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PubMed2001

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Animals↗

PubMed2004

The use of cytological examination of vaginal smears (CEVS) in the selection of rabbits for superovulation.

In the present study, we examined two factors associated with the reproduction of rabbit does, cytology of vaginal smears and color of vulva, as potential predictors of the success of superovulation treatment. Vulval color and vaginal smear cytology were assessed in 55 young New Zealand does. Superovulation was then induced by a single administration of eCG (20 IU/kg BW i.m.). Does were artificially inseminated 3 days later, followed by intravenous administration of hCG (120 IU per animal). Does were classified with regard to vulval color (white, rose, red, purple), and the predominant cell type in the vaginal smear (i.e. parabasal, intermediate, superficial, or anuclear). Furthermore, we categorized cells into two groups because we usually observed parabasal and intermediate cells (Group A), and superficial and anuclear cell (Group B) in the same smear. Does were humanely killed 19 h after administration of hCG and the total numbers of corpora lutea (CLs), oocytes, and zygotes (i.e. 1-cell embryos) were determined. The zygotes were assessed by morphological appearance and classified as normal or abnormal. The color of the vulva at the time of eCG treatment did not predict the success of superovulation in terms of the number of CLs, oocytes or zygotes. Does with predominantly superficial cells in vaginal smears yielded significantly fewer CLs and oocytes-zygotes (OZ) compared to does with predominantly parabasal, intermediate, or anuclear cells (P<0.05). Does with predominantly superficial cells in vaginal smears tended to yield fewer normal zygotes (nZ), but this reached significance only when compared to does with predominantly parabasal cells (P<0.05). Does in Group A yielded significantly more (P<0.05) CLs, OZ and nZ compared to does in Group B. Does with predominantly parabasal and intermediate cells in vaginal smears and rose color vulva tended to yield more OZ and nZ (P<0.05). These results suggest that the cytology of vaginal smears may help identify does with a significantly higher likelihood of yielding low numbers of CLs, oocytes, or nZ.

Animals↗

PubMed1998

Superovulation of rabbits with FSH alters in vivo development of vitrified morulae.

Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation.

Animals↗

PubMed1998

The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers.

Acute decreases in nutrient intake can improve embryo quality in sheep, although reductions in ovulation rate can also occur. In cattle, short-term nutrient restriction prior to ovulation has been shown to increase subsequent pregnancy rates. Thus, the objective was to determine the effect of a severe reduction in food intake on follicle growth and embryo quantity and quality in heifers superovulated with FSH. Beef heifers (n = 61) were offered a diet of grass silage and concentrates (ratio of 5:1, on a fresh weight basis), which was adjusted to provide a predicted intake of 28.6 Mcal/kg ME/d (H) or 9.6 Mcal/kg ME/d (L). Heifers were synchronized with a progesterone-releasing device for 7 d. They were allocated to oocyte recovery (n = 16/treatment) after 3 (225 IU) or 8 (600 IU) injections of FSH given at 12-h intervals. Oocytes were matured, fertilized and cultured individually in vitro. The remaining heifers (n = 14/treatment) were superovulated using FSH (600 IU), and embryos were recovered 7 d after breeding. The embryos were morphologically graded and subsequently cultured for 24 h before differential staining to determine inner cell mass and trophectoderm cell numbers. Follicle numbers increased following 8 (16.6 +/- 2.0) compared with 3 (6.7 +/- 0.6) injections of FSH (P < 0.0001). Heifers on the L diet had more follicles than those on the H diet (13.5 +/- 2.4 vs 9.6 +/- 1.2; P < 0.06), which was predominantly due to an increase in the number of 7- to 10-mm follicles. However, this effect was only evident after 8 injections of FSH. There was no nutritional effect on cleavage rates in vitro (55.6 +/- 8.1 vs 53.8 +/- 9.0 for H vs L diets, respectively). However, cleavage rates were lower in oocytes collected after 8 than after 3 injections of FSH (31.3 vs 69.2%; P < 0.0001). There was no significant effect of nutrition on ovulation rate after FSH (14.4 +/- 1.9 vs 16.3 +/- 3.0 for H vs L diet, respectively). The number of embryos recovered was not different between heifers on H (10.4 +/- 1.3) and L (11.3 +/- 2.4) diets. Following culture for 24 h, a significantly higher proportion of embryos from heifers on the L diet developed to the blastocyst stage (72.9 vs 41.5%; P < 0.01). Total cell numbers on Day 8 were greater in embryos from heifers on the L diet (98.3 vs 75.4; P < 0.0001); yet the inner cell mass as a percentage of total cells was not different (21 vs 20%). These data indicate that low energy intake prior to and during superovulation resulted in more follicles and in improved embryo quality, as evident from the increased number of blastocysts formed and higher cell numbers.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Effect of type and quantity of concentrates on superovulation and embryo yield in beef heifers.

Embryo yield and quality can be decreased following superovulation of cattle on high levels of concentrates. Concentrate type can alter rumen fermentation patterns and so affect energy availability and thus embryo quality. The objectives of this experiment were to examine the effect of 2 levels and 2 types of concentrate on superovulatory response and embryo quality in beef heifers. Beef heifers received grass silage as a basal diet and barley at 3 kg (n = 20) or ad-libitum (n = 19), or citrus/beet pulp at 3 kg (n = 18) or ad-libitum (n = 19) as the source of concentrate supplement. Silage was available ad-libitum for heifers offered 3 kg but was restricted to 1 kg DM/day for heifers on ad-libitum concentrate intake. Both concentrates contained 14% crude protein. After 100 d, heifers were treated with an intravaginal progesterone releasing device (CIDR) for 7 d, and superovulation was initiated 60 h before CIDR withdrawal. Heifers received pFSH (a total of 265 mg NIH-FSH-P1 equivalent) administered over 8 injections at 12 h intervals with the last 2 injections administered at 12 and 24 h after CIDR withdrawal; they were inseminated at 56 and 72 h after CIDR withdrawal without reference to estrus. Heifers were slaughtered 6, 7 or 8 d after the first insemination, and embryos were flushed from the uterus with PBS and were graded morphologically. Statistical analyses were performed using analysis of variance. There was no interaction between level and type of diet, and thus data for the main effects are presented separately. Final live weights at the end of the experiment and carcass weights for heifers offered ad-libitum concentrate (643.8+/-6.9 kg; 354+/-8 kg, respectively) were higher (P<0.01) than those of heifers offered 3 kg concentrate (613.1+/-7.5 kg; 338.4+/-3.2 kg, respectively) while live weights and carcass weights of heifers offered barley (625.6+/-8.1 and 345.4+/-4.1 kg, respectively) or citrus/beet pulp concentrate (631.4+/-7.0 and 348.0+/-3.1 kg, respectively) were not different (P>0.05). Superovulatory responses (number of corpora lutea at slaughter) was greater (P<0.06) when heifers were offered 3 kg (15.5+/-1.6) than when offered ad-libitum concentrates (12.3+/-1.4). However, the superovulatory response for both citrus/beet pulp (14.4+/-1.5) and barley (13.4+/-1.5) diets were not different (P>0.05). Heifers offered 3 kg concentrates produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) compared with heifers fed ad-libitum concentrates (2.8+/-0.4). Heifers offered citrus/beet pulp produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) than heifers offered barley (2.9+/-0.5). These data indicate that high concentrate intake has a negative effect on embryo yield and quality and that a barley compared with citrus/beet pulp based concentrate has a negative effect on embryo quality following superovulation in beef heifers.

Animal Feed↗

PubMed1999

In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs.

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.

Animals↗

PubMed1999

Bull-specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis).

This study was conducted to compare fertilization rate and viable embryo recovery rate in superovulated buffalo (n = 64) following insemination with semen from buffalo bulls (n = 5) having different fertility rates as determined by AI. Frozen-thawed semen from fertile bulls with similar post-thaw progressive motility and sperm morphology was used to inseminate buffalo at superovulatory estrus. Fertilization and viable embryo recovery rates differed among bulls, but this bull-specific effect was not related to the overall herd fertility rate as determined by AI in normal cyclic animals. These results indicate that individual bulls differ in their contribution to fertilization of superovulated donors and also to embryonic development, as determined by viable embryo recovery. Moreover, the results also suggest that buffalo bulls can be screened for optimal fertility and embryo recovery rates in superovulated donors. Further studies are warranted to ascertain the factors which contribute to such bull-specific effects.

Animals↗

PubMed2000

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination.

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.

Animals↗

PubMed2001

Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment.

Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF(2alpha) (Cloprostenol, 50 microg, 48h before sponge removal - s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH-PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH-PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P>0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) and four doses (from 24h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH-PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P <0.01) compared to the pFSH-PVP treatment carried out i.m. 48 h before s.r. These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight.

Animals↗

PubMed2002

Use of steroid hormone treatments prior to superovulation in Nelore donors.

The objective of this study was to evaluate the effectiveness of synchronization of follicular wave emergence using steroid hormone treatments in Nelore cows. Donors were placed into three groups. Those that were between days 9 and 12 of their cycle (estrus=day 0) formed the TI group (n=60), whilst those that were in any other stages of their estrus cycle constituted groups TII (n=60) and TIII (n=60). TI donors were submitted to a standard protocol of superovulation, however, TII and TIII donors were treated with the Syncro-Mate-B (SMB) or Controlled Internal Drug Releasing Device (CIDR-B) programs, respectively. Superovulation was induced with p-FSH, divided into eight decreasing doses at intervals of 12h. The donors received cloprostenol 48h after the beginning of the treatment and progestagens were removed 12h later. Artificial inseminations (AI) were done at 12 and 22h after the initiation of estrus and the embryo collections were done 7 days after AI. In the donors which displayed behavioral estrus, mean (+/-S.E.M.) total ova and viable (transferable) embryos were 15.8+/-1.4 and 8.3+/-1.0 (TI, n=56); 15.6+/-1.3 and 8.9+/-1.0 (TII, n=56); 17.3+/-1.0 and 9.9+/-0.9 (TIII, n=57), respectively, with no significant difference (P > or =0.05) among groups. In those animals that did not displayed behavioral estrus, the mean values of total ova and viable embryos were 3.5+/-1.6 and 0.7+/-0.5 (TI, n=4); 11.5+/-3.9 and 9.0+/-4.4 (TII, n=4); 8.7+/-5.0 and 5.0+/-2.9 (TIII, n=3), respectively, with no significant differences (P > or =0.05) among groups. Pregnancy rates of 62.2% (TI, n=235); 66.4% (TII, n=284) and 65.1% (TIII, n=244) were obtained with embryos transferred from these collections and did not differ significantly (P > or =0.05) among groups. It was concluded that the synchronization of the emergence of follicular waves in Nelore donors is usable and does not harm the efficiency of embryo transfer programs. In addition, in contrast to the standard superovulation protocol, this method permits the use of a large number of donors in a short time period, at any stage of the estrus cycle, minimizing the costs of embryo transfer.

Animals↗

PubMed2003

The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle.

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.

Administration, Intravaginal↗

PubMed1999

Superovulation induction in Japanese Black cattle by a single intramuscular injection of hMG or FSH dissolved in polyvinylpyrrolidone.

The efficacy of a single intramuscular dose of 450 or 600 international units (IU) of human menopausal gonadotropin (hMG) or 30 mg of follicle stimulating hormone (FSH), each dissolved in 30% polyvinylpyrrolidone K-30 (PVP), for superovulation treatment was compared to that of superovulation induction by administration of a total dose of 600 IU hMG given in declining doses twice daily over a 3-day period. A total of 48 Japanese Black cows were used for the investigation. Oestrus was observed within 60 h after PGF2alpha administration in all cows in the hMG groups. In the hMG group that received a single dose of 600 IU hMG (n = 12), oestrus was observed less than 36 h after treatment in one cow. In contrast, oestrus was not observed in 3 of the 12 cows (25%) in the FSH group. Neither the average number of recovered ova/embryos nor the number of transferable embryos per collection differed significantly among the hMG groups. However, the average number of transferable embryos was not significantly higher in cows treated with a single dose of 600 IU of hMG than in cows treated with a single 30 mg dose of FSH (7.5+/-4.5 vs. 2.1+/-2.8). The number of cows from which more than three excellent grade embryos were collected was highest in the group that received a single dose of 600 IU hMG (9/12, 75%) and lowest in the group that received a single 30 mg dose of FSH (2/9, 22%). The differences between groups in the percentages of cows with three or more excellent embryos between treatments were not statistically significant. The proportion of recovered ova/embryos classified as excellent was highest in the group that received 600 IU hMG in declining doses and lowest in the group that received a single 30 mg dose of FSH (55.2% vs. 30.2%; P < 0.05). The recovery rate of unfertilized ova was lowest in the group that received a single dose of 600 IU hMG and highest in the group received a single 30 mg dose of FSH (18.3% vs. 48.8%; P < 0.05). Although the differences in recovery results between the groups were not statistically significant, the recovery rates in hMG groups were higher than that the FSH group. These findings suggest that superovulation can be induced adequately in Japanese Black cows using one injection of 450 to 600 IU hMG dissolved in PVP.

Animals↗

PubMed2001

Superovulation strategies for assisted reproductive technologies.

Assisted reproductive technologies (ART) pregnancy rates improve with the replacement of multiple embryos. The specific number of embryos to be transferred (typically between two and four) is based on a patient's age and past history and a morphologic assessment of embryo quality. Superovulation, with the goal of multifollicular recruitment and the harvest of multiple preovulatory oocytes, is therefore an integral aspect of in vitro fertilization and related techniques. Given the considerable interindividual variability in response to superovulation, the selection of an appropriate ovarian stimulation protocol is critical both for the safety and success of ART. Specific superovulation strategies have therefore been developed for treatment of abnormal response patients as well as for patients expected to manifest a "normal" response to ovulation inducing agents.

Age Factors↗

PubMed1980

Triacylglycerols and glycerophospholipids in ovaries from maturing and superovulated immature rats.

The concentration and composition of triacylglycerols and glycerophospholipids in the rat ovary were measured during the development of follicles to corpora lutea. Changes occurring during normal development, as the rats aged from 20 to 41 days, were compared with those following treatment of 20-day-old rats with gonadotrophin to stimulate superovulation. In the ovaries of immature superovulated rats, where many follicles developed into corpora lutea in synchrony, the glycerophospholipids increased in concentration throughout growth and luteinization. At the same time the total amount of triacylglycerol rose 10-fold but only increased in concentration during the 24 h after administration of pregnant mare's serum gonadotrophin. In both classes of lipid the proportion of polyunsaturated fatty acids rose throughout. Six days after administration of gonadotrophin 20% of the fatty acid in glycerophospholipid was arachidonic acid (20:4), while the amount of 22:5 acid had risen from 3 to 17% in the triacylglycerols. During normal aging, changes in the developing follicles were masked by the number of other cell types present. In the whole ovary the glycerophospholipids did not change between 20 and 41 days, but the triacylglycerol concentration rose. However, there was a marked similarity between the concentrations of both lipid classes in corpora lutea dissected out of the 41-day-old ovary and those of the superovulated rats. The changes in proportion of the polyunsaturated fatty acids were also the same during normal and stimulated development of the corpora lutea. In the day-41 corpus luteum the phospholipid contained 22% arachidonic acid (20:4), and 20% of the fatty acids in the triacylglycerol was 22:5. The changes in composition of fatty acids suggests that when a follicle begins to develop there is transfer of polyunsaturated fatty acids from cholesterol esters in the interstitial tissue to the triacylglycerols.

Animals↗

PubMed1982

Superovulation in mice in relation to their age.

Superovulation in mice is most likely to be successful if they are treated at the age of 20-32 days. Both the number of ovulations and the quality of the embryos are significantly better in comparison with superovulated older mice (32-60 days). In this investigation the production of normally developed embryos in young mice of 20-32 days old was 28.7 per superovulated animal and 11.3 or 14.0 in the groups of older mice respectively (32-46 days and 46-60 days).

Age Factors↗

PubMed2001

Superovulation of female mice delays embryonic and fetal development.

Mouse and human embryos, cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded. The objective of this study was to test the hypothesis that both in vitro and in vivo, preimplantation embryonic development and postimplantation fetal development are impaired in superovulated female mice when compared with naturally cycling controls. A delay in in-vitro blastocyst hatching and in-vivo blastocyst formation (P < 0.03 and P < 0.0001 respectively) and a 40% fetal growth retardation (P < 0.0001) were observed after superovulation in comparison with naturally cycling controls. After transfer to non-stimulated foster mothers, blastocysts from stimulated females had a lower implantation rate (P < 0.005), and developed into fewer living fetuses (P < 0.02), more resorption sites (P < 0.02) and had more pronounced growth retardation (P < 0.0001) when compared with blastocysts from naturally cycling controls. In conclusion, superovulation in the mouse causes a delayed embryonic development in vitro and in vivo, an increased abnormal blastocyst formation, a pronounced fetal growth retardation, and an increased number of resorption sites. If this observation in mice can be extrapolated to humans, it may offer an explanation for the delay in embryonic development and the low birth weight observed after IVF.

Animals↗

PubMed2004

A new alternative method for superovulation using passive immunization against inhibin in adult rats.

The present study was undertaken to investigate the effects of passive immunoneutralization of endogenous inhibin on ovulation rate and embryo development in vivo and in vitro to establish a new alternative superovulation method in the adult rat. Female adult rats of Wistar strain were superovulated with a single injection of inhibin antiserum (inhibin-AS; 100 or 400 microl) or an injection of 20 IU eCG followed by an injection of 10 IU hCG. Untreated animals served as controls. Embryos were collected from oviducts or uteri on Days 1-5 of pregnancy, and the number of embryos and implantation sites were observed. On Day 1 of pregnancy, the two-cell-stage embryos were cultured and embryos from the 100-microl inhibin-AS group and the control group were transferred to recipient females to determine developmental competence. There were no significant differences between groups in fertilization rate. The numbers of normal embryos in the inhibin-AS-treated groups were significantly higher than the control and the eCG-hCG-treated groups throughout Days 1-4 of pregnancy. The number of implantation sites observed on Day 5 of pregnancy in the inhibin-AS-treated groups was significantly higher than both the control and the eCG-hCG-treated groups. Furthermore, the rate of blastocyst development in vitro in the inhibin-AS-treated groups and posttransfer viability in the 100-microl-inhibin-AS group were comparable with those of the control group. These results indicate that immunoneutralization of endogenous inhibin is a new practical alternative for induction of superovulation as a substitution for eCG-hCG method in the adult rat.

Animals↗

PubMed1985

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation.

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.

Androstenedione↗