in treatment group than in control group (5.6±2.2 vs 1.5±1.5; P<0.05). Similarly, the number of unovulated large follicles were also more in the treatment group (5.5±1.2 vs 2.0±0.9). The total number of embryos recovered, significantly decreased in treatment group compared to the control group (0.7±0.3 vs 2.8±0.5; P<0.05). Plasma progesterone levels on the day of oestrus in treatment and control group was almost similar (0.6±0.1 vs 0.6±0.1 ng/ml). Plasma progesterone levels on other corresponding days in both groups did not vary significantly. The injection of oxytocin in addition to PGF2a in goats pretreated with hCG could not increase ovarian response in terms of the number of normal functional corpora lutea and number of embryos recovered.

In vitro developmental ability of sheep nuclear transplant embryos

Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be covenient 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-17β (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 application 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.

Embryo production by SUPER-OV ® and FSH-P ®

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted.No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.

Improved embryo yield and condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted. No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.

Superovulatory response in merino ewes to three PMSG dose levels

The effect of oxytocin in addition to an prostaglandin (PGF2a) injection, given on day 12 of the oestrous cycle (at the time of fifth and sixth FSH injection) in goats pretreated with human chorionic gonadotrophin (hCG) on day 7 of the oestrous cycle was studied. Sixteen cyclic local nondescript goats were randomly allocated to control (without oxytocin, n=8) and treatment (oxytocin, n=8) superovulation groups. All goats in the control group received a total of 20 NIH FSH S1 units divided into eight doses (4/4, 3/3, 2/2 and 1/1 units) over 4 days (day 10 to 13 of the cycle) and 10 mg dinoprost tromethamine (PGF2a) on day 12. Goats in the treatment group in addition also received 500 IU hCG on day 7 of the oestrous cycle and 50 IU oxytocin intramuscularly with the fifth and sixth FSH injection on day 3 of the superovulatory treatment. Goats in both the groups were administered 500 IU hCG, 10–16 h after the onset of oestrus and naturally serviced at 12 hourly intervals till the end of the oestrus. Laparotomies were performed on all goats on day 6 for assessment of ovarian response and embryo recovery. Blood samples were taken on day 7, (before hCG or any pretreatment), day 10 (before beginning of FSH administration), day 12 (before PGF2a administration), day 0 (day of superovulatory oestrus) and day 6 (day of flushing) for the estimation of the progesterone. There was a significant decrease in the number of corpora lutea/donor in the treatment group compared to the control group (0.8±0.3 vs 5.1±0.9; P<0.01). The number of white avascular corpora lutea were significantly more in treatment group than in control group (5.6±2.2 vs 1.5±1.5; P<0.05). Similarly, the number of unovulated large follicles were also more in the treatment group (5.5±1.2 vs 2.0±0.9). The total number of embryos recovered, significantly decreased in treatment group compared to the control group (0.7±0.3 vs 2.8±0.5; P<0.05). Plasma progesterone levels on the day of oestrus in treatment and control group was almost similar (0.6±0.1 vs 0.6±0.1 ng/ml). Plasma progesterone levels on other corresponding days in both groups did not vary significantly. The injection of oxytocin in addition to PGF2a in goats pretreated with hCG could not increase ovarian response in terms of the number of normal functional corpora lutea and number of embryos recovered.

Viability of mouse oocytes ovulated within 14 hours of an injection of pregnant mares' serum gonadotrophin.

The viability and development capacity of oocytes ovulated within 13-14 hours after a single injection of pregnant mares serum gonadotrophin (PMSG) were examined and compared with those of oocytes obtained by superovulation. By means of fertilization in vitro embryo culture and transfer of oocytes to foster mothers viable fetuses were obtained. Ip injection of 10 IU PMSG was given to adult F1 hybrid mice (C57B1 x A2G). Oocytes were recovered 13-14 hours later. A second group of mice received an ip injection of 10 IU HCG and oocytes recovered following superovulation 13-14 hours later. This second group had received the PMSG 10 IU 48 hours before receiving the HCG. Spermatozoa were collected from CFLP strain mice and incubated with ova for 5 hours at 37 degrees C in a culture medium in an atmosphere of 5% carbon dioxide in air. After washing with a change of medium to remove excess spermatozoa ova were cultured in a similar medium for 19-20 hours. The number of 2-cell ova was then recorded. In 3 such experiments 72 of 122 oocytes (58.2%) from groups receiving PMSG only developed through the first cleavage division compared with 127 of 277 (45.9%) of the superovulation group. The 2-cell embryos were further cultured for 72 hours after which time 35.3% of the PMSG-alone group developed to the blastocyst stage. To test the viability of the oocytes some were transferred at the 2- and 4-cell stages to the oviducts of suitably prepared recipient foster mothers of the CFLP strain. After 15 days viable fetuses were found in the uterine horns of the foster mother s.