Historical perspectives and recent research on superovulation in cattle

Superovulation protocols have evolved greatly over the past 40 to 50 years. The development of commercial pituitary extracts and prostaglandins in the 1970s, and partially purified pituitary extracts and progesterone-releasing devices in the 1980s and 1990s have provided for the development of many of the protocols that we use today. Furthermore, the knowledge of follicular wave dynamics through the use of real-time ultrasonography and the development of the means by which follicular wave emergence can be controlled have provided new practical approaches. Although some embryo transfer practitioners still initiate superstimulatory treatments during mid-cycle in donor cows, the elective control of follicular wave emergence and ovulation has had a great effect on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. The most common treatment for the synchronization of follicular wave emergence for many years has been estradiol and progestins. In countries where estradiol cannot be used, practitioners have turned to alternative treatments for the synchronization of follicle wave emergence, such as mechanical follicle ablation or the administration of GnRH to induce ovulation. An approach that has shown promise is to initiate FSH treatments at the time of the emergence of the new follicular wave after GnRH-induced ovulation of an induced persistent follicle. Alternatively, it has been suggested recently that it might be possible to ignore follicular wave status, and by extending the treatment protocol, induce small antral follicles to grow and superovulate. Recently, the mixing of FSH with sustained release polymers or the development of long-acting recombinant FSH products have permitted superstimulation with a single or alternatively, two gonadotropin treatments 48 hours apart, reducing the need for animal handling during superstimulation. Although the number of transferable embryos per donor cow superstimulated has not increased, the protocols that are used today have increased the numbers of transferable embryos recovered per unit time and have facilitated the application of on-farm embryo transfer programs. They are practical, easy to administer by farm personnel, and more importantly, they eliminate the need for detecting estrus.     

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers.Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH.In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.     

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF(2α) was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means±standard error. The FSH/eCG group had higher superstimulatory (33.8±3.9 compared to 23.8±2.6 follicles; P=0.03) and SOV (16.8±2.9 compared to 10.8±2.1 CL; P=0.10) responses. Although the number of total ova/embryos was not different between groups (8.2±1.8 compared to 5.9±1.4 for FSH/eCG and FSH groups, respectively; P=0.25), the number (5.8±1.3 compared to 2.6±0.7; P=0.02) and percentage (75.6±5.7 compared to 53.2±9.7%; P=0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.     

Induction of superovulation in South American camelids

The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular growth.     

Lengthening the superstimulatory treatment protocol increases ovarian response and number of transferable embryos in beef cows

This study determined if lengthening the superstimulation protocol from 4 to 7 days would result in an increase in the superovulatory response with no adverse effects on oocyte/embryo competence in beef cows. Follicular ablation was performed, a progesterone-releasing intravaginal device (PRID) was inserted, and cows were assigned to one of two treatment groups 5 to 8 days after ovulation: Control (4 days of follicle stimulating hormone (FSH)) or Long (7 days of FSH; n = 12 per group). The FSH treatments were initiated 1.5 days later (Day 0). A dose of 400 mg NIH-FSH-P1 (Folltropin-V) was distributed equally over 8 (Control) or 14 (Long) im injections at 12-h intervals. Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 2 (Control) or Day 5 (Long), and PRID were removed 12 h after the second PGF. Both groups were given 25 mg pLH (lutropin-V) im 24 h after PRID removal and AI was done 12 and 24 h later. Ova/embryos were collected 7 days after the pLH injection. The mean (± SEM) number of ≥ 9 mm follicles at the time of first AI did not differ (P = 0.24) between groups, but more ovulations (30.9 ± 3.9 vs. 18.3 ± 2.9, P = 0.01) and CL (27.2 ± 2.1 vs. 20.8 ± 2.2, P = 0.04) occurred in the Long group. A higher proportion of the ≥ 9 mm follicles ovulated between 12 and 36 h after pLH in the Long group (93 vs. 69%; P = 0.001). Although numerically higher in the Long group, mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos did not differ. In conclusion, a lengthened superstimulatory treatment protocol resulted in more follicles acquiring the capacity to ovulate with an increased number of ovulations, and without a decrease in oocyte/embryo competence.     

Progesterone treatment, FSH plus eCG, GnRH administration, and Day 0 Protocol for MOET programs in sheep

The effect of various superstimulatory treatments on the number of corpora lutea, fertilization rate, and embryo yield was studied in sheep. Overall, data from 708 Merino donors and 4262 embryos were analyzed in four experiments. In Experiment 1, varying intervals of progesterone treatment (5 to 14 d) before follicle-stimulating hormone (FSH) administration did not significantly affect the proportion of responding donors, the mean number of corpora lutea, or the mean number of recovered and transferable embryos per donor. In Experiment 2, a single dose of equine chorionic gonadotropin (eCG, 200 or 300 IU) combined with the FSH treatment (i.e., given at CIDR removal) reduced the number and the quality of embryos compared with that for not giving eCG (P < 0.05). In Experiment 3, one dose of gonadotropin-releasing hormone (GnRH) given 24 h after CIDR removal improved the number of transferable embryos compared with that for not giving GnRH (P < 0.05). In Experiment 4, the new superstimulatory Day 0 Protocol, which includes starting FSH treatment at the emergence of Wave 1 (i.e., soon after ovulation, in the absence of a large follicle), improved ovarian response, with a tendency to produce more embryos compared with that for the Traditional Protocol. In summary, this study, analyzing data from various pharmacologic treatments, allows an improvement from four to eight transferable embryos per treated donor in multiple ovulation and embryo transfer programs in sheep.     

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.     

Number of oocytes obtained from cows by OPU in early, but not late lactation increased with plasma insulin and estradiol concentrations and expression of mRNA of the FSH receptor in granulosa cells

The effects of lactation stage and hormonal profile on the quality and quantity of oocytes and the gonadotrophic sensitivity of granulosa cells (GC) from small antral follicles obtained by sequential aspirations from ovaries of high producing dairy cows were examined. Cows in late lactation (263(+/- 60) days postpartum) and 98(+/- 16) days pregnant in positive energy balance (EB) showed no significant changes in plasma concentrations of estradiol, progesterone, insulin, IGF1 or expression of mRNA of the FSH receptor in GC from small antral follicles during the 49 days experimental period. There were no changes in the number and quality of oocytes obtained from each aspiration. In cows in early lactation (72.8 +/- 6 days postpartum), plasma insulin concentrations increased and were positively correlated with plasma estradiol concentration. Due to the sequential aspirations progesterone blood concentrations were low in early lactation cows. Expression of mRNA of the FSH receptor increased in GC from small antral follicles of early lactation cows together with the number of oocytes obtained with aspiration sessions. No differences were found in morphological quality or function between oocytes obtained from small antral follicles from cows in early or late lactation. In early, but not late lactation, the number of oocytes was correlated with both insulin and E2 plasma concentrations. Improved EB and sensitivity of GC to FSH may be involved in oocyte recruitment in early lactation.     

Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in vitro

The aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.     

Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers

One of the primary limiting factors to superovulation and embryo transfer in cattle has been the large variability in response, both between and within animals. It appears that the primary source of this problem is the variability in the population of gonadotropin-responsive follicles present in ovaries at the time of stimulation. We have shown that treatment of heifers with recombinant bovine somatotropin (rbGH) increases the number of small antral follicles (2 to 5 mm) and, therefore, enhances the subsequent superovulatory response to eCG. To investigate further the potential of using this approach to improve superovulatory regimens in cattle, the effect of rbGH pretreatment on the response to pituitary FSH was studied. The estrous cycles of 16 heifers were synchronized using PGF2alpha. On Day 7 of the synchronized cycle, half of the animals were injected with 320 mg sustained-release formulated rbGH, while the other half received 10 ml saline. Five days later, all heifers were given a decreasing-dose regimen of twice daily injections of oFSH for 4 d, incorporating an injection of PGF2alpha with the fifth FSH treatment, to induce superovulation. All animals were artificially inseminated twice with semen from the same bull during estrus. Ova/embryos were recovered nonsurgically on Days 6 to 8 of the following estrous cycle, and the ovulation rate assessed on Day 9 by laparoscopy. Using the same animals as described above, the experiment was repeated twice, 3 and 6 mo later, with no laparoscopy in the third experiment. The animals were randomized both between experiments and for the day of ova/embryo collection. Pretreatment of heifers with rbGH significantly (P < 0.01) increased the number of ovulations, total number of ova/embryos recovered and the number of transferable embryos. The percentage of transferable embryos was significantly (P < 0.05) increased by rbGH pretreatment. In addition, the incidence (2/16) of follicular cysts with a poor ovulatory response (< 6 ovulations) for the rbGH-pretreated heifers was significantly lower (P < 0.05) when compared with the incidence (7/16) in the control animals. It is concluded that pretreatment with rbGH may provide a useful approach for improving superovulatory response in cattle.     

The day of the estrous cycle that FSH is started and superovulation in cattle

A total of 993 commercial donor cows were superovulated with 28 mg FSH-P. The first injection of FSH-P was administered on one of days 9 through 13 of the donor's estrous cycle. The day that FSH was started did not affect total embryos collected, the number of transferable embryos or the percent transferable. These results did not support the current hypothesis that because the population of antral and preantral follicles in the ovaries on days 9 and 10 is higher, best embryo production should be achieved by starting donor cows on day 9 or 10 of the estrous cycle.     

Measurement of inhibin A and follicular status predict the response of ewes to superovulatory FSH treatments

Variability in superovulatory response to FSH stimulation is common to most mammals and imposes practical problems for assisted reproduction. In sheep, we have studied if this response is related to the ovarian follicular population and activity before the stimulation. During the breeding season, 30 ewes were treated with 40 mg FGA sponges for 14 days and 125 microg cloprostenol injection on Day 12, considering Day 0 as the day of progestagen insertion. Superovulatory response was induced with two different FSH regimes using the same total dose (8.8 mg), administered twice daily from 60 h before to 24 h after progestagen withdrawal. At the first FSH injection, all follicles > or = 2 mm were observed by transrectal ultrasonography and plasma FSH and inhibin A levels were determined. The number of corpora lutea and the number of and viability of recovered embryos in response to the treatment were determined on Day 7 after sponge withdrawal. No significant differences were found between treatments. The total mean number of corpora lutea (11.5 +/- 1.2) and recovered embryos (7.9 +/- 1.1) were positively correlated (P < 0.05 and <0.01, respectively) with the number of small antral follicles (2-3 mm: 9.2 +/- 0.7) and inhibin A concentration (240 +/- 18 pg/ml; P < 0.05 for corpora lutea and P < 0.005 for recovered embryos) observed at the onset of the superovulatory treatment, which was also positively correlated with the number of viable embryos (5.8 +/- 0.9, P < 0.005). In 18 ewes with follicles > or = 6 mm prior to FSH treatment, the ovulation rate was unaffected but the number of embryos (6.1 +/- 0.9 versus 11.6 +/- 2; P < 0.05) and their viability (4.5 +/- 0.8 versus 8.5 +/- 2; P < 0.05) was reduced. The lower number of embryos produced when a large follicle is present suggest that a proportion of the smaller follicles are in early stages of atresia and the developmental competence of their oocyte is compromised.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.     

Superovulation and embryo transfer in wood bison (Bison bison athabascae)

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.