Search

Induced ovulation maximizes captive breeding success, increasing productivity and facilitating the contribution of otherwise infertile animals to the genetic pool. In marsupials, induced ovulation to produce fertile young is unknown. Here we present an induction protocol efficient in inducing non-cycling and non-reproductive females to cycle, mate, ovulate, and conceive. Ovulation was induced in Sminthopsis macroura using an initial injection of 0.06 IU equine serum gonadotropin (eSG)/g (time 0), followed on day 4 by 0.04 IU eSG/g. Using this induction regime, the timing of follicular and embryonic development mimics natural cycles and results in the birth of viable, fertile young. Response to induction is not significantly affected by animal age, making this protocol an effective conservation tool. We have established a time-table of development following induction, providing a source of precisely timed research material. This is the first induced ovulation protocol in any marsupial to result in demonstrated fertile offspring and to allow the reliable collection of known-age samples during both the follicular phase and the gestation period.

We have previously shown the presence of immunoreactive angiotensin-(1-7) [Ang-(1-7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1-7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1-7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1-7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1-7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1-7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.

Two experiments were designed to evaluate strategies to increase fertility of Bos indicus postpubertal heifers and nonlactating cows submitted to a fixed-time artificial insemination (TAI) protocol consisting of an intravaginal device containing 1.9 g of progesterone (CIDR) insertion+estradiol benzoate on Day 0, CIDR withdrawal+estradiol cypionate on Day 9, and TAI on Day 11. In Experiment 1, heifers (n=1153) received a new or an 18-d previously used CIDR and, on Day 9, prostaglandin F(2 alpha) (PGF(2 alpha))+0, 200, or 300 IU equine chorionic gonadotropin (eCG). Heifers treated with a new CIDR had greater (least squares means+/-SEM) serum concentration of progesterone on Day 9 (3.06+/-0.09 ng/mL vs. 2.53+/-0.09 ng/mL; P<0.05) and a smaller follicle at TAI (11.61+/-0.11 mm vs. 12.05+/-0.12 mm; P<0.05). Heifers with smaller follicles at TAI had lesser serum progesterone concentrations on Day 18 and reduced rates of ovulation, conception, and pregnancy (P<0.05). Treatment with eCG improved (P<0.05) follicle diameter at TAI (11.50+/-0.10mm, 11.90+/-0.11 mm, and 12.00+/-0.10mm for 0, 100, and 200 IU, respectively), serum progesterone concentration on Day 18 (2.77+/-0.11 ng/mL, 3.81+/-0.11 ng/mL, and 4.87+/-0.11 ng/mL), and rates of ovulation (83.8%, 88.5%, and 94.3%) and pregnancy (41.3%, 47.0%, and 46.7%). In Experiment 2, nonlactating Nelore cows (n=702) received PGF(2 alpha) treatment on Days 7 or 9 and, on Day 9, 0 or 300 IU eCG. Cows receiving PGF(2 alpha) on Day 7 had lesser serum progesterone concentrations on Day 9 (3.05+/-0.21 ng/mL vs. 4.58+/-0.21 ng/mL; P<0.05), a larger follicle at TAI (11.54+/-0.21 mm vs. 10.84+/-0.21 mm; P<0.05), and improved (P<0.05) rates of ovulation (85.4% vs. 77.0%), conception (60.9% vs. 47.2%), and pregnancy (52.0% vs. 36.4%). Treatment with eCG improved (P<0.05) serum progesterone concentration on Day 18 (3.24+/-0.14 ng/mL vs. 4.55+/-0.14 ng/mL) and the rates of ovulation (72.4% vs. 90.0%) and pregnancy (37.5% vs. 50.8%). In conclusion, giving PGF(2 alpha) earlier in the protocol in nonlactating cows and eCG treatment in postpubertal heifers and nonlactating cows improved fertility in response to a TAI (progesterone+estradiol) protocol.

Within gilt pools, incidences of delayed puberty attainment, failure to exhibit regular oestrous cycles and low first litter size are often high. Boar exposure is an effective method of accelerating puberty; however, the timing of gilt response can vary greatly. Although, PG600 (400 IU of PMSG and 200 IU of hCG; Intervet) can induce a rapid and synchronous ovulatory response, thus providing an alternative to boar contact, the quality of the response is often variable. This study compared the effect of PG600, either alone (NBC) or in conjunction with boar exposure (BC), on puberty attainment and maintenance of oestrous cyclicity. The effects of first mating these gilts at the hormonally induced (pubertal) or second oestrus on ovulation rate and early embryo survival were also studied. Eighty Large White cross terminal (Duroc) line gilts were used in this study. The study was conducted in two blocks, with 10 gilts allocated to each of the four treatments in each block. Gilts were artificially inseminated at the allocated oestrus, with the reproductive tracts collected at 26.5+/-0.29 days after first mating (mean+/-S.E.M.), and the number of corpora lutea and viable embryos recorded. Mean days-to-puberty was significantly reduced (P<0.05) when gilts received both PG600 and boar exposure as opposed to PG600 alone (5.7+/-0.15 versus 6.9+/-0.37 days; P<0.01). The proportion of gilts exhibiting an ovulatory response to PG600 was similar for the BC and NBC treatment groups (0.88 and 0.84); however, the proportion of gilts exhibiting visible signs of oestrus in response to PG600 was significantly higher for the BC compared to the NBC treatment groups (0.81 versus 0.49; P<0.05). Boar contact resulted in a numerical, but not significant, increase in the proportion of gilts exhibited a second oestrus (1.00 versus 0.76). There was no significant effect of boar contact on ovulation rate, embryo number or survival. Although ovulation rate was unaffected by oestrus at mating, embryo number was significantly increased (P<0.05) following mating at the second compared to the first oestrus (11.2+/-0.96 versus 7.8+/-1.17). In conclusion, the current data indicate that the timing of puberty attainment and oestrus detection are significantly improved when PG600 treated gilts receive full boar contact. Further, it is evident that mating gilts at their second as opposed to the hormonally induced oestrus significantly increases embryo number at day 26 post-mating.

The objectives were to evaluate the effects of equine chorionic gonadotropin (eCG) supplementation (with or without eCG) and type of ovulatory stimulus (GnRH or ECP) on ovarian follicular dynamics, luteal function, and pregnancies per AI (P/AI) in Holstein cows receiving timed artificial insemination (TAI). On Day 0, 742 cows in a total of 782 breedings, received 2mg of estradiol benzoate (EB) and one intravaginal progesterone (P4) insert (CIDR). On Day 8, the CIDR was removed, and all cows were given PGF2 alpha and assigned to one of four treatments in a 2 x 2 factorial arrangement: (1) CG: GnRH 48 h later; (2) CE: ECP; (3) EG: eCG+GnRH 48 h later; (4) EE: eCG+ECP. There were significant interactions for eCG x ovulatory stimulus and eCG x BCS. Cows in the CG group were less likely (28.9% vs. 33.8%; P<0.05) to become pregnant compared with those in the EG group (odds ratio [OR]=0.28). There were no differences in P/AI between CE and EE cows (30.9% vs. 29.1%; OR=0.85; P=0.56), respectively. Thinner cows not receiving eCG had lower P/AI than thinner cows receiving eCG (15.2% vs. 38.0%; OR=0.20; P<0.01). Treatment with eCG tended to increase serum progestesterone concentrations during the diestrus following synchronized ovulation (P<0.10). However, the treatment used to induce ovulation did not affect CL volume or serum progesterone concentrations. In conclusion, both ECP and GnRH yielded comparable P/AI. However, eCG treatment at CIDR removal increased pregnancy rate in cows induced to ovulate with GnRH and in cows with lower BCS.

Glucocorticoids have heterogeneous effects on reproductive function. We used a gonadotropin-primed, immature rat model to study the influence of dexamethasone (1 mg/kg), given during the latter stages of follicular development, on litter size, the number of oocytes released, and pituitary hormone levels. Dexamethasone-treated females released a larger number of oocytes at ovulation and gave birth to larger litters indicating the oocytes were viable. Survival to weaning age was not affected but average weight at weaning was lower for pups born to DEX-treated females. Serum FSH and LH were assayed at 12, 24 and 48 h following eCG and did not differ between dexamethasone-treated and control animals, but prolactin showed a prolonged pattern of elevation in DEX-treated females. Prolactin, which normally exhibits an elevation on proestrous, may modulate follicular development. Dexamethasone enhances fertility and fecundity possible through an effect of prolactin on follicle development, or by other direct effects on the ovary. These results may improve our understanding of the usefulness of DEX in assisted reproductive therapies for women.

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36h later. Sorted (S) and non-sorted (NS) doses of 1 or 15x10(6) motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66h after progestagen withdrawal. An additional treatment dose of 0.5x10(6) S or NS sperm was inseminated at the 58h time point (n=60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1x10(6) and 15x10(6) sperm achieved similar pregnancy rates, regardless of sperm type, at 58h (S1: 46+/-9.4%; S15: 43+/-9.3%; NS1: 41+/-9.2%; NS15: 49+/-9.4%). However, pregnancy rates were lower (P<0.05) for doses of 1 than 15x10(6) sperm inseminated at 50 (15+/-6.3% vs. 36+/-9.1%), 54 (14+/-4.4% vs. 55+/-7.3%), 62 (33+/-6.9% vs. 54+/-7.3%), and 66h (29+/-8.6% vs. 56+/-9.5%). There was no difference between S and NS sperm for inseminations with 0.5x10(6) motile sperm at 58h after PR (15+/-3.6% vs. 14+/-3.3%), nor with 15x10(6) motile sperm at all insemination times (49+/-6.3% vs. 49+/-6.3%). However, fertility was higher for S than NS sperm at the 1x10(6) dose level (37+/-6.1% and 16+/-4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.

The blackbuck (Antilope cervicapra) is a small (20-30 kg) Indian antelope that is listed on Schedule I of the Indian Wildlife Protection Act, 1972. Studies were undertaken to develop assisted reproductive technologies, such as synchronisation of oestrus and non-surgical AI, to support the conservation and genetic management of this Indian antelope. Semen characteristics, testosterone levels and the feasibility of short-term cold storage of semen were investigated. Furthermore, different oestrous synchronisation protocols (norgestomet implants and prostaglandin injections) were evaluated for successful AI, defined as the birth of live young. Norgestomet ear implants and i.m. administration of pregnant mare's serum gonadotropin (PMSG) resulted in successful pregnancies in two of five inseminated females, but both had twin pregnancies that were delivered prematurely. In contrast, two injections of prostaglandin 11 days apart were effective in synchronising oestrus in the blackbuck. Transcervical AI in oestrous-synchronised animals 72 and 96 h after the second prostaglandin injection resulted in successful pregnancies in four of six inseminated females (67%) and resulted in the delivery of three live fawns. These studies demonstrate the potential application of AI technology for the conservation of endangered ungulates. To our knowledge, this is the first report regarding the synchronisation of oestrus and successful non-surgical AI in blackbuck.

The follicular development in the cow occurs in a wave-like pattern, and it takes place also during pregnancy. In the cow, Equine Chorionic Gonadotropin (eCG) is used for superovulation, but a decrease in total fertility has been reported, likely because of its immunogenic properties in species other than equine. In this regard, immune response has been implicated in follicular growth, ovulation, and placental development. So, aims of our study are to test the safety of eCG administered during pregnancy and characterize the ovarian activity, the quality of oocytes, the hormonal status, and interleukin levels in eCG-treated pregnant cows.

The effects of melengestrol acetate (MGA) and P.G. 600 on ewe fertility outside the natural breeding season were evaluated. Rambouillet ewes were assigned to one of four groups: (1) control (C; n=92); (2) PG600 (n=86); (3) MGA (n=99); and (4) MGA+PG600 (n=92). A pellet with or without MGA (0.3mg/ewe/d) was fed at 0.15kg/ewe/d for 7d. On the last day of pellet feeding, ewes were given either saline or 5mL of P.G. 600 i.m. (400IU equine chorionic gonadotropin (eCG) and 200IU human chorionic gonadotropin (hCG)). Ultrasonography was performed between Days 20 and 25 of gestation for ewes that were mated during the first 6 d of the breeding period from the MGA (n=15) and MGA+PG600 (n=8) groups, and the number of luteal structures and embryos were counted. During the first 6d of the breeding period, MGA increased (P<0.05) the percentage of ewes that mated and conceived when compared to C and PG600 (24.2% vs. 3.3% and 10.5%, respectively). Relative to MGA, the mean (+/-S.E.M.) number of luteal structures per ewe was enhanced (P<0.03) in MGA+PG600 (1.53+/-0.13 vs. 2.38+/-0.42, respectively), however as pregnancy progressed, the number of embryos (1.5+/-0.13 vs. 1.8+/-0.16, respectively) and lambs born (1.3+/-0.15 vs. 1.5+/-0.27, respectively) did not differ. Treatment with MGA reduced (P<0.01) the interval from ram introduction to lambing relative to groups that did not receive MGA (168+/-0.8d vs. 171+/-0.6d, respectively). In conclusion, treatment with MGA increased the percentage of ewes conceiving early in the breeding period. Although P.G. 600 increased the number of luteal structures present per ewe, it did not significantly enhance ewe prolificacy.

To control postpartum anestrus and reduce calving to conception interval, 167 crossbred non-pregnant cows that were 90-130 days postpartum were allotted randomly to one of the following treatments: PH (n=59), intra-vaginal sponge with 250 mg of medroxyprogesterone acetate (MAP) for 7 days plus 50mg of MAP and 5mg 17-beta estradiol (17beta-E) in the first day of treatment (day -8), 500 UI eCG (day -3) and 1.5mg 17beta-E in 24h after sponge removal (day 0); CR (n=57), temporary calf removal for 120 h; CG (n=51), control group without treatment. Estrus rate differed among treatments (P<0.01) being greater in PH (78.2%), followed by CR (52.0%) and CG (22.9%). A greater proportion of cows in the PH (80.0%) and CR (54%) groups had ovulations when compared to CG (35.4%). Intervals to first estrus were 13.5+/-6.3 days, 26.1+/-6.4 days and 52.5+/-7.5 days for the PH, CR and CG groups, respectively. First insemination conception was similar in the three groups. Postpartum intervals to first breeding (PFS) and to conception (PCI) were longer in CG than PH and CR groups (P<0.05; P<0.01). The PH and CR groups had a similar PFS but PCI was different (P<0.02). Accumulated pregnancy rate at 30 and 60 but not at 90 days were different (30 days: P<0.09; P<0.01; P<0.09; 60 days: P<0.06; P<0.01; P<0.03) among treatments. After 90 days post-treatment, 9%, 18% and 33% of cows from the PH, CR and CG groups had not conceived. Similarly, 5.4%, 6.0% and 12.5% of cows from the PH, CR and CG groups, respectively, were culled from the herd because of lack of pregnancy after 180 days post treatment. In the group of cows evaluated by ultrasonography, only those cows having larger ovaries and dominant follicles had ovulations. It was concluded that the hormonal treatment was more efficient in inducing a fertile estrus and reducing calving to conception interval followed by the calf removal for 120 h. Each method can be considered as an important tool to reduce the postpartum anestrous period in dual purpose herds when AI is conduct in the tropics.

The efficiency of cronolone sponges in combination with either pregnant mare serum gonadotrophin (PMSG) or cloprostenol (PGF2alpha) for inducing and synchronizing the estrous cycle in Turkish Saanen does was investigated during the transition from non-breeding to breeding season. All does (n = 80) were treated with 20 mg cronolone sponges for 11 days and divided into 4 equal groups. In addition, each doe received an intramuscular injection of either 1.5 ml sterile saline solution, 0.075 mg PGF2alpha, 500 IU PMSG or 500 IU PMSG and 0.075 mg PGF2alpha, 24 h before the sponge removal. Cervical artificial insemination (AI) with frozen-thawed semen was performed once 16 h after the detection of the first accepted mount. The total estrous response for the first 24 +/- 4 h, total estrous response within 96 h, time to onset of the induced estrus, duration of the induced estrus and pregnancy rate was found to be 75.0%, 97.5%, 31.4 +/- 1.2 h, 29.3 +/- 1.2 h, and 33.3%, respectively. There were significant differences between the first two groups and the last two groups in terms of the onset of induced estrus and estrous response at the first 24 +/- 4 h (P < 0.05). These results indicate that the use of cronolone/PMSG was more effective than cronolone/PGF2alpha in the attainment of early and compact induction of estrus in Turkish Saanen does.

The aim of this study was to assess the ultrasonographic characteristics of ovulatory follicles in cyclic Western White Face ewes (December) that had received intravaginal sponges containing medroxyprogesterone acetate (MAP; 60 mg) for 12 days, with or without an injection of 500 IU of equine chorionic gonadotropin (eCG) at sponge removal. We hypothesized that quantitative echotextural attributes of the follicles in ewes treated only with MAP would differ from those in MAP/eCG-treated ewes, reflecting the increased antral follicular growth and secretory function under eCG influence. Digital images of ovulatory follicles obtained at 0 and 24 h after MAP sponge removal and at 24 h before ovulation in the eCG-treated (five ewes, 13 follicles) and control (six ewes, 9 follicles) animals, were subjected to computerized analyses. The mean diameter of ovulatory follicles increased (p < 0.001) 24 h after eCG treatment. The mean pixel intensity and heterogeneity of the follicular antrum (p < 0.001), as well as mean pixel intensity of the follicular wall and perifollicular ovarian stroma (p < 0.05), were greater in eCG-treated animals compared with control ewes 24 h after sponge removal and at 24 h before ovulation. Mean serum concentrations of oestradiol-17beta tended to increase (p = 0.06) 24 h after eCG treatment and the eCG-treated ewes exceeded (p < 0.05) control animals in progesterone concentrations from days 9-15 after ovulation. Our results support the hypothesis that large antral follicles in eCG-treated ewes exhibit distinctive echotextural characteristics. Follicular image attributes in eCG-treated ewes appear to be indicative of the changes in follicular morphology and secretory activity caused by the administration of the exogenous gonadotropin, which has both FSH- and LH-like activities.

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F(1) hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.

This study evaluated the effects of altering dose of PG600 on estrus and ovulation responses in prepubertal gilts and weaned sows. Experiment 1 tested the effects of one (1.0x, 400IU eCG+200IU hCG, n=74), one and a half (1.5x, n=82), or two (2.0x, n=71) doses of PG600 for prepubertal gilts. Estrus (58%) and ovulation (90%) were not affected (P>0.10) by dose. Higher doses increased (P<0.01) numbers of corpora lutea (17, 24, and 25), but not (P>0.10) the proportion of gilts with cysts (26, 36, and 46% for 1.0x, 1.5x, and 2.0x, respectively). Experiment 2 tested the effects of 0x (n=30), 0.5x (n=32), 1.0x (n=29), or 1.5x (n=30) doses of PG600 in weaned sows. Dose did not influence return to estrus (90%, P>0.10). There was an effect of dose (P<0.05) on incidence of cysts (3.4, 1.8, 6.4, and 29.8%, for 0x, 0.5x, 1.0x, and 1.5x doses, respectively). The 0.5x dose increased (P<0.01) farrowing rate (83.2%) compared to 0x (72.1%) and 1.5x (58.6%), but was not different from 1.0x (76.4%). Total pigs born (10.5+/-0.8) did not differ (P>0.10) among treatments. These data suggest that increasing dose of PG600 to 1.5x for gilts increases the number of corpora lutea but does not alter the proportion expressing estrus or ovulating. Reducing dose of PG600 for weaned sows did not alter estrus or ovulation, but the 0.5x dose increased farrowing rate compared to no PG600.

The objective of this investigation was to examine the effects of 6-methoxy-benzoxazolinone (MBOA), a plant compound that resembles melatonin and alters ovarian function in rodents, in combination with PMSG on superovulatory responses in the cycling ewe. In Experiment I, St. Croix White ewes (n = 44) were synchronized (intra-vaginal progestin sponge) for 14days followed by hCG (750 IU) at 1 day after sponge removal (day 0). Ewes were assigned to one of six treatments administered on day -1: Control (no PMSG or MBOA; n = 7); PMSG (1000 IU i.m.; n = 7); Low MBOA (0.43 mg/kg i.m.; n = 7); High MBOA (1.15 mg/kg i.m.; n = 7); Low MBOA + PMSG (n = 8); High MBOA + PMSG (n = 8). In Experiment II, St. Croix White ewes (n = 24) were synchronized (progestin CIDR) for 14 days followed by hCG on day 1 after CIDR removal (day 0). Ewes were assigned to one of three treatments administered on day -1: Control (n = 8); PMSG (n = 8); Low MBOA+PMSG (n = 8). Laparoscopy was performed on day 9 to assess numbers of corpora lutea (CL) and visible follicles on each ovary. Blood samples were collected on day -13, -1, 0, 1, and days 6 or 7-12 for analysis of serum progesterone (P4) by RIA. Treatment groups receiving PMSG (alone or with MBOA) exhibited greater (P < 0.05) serum concentrations of P4 post-synchrony than Control and MBOA-only groups. Ovulation rate was lower (P < 0.05) for Control and MBOA-only treated ewes than ewes receiving PMSG. Ovulation rate in ewes treated with MBOA alone was similar (P > 0.10) to Controls, and PMSG treatment alone did not differ (P > 0.10) from MBOA + PMSG treatment. Ewes treated with PMSG alone did not differ (P > 0.10) in follicle number from High MBOA + PMSG treated ewes, however, Low MBOA + PMSG treated ewes had greater numbers of follicles at day 9 (P < 0.05) than the PMSG or High MBOA + PMSG groups in Experiment I; although, this was not replicated in Experiment II with numbers of follicles in the Low MBOA + PMSG group being similar (P > 0.10) to PMSG alone. In summary, the addition of MBOA in combination with PMSG as part of a synchronization-superovuation protocol in the ewe did not increase ovulation rate.

Ketosis was diagnosed in a flock of Merino ewes that conceived from synchronised oestrus in the early autumn period. On day 140 of pregnancy the ewes were sampled for determination of betaOH-butyrate (BHB), AST, glucose, non-esterified fatty acids (NEFA), total cholesterol (TCH), insulin, T4, T3, cortisol, IGF-1 and leptin. The results were evaluated according to the number of fetuses born some days later and the presence of hyperketonaemia (BHB: > or = 1.60 mmol/l). In May, about 3 months after lambing, cyclic ovarian function was induced (Cronolone + eCG), and the ewes were inseminated artificially (AI) 48 h after the removal of gestagen-containing sponge. At the time of AI and 10 days later blood samples were collected again to check the plasma levels of the same constituents as previously (in samples taken at AI), and to monitor the ovarian response by assaying progesterone (in both samples). On day 140 of gestation significantly lower BHB levels were detected in dams with single (n = 41) than in those with twin (n = 57) pregnancies. Hyperketonaemia was found only in ewes bearing twins (n = 27). These animals had higher NEFA and cortisol, and lower TCH, insulin, IGF-1, leptin and T3 levels than their normoketonaemic twin-bearing flock-mates, and those with single pregnancy. The blood glucose concentrations varied within a wide range, and the means of groups did not exhibit any significant differences. The formerly hyperketonaemic individuals were characterised by lower leptin level 3 months after lambing, and they showed a poorer response to the cycle-induction procedure than the others. The non-responders had lower IGF-1 and leptin levels than those ovulated after this treatment. It was concluded that the subclinical form of ovine ketosis is characterised by complex endocrine alterations, reflecting an obvious form of negative energy balance. If attempts to induce cyclic ovarian function outside the breeding season are made soon after lambing, the ovarian response and fertility of these ewes may also be depressed.

In this study the reproductive performance of weaned sows in a large production unit was evaluated during periods of high (> 35 degrees C) summer temperatures. Only weaned sows of parity 2-6 (a total of n = 3386) were evaluated in two groups in 13 replicates: Group 1 (n = 1694): The sows were treated subcutaneously with 400 I.U. of Gonadotropinum sericum (pregnant mare serum gonadotropin, PMSG [eCG]) and 200 I.U. of Gonadotropinum chorionicum (human choriongonadotropin, HCG [hCG]) on the day of weaning. Group 2: (n=1692). The sows received subcutaneously 4 ml of physiological NaCl solution on the day of weaning. Weaning to service intervals, regular and irregular returns to service, farrowing rates, and subsequent litter sizes were evaluated. Control sows had longer (P < 0.01) weaning to service intervals and more (P < 0.01) regular returns to service during hot weather periods compared with the gonadotropin treated sows. Irregular returns to service were low and showed no significant differences between the treated and non-treated sows (1.4+/-0.3 % vs. 1.5+/-0.4 %). Farrowing rates and litter sizes were significantly (P < 0.01) better in the treated compared with the non-treated sows.

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.

Rationale: Allopregnanolone, a neurosteroid-reduced metabolite of progesterone, is a well-documented positive modulator of the gamma-aminobutyric type A (GABA(A)) receptor. As has been reported for other positive modulators of the GABA(A) receptor, chronic exposure to neurosteroids is hypothesized to decrease GABA(A) receptor function. Drawing from the literature on chronic exposure to benzodiazepines or alcohol, putative changes in N-methyl-D-aspartate (NMDA) receptor function are also expected after chronic neurosteroid exposure., Objectives: To assess the sensitivity of the GABA(A) and NMDA receptors after chronic elevation of neurosteroid produced by termination of pseudopregnancy in behavioral tests of anxiety and sensorimotor coordination., Methods: Female rats ovariectomized on day 10 of pseudopregnancy were tested in the elevated plus-maze and on the rotor rod after an acute injection of progesterone (4 mg/0.2 ml, s.c.), chlordiazepoxide (5 or 15 mg/kg, i.p.), or MK-801 (0.025, 0.05, or 0.1 mg/kg, i.p.)., Results: Pseudopregnancy termination produced an anxiogenic-like response in the plus-maze; an acute injection of progesterone restored baseline levels of behavior in this test. Pseudopregnancy termination eliminated the anxiolytic-like, sedative, and ataxic effects of chlordiazepoxide. In contrast, pseudopregnancy termination produced an increased sensitivity to the anxiolytic-like and ataxic effects of MK-801., Conclusions: The effects of pseudopregnancy termination on the behavioral response to positive modulators of the GABA(A) receptor are consistent with results from studies in which chronic exposure to neurosteroids decreases the response to acute neurosteroid and benzodiazepine administration. However, unlike the enhanced glutamatergic tone resulting from discontinuation of chronic benzodiazepine or alcohol exposure, the termination of pseudopregnancy apparently decreases NMDA receptor function.

We previously reported that ovulation rate, but not pregnancy rate or litter size at d 30 after mating, was enhanced by treatment with P.G. 600 (400 IU of PMSG and 200 IU of hCG, Intervet America, Inc., Millsboro, DE) in gilts fed the orally active progestin, altrenogest (Matrix, Intervet America, Inc.) to synchronize estrus. We hypothesized that in addition to increasing ovulation rate, P.G. 600 may have altered the timing of ovulation. Therefore, mating gilts 12 and 24 h after first detection of estrus, as is common in the swine industry, may not have been the optimal breeding regimen, and as a consequence, pregnancy rate and litter size were not altered. The objective of the present study was to determine the effect of P.G. 600 on the timing of ovulation in gilts treated with altrenogest. Randomly cycling, crossbred gilts (5.5 mo old, 117 kg BW, and 14.7 mm of backfat) were fed a diet containing altrenogest (15 mg/d) for 18 d. Twenty-four hours after altrenogest withdrawal, gilts received i.m. injections of P.G. 600 (n = 25) or saline (n = 25). Gilts were checked for estrus at 8-h intervals. After first detection of estrus, transrectal ultrasonography was performed at 8-h intervals to determine the time of ovulation. Gilts were killed 9 to 11 d after the onset of estrus to determine ovulation rate. All gilts displayed estrus by 7 d after treatment with P.G. 600 or saline. Compared with saline, P.G. 600 increased (P = 0.07) ovulation rate (14.8 vs. 17.5, respectively; SE = 1.1). The intervals from injection to estrus (110.9 vs. 98.4; SE = 2.7 h; P < 0.01) and injection to ovulation (141.9 vs. 128.6; SE = 3.2 h; P < 0.01) were greater in gilts treated with saline than in gilts treated with P.G. 600. Duration of estrus (54.4 vs. 53.7; SE = 2.5 h), the estrus-to-ovulation interval (30.2 vs. 31.7; SE = 2.2 h), and the time of ovulation as a percentage of estrus duration (55.8 vs. 57.5; SE = 3.0%) did not differ for the P.G. 600 and saline-injected gilts, respectively. In summary, P.G. 600 advanced the onset of estrus and ovulation following termination of altrenogest treatment and increased ovulation rate; however, treatment of gilts with P.G. 600 had no effect on the timing of ovulation relative to the onset of estrus.

Background: Germinal vesicle (GV) oocytes constitute a potential resource but their developmental competence is questionable especially when surrounding cumulus cells are removed. The intercellular factors/mechanisms underlying such poor embryonic competence may originate at a nuclear and/or ooplasmic level., Methods: Immature or mature oocytes were obtained from three mouse strains following pregnant mare serum gonadotropin (PMSG) or PMSG+ human chorionic gonadotropin (hCG) treatment. Immature oocytes were denuded of cumulus cells prior to in vitro maturation. Pronuclear (PN) transfer was used to examine nuclear-ooplasmic interplay on resultant embryonic development and Oct-4 immuno-staining patterns., Results: Embryos arising from ooplasts of in vivo matured oocytes displayed significant increases in blastocyst formation rates and total blastomere numbers when compared to those created from ooplasts of denuded oocytes. Oct-4 staining was more pronounced and restricted to the inner cell mass (ICM) in blastocysts arising from the ooplasm of in vivo matured zygotes than in those created from denuded oocytes., Conclusions: Developmental defect(s) appear to develop primarily in the ooplasm of oocytes that are denuded of their cumulus cells prior to in vitro maturation. Such oocytes result in embryos with poor developmental competence. These defects result in anomalies in cell number and Oct-4 expression during the morula-blastocyst developmental transition.

Synchronization and superovulation are commonly used to obtain large numbers of embryos for experimental and practical purposes. This study compared the number, quality, and in vitro development of embryos recovered from gilts following single or double estrus synchronization and superovulation. Prepubertal gilts from the single synchronization group were injected with 1500 I.U. PMSG and 1000 I.U. hCG 72 h later. The double synchronized group of gilts was treated with 750 I.U. PMSG and 500 I.U. hCG 72 h later. After 17 days, 1500 I.U. PMSG followed by 1000 I.U. hCG was administered. Five days after insemination embryos were recovered and cultured for 6 days. Both single and double hormonal stimulation schedules resulted in recovery of elevated numbers of embryos (28.4 and 23.4 vs. 11.3; p<0.01 and p

The use of exogenous gonadotrophins in puberty inducement and ovulation synchronization is a technique that has a positive influence on the management of swine. The purpose of this study was to verify the effects of a second gonadotrophin treatment [equine chorionic gonadotrophin (eCG) and luteinizing hormone (LH), intramuscularly (i.m.)] upon the second oestrus synchronization and fertility in gilts. Seventy-one NAIMA (Pen Ar Lan) gilts had their first oestrus (puberty inducement) induced by a hormonal treatment (eCG and LH). Then, they were randomly distributed into two treatments, with (T1) and without (C) gonadotrophin treatment at the second oestrus. The animals were fed with a single ration (16% of crude protein and 3286.73 kcal ME/kg), and timed artificial insemination performed at the second oestrus. Gilts were slaughtered for embryo recovery and ovary examination about 5 days after insemination. There was no evidence of a difference in the percentage of the second oestrus expression (T1 - 90.90% and C - 86.84%), the duration of the oestrus cycle (T1 - 19.62 +/- 0.82 days and C - 19.67 +/- 4.14 days), the percentage of follicular cysts (T1 - 15.15% and C - 18.42%) and number of ovulations (T1 - 14.60 +/- 5.7 and C - 13.23 +/- 4.8) between treatments (p > 0.05). However, the hormonal treatment (T1) showed minor oestrus dispersion and embryo viability (T1 - 8.4 +/- 5.6 and C - 11.2 +/- 4.6) (p < 0.05). These results indicate that the better synchronization and expression of the second oestrus when using gonadotrophins (eCG and LH) is followed by a lower embryo viability, which is probably the consequence of the heterogeneous follicle recruitment during the injection of eCG.

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

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

The success rates of assisted reproduction techniques are still unsatisfactory. Relatively few in vitro cultured embryos reach the blastocyst stage. The purpose of the study was to evaluate the protective potential of epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF-I, IGF-II) and stem cell factor (SCF) on in vitro development of pre-implantation mouse embryos exposed to tumor necrosis factor alpha (TNFalpha). C3B6F1 female mice were superovulated with 5 IU of pregnant mare serum gonadotropin (PMSG) and 48 h later with 5IU of equine chorionic gonadotropin (eCG). Following the second injection females were mated with DBA males. Two cell embryos were flushed out from the fallopian tubes 40 h after eCG administration. After retrieval, the embryos were divided into control and experimental media and incubated in groups of ten for 96 h (37 degrees C, 5%CO(2), in droplets of 50 microl under mineral oil). In the first part of experiment, the embryo development was tested in media containing EGF, IGF-I, IGF-II, SCF, TNF-alpha (1 to 1000 ng/ml). In the second part of the study, the development of embryos was examined in medium containing 100 ng/ml TNFalpha and one of following factors: IGF-I, IGF-II; EGF or SCF (100 ng/ml). During the culture embryos were examined at 24 hours intervals to assess the embryo development. Blastocyst rate was determined following 96 hours of culture. Evaluation of total blastocyst cell number (TB) and inner cell mass (ICM) was also performed. TNFalpha significantly reduced (p<0.05) the blastocyst rates as well as TB and ICM. The examined growth factors improved the development of embryos exposed to TNFalpha. Thus, in this study, the protective action of IGF-I and II, EGF and SCF against the detrimental influence of TNFalpha was demonstrated.

Three experiments were undertaken to investigate the effect of a pre-mating ram exposure during progestagen synchronisation treatment on time of breeding, ovulation rate, embryo quality and fertility and any interaction with time of ram introduction for breeding post sponge withdrawal. Crossbred ewes in experiment 1a (n = 348), 1b (mule; n = 133) and 2 (n = 58) underwent a 12-14 days synchronisation protocol. Three days prior to sponge withdrawal ewes were divided into Control (ewes in continued isolation from rams) or +Ram (ram-exposed) groups. Rams were introduced to +Ram ewes and remained with ewes until sponge withdrawal. Ewes in experiments 1a and 2 received eCG at sponge withdrawal and were reintroduced to rams at either 36 or 48 h post sponge removal (PSR). In experiment 1b, ewes did not receive eCG and were reintroduced to rams at 24 h PSR. In experiments 1a and 1b time of breeding, date of lambing and litter size were recorded. In experiment 2, ewes were slaughtered 5 days post breeding, reproductive tracts flushed and corpora lutea, ova and embryos assessed. Fewer +Ram ewes were mated by 96 h PSR (P < 0.001) than Control ewes in experiment 1a but not when rams were introduced earlier in experiment 1b. In experiment 1a, ram introduction at 36 h PSR improved conception to first service compared to introduction at 48 h PSR (P < 0.01) in both +Ram and Control groups. In experiments 1a and 1b, +Ram ewes had reduced litter size caused by more single births (1a; P < 0.001, 1b; P < 0.01). In experiment 2, +Ram ewes had fewer corpora lutea than Control ewes (P < 0.001) but embryo quality was similar. However, more good embryos were produced when rams were introduced for breeding at 36 h compared to 48 h PSR (P < 0.001). We conclude that a pre-mating ram exposure during the synchronisation treatment reduced the number of ewes mated at and conceiving to the first service. This was partially overcome by introducing rams for breeding earlier (24 or 36 h compared to 48 h PSR) but the most dramatic decrease in fertility was due to a reduction in ovulation rate in the ram-exposed ewes.

Boar exposure has been used for estrus induction of prepubertal gilts, but has limited effect on estrus synchronization within 7 d of introduction. In contrast, PG600 (400 IU of PMSG and 200 IU of hCG; Intervet, Millsboro, DE) is effective for induction of synchronized estrus, but the response is often variable. It is unknown whether boar exposure before PG600 administration might improve the efficiency of estrus induction of prepubertal gilts. In Exp. 1, physical or fence-line boar contact for 19 d was evaluated for inducing puberty in gilts before administration of i.m. PG600. Exp. 2 investigated whether 4-d boar exposure and gilt age influenced response to PG600. In Exp. 1, 150-d-old prepubertal gilts were randomly allotted to receive fence-line (n = 27, FBE) or physical (n = 29, PBE) boar exposure. Gilts were provided exposure to a mature boar for 30 min daily. All gilts received PG600 at 169 d of age. Estrous detection continued for 20 d after injection. In Exp. 2, prepubertal gilts were allotted by age group (160 or 180 d) to receive no boar exposure (NBE) or 4 d of fence-line boar exposure (BE) for 30 min daily before receiving PG600 either i.m. or s.c. Following PG600 administration, detection for estrus occurred twice-daily using fence-line boar exposure for 7 d. Results of Exp. 1 indicated no differences between FBE and PBE on estrus (77%), age at puberty (170 d), interval from PG600 to estrus (4 d), gilts ovulating (67%), or ovulation rate (12 corpora lutea, CL). Results from Exp. 2 indicated no effect of age group on estrus (55%) and days from PG600 to estrus (4 d). A greater (P < 0.05) proportion of BE gilts expressed estrus (65 vs. 47%), had a shorter (P < 0.05) interval from PG600 to estrus (3.6 vs. 4.3 d), and had decreased (P < 0.05) age at estrus (174 vs. 189 d) compared with NBE. Ovulation rate was greater (P < 0.05) in the BE group for the 180-d-old gilts (12.7 vs. 11.9 CL) compared with the NBE group. However, age group had no effect on ovulation (77%) or ovulation rate (12 CL). Collectively, these results indicate that physical boar contact may not be necessary when used in conjunction with PG600 to induce early puberty. The administration of PG600 to 180-d-old gilts in conjunction with 4 d prior fence-line boar exposure may improve induction of estrus, ovulation, and decrease age at puberty.

The objective was to evaluate the effects of 400 IU of eCG given on Days 5 or 8 of an estrus synchronization protocol with progesterone-releasing intravaginal devices (PRID) and estradiol benzoate (EB), in recipients for fixed-time embryo transfer. A secondary objective was to determine the effects of injectable progesterone (given concurrent with EB treatment). Three-hundred-and-four crossbred Bos taurus x Bos indicus beef heifers were randomly assigned to one of four treatment groups (2 x 2 factorial design). At unknown stages of the estrous cycle (Day 0), all heifers received a progesterone-releasing intravaginal device (PRID), plus 2mg of EB i.m., with or without a concurrent treatment of 50mg of progesterone i.m. Heifers were further subdivided to receive 0.15 mg of d-cloprostenol (PGF) i.m. and 400 IU of eCG i.m. on Days 5 or 8. In all heifers, intravaginal devices were removed on Day 8 and 1mg of EB was given i.m. on Day 9 (Day 10 was arbitrarily considered the day of estrus). On Day 17, all heifers with >1 CL or a single CL with a diameter > or =18 mm (based on ultrasonographic examination), received an in vitro produced (IVP) embryo by non-surgical transfer. On Day 17, there was an effect of day of eCG administration on the number of CL (1.35 +/- 0.08 versus 1.13 +/- 0.04, for Day 5 versus Day 8, respectively; P = 0.02) and (in a subset of 154 heifers) mean (+/-S.E.M.) plasma progesterone concentrations (2.41 +/- 0.26 versus 1.74 +/- 0.19 ng/mL; P = 0.03). Although the proportion of recipients transferred/treated and pregnant/transferred did not differ among groups, the proportion of recipients pregnant/treated tended (P = 0.1) to be higher in heifers treated with eCG on Day 5 versus Day 8 (47.0% versus 40.7%, respectively). Progesterone treatment had no significant effect. In conclusion, treatment with eCG (and D-cloprostenol) on Day 5 significantly increased the number of CL and plasma progesterone concentrations and tended to increase pregnancy rates, although progesterone treatment had no significant effect.

Neurokinin B (NKB), a member of the tachykinin family, and its neurokinin 3 receptor (NK3-R) are preferentially found in the central nervous system. Others have recently reported on mRNA from this ligand-receptor system in the uterus and on NK3-R expression increasing with age. NKB and NK3-R mRNAs have also been noted in cumulus cells and oocytes from superovulated rats. Intact ovaries before and after puberty have not been studied. In this study, we stimulated 29-day-old rats by s.c. injections with gonadotropins for estrous cycle synchronization in order to elucidate the NKB-NK3-R system's expression and function in the ovary. Simultaneously, NaCl, the NK3-R agonist (Pro(7))-NKB, the antagonist SB 218795, or thiorphan, a neutral endopeptidase inhibitor of tachykinin degradation, were injected intraperitoneally (i.p.) for 3 1/2 consecutive days. First, we demonstrated NKB and NK3-R transcripts in one rat ovary by RT-PCR. No significant mRNA differences were noted between immature ovaries and superovulated ovaries in any of the i.p. applications. Second, the possible role of NK3-R on the ovulatory process was verified by counting corpora lutea (CL) and CL cysts in serial sections of the other ovary derived from the four different groups and embedded in paraffin wax. CL and CL cysts were noted in greater numbers in the pharmacologically treated groups than in the saline-treated group. To validate possible drug effects on the peritoneum, we additionally studied pieces of the omentum majus and retroperitoneal fat tissue. Both tissues were heavily infiltrated by granulocytes similar to a non-specific inflammatory response. The saline-treated group as well as the pharmacologically treated groups appeared to develop this unexpected side effect to a similar degree. We conclude that transcripts of NKB and NK3-R are present before and after puberty in the rat ovary and appear to be expressed at similar levels which may indicate a role for the NKB-NK3-R system in follicle growth. The effect of increased CL formation after application of the NK3-R agonist i.p. is related to a non-specific response.

A less competent murine in vitro maturation (IVM) model was achieved by shortening the standard duration of in vivo PMSG stimulation from 48 to 24 h and selecting only naked/partially naked GV oocytes from a mixture of large and small follicles. Porcine granulosa coculture enhanced meiotic maturation within such a less competent model (37.3% versus 23.1%, P<0.05), while no significant enhancement was observed with macaque and murine granulosa coculture. Culture of porcine granulosa on extracellular matrix (ECM) gel resulted in a more differentiated morphology, but did not significantly further enhance the beneficial effects it already had on meiotic maturation. Increased concentrations of serum as well as the supplementation of gonadotrophins and follicular fluid within the culture milieu did not enhance IVM under both cell-free and coculture conditions. Porcine granulosa-conditioned medium also enhanced meiotic maturation (36.5% versus 26.7%, P<0.05), which was not diminished upon freeze-thawing (35.8% versus 22.6%, P<0.05). Enhancement of meiotic maturation by porcine granulosa coculture did not however translate to significant improvements in developmental competence, as assessed by in vitro fertilization (IVF) and embryo culture to the blastocyst stage, followed by total cell counts. ECM gel had a detrimental effect on fertilization and developmental competence, even though it had no detrimental effect on meiotic maturation itself.

The objective of the current study was to develop an ovine animal model for consistent study of uterine blood flow (UBF) changes during synchronized ovarian cycles regardless of season. Sheep were surgically bilaterally instrumented with uterine artery blood flow transducers and 5-7 days later implanted with a vaginal progesterone (P(4))-controlled internal drug-releasing device (CIDR; 0.3 g) for 7 days. On Day 6 of P(4), sheep were given two prostaglandin F(2 alpha) injections (7.5 mg i.m. 4 h apart). At CIDR removal, Experimental Day 0, zero (n = 9), 500 IU (n = 8), or 1000 IU (n = 7) eCG was injected i.m.; UBF was monitored continuously for 55-75 h. Jugular blood was sampled every 8 h to evaluate levels of P(4), estradiol-17 beta (E(2)beta) and luteinizing hormone (LH). The inhibitor of nitric oxide synthase, L-nitro-arginine methyl ester (L-NAME) was infused in a stepwise fashion unilaterally into one uterine artery at 48-50 h after 500 IU eCG and the effects on UBF were examined (n = 7). The zero-eCG group gradually increased UBF from a baseline of 17.4 +/- 3.9 to 80.5 +/- 1.1 ml/min. The 500-IU-eCG group increased UBF between 10 and 15 h from a baseline of 11 +/- 3.3 to 83.3 +/- 1.0 ml/min, whereas UBF for the 1000-IU-eCG group was higher (100.1 +/- 1.7 ml/min) than that seen in either of the other groups. Plasma P(4) fell to baseline within 8 h of CIDR removal, while E(2)beta rose gradually in association with elevations in UBF. LH surges occurred between 32 and 56 h after CIDR removal and the LH surge occurred earlier in the 1000-IU-eCG group than the other two groups (P < 0.01). L-NAME infusion dose dependently reduced maximum levels of UBF ipsilaterally by 54.6% +/- 6.2%, but contralaterally only by 27.4% +/- 8.5%. Regardless of season, either dose of eCG will result in analogous UBF responses. During the follicular phase, elevations in UBF are in part locally controlled by the de novo production of nitric oxide.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.

This study was undertaken to investigate the effects of three media, volume and type of oil and frequency of observation on the in vitro development of mouse zygotes. B6CBF1 female mice (4 to 6 wk old) were superovulated using PMSG/hCG and mated with a proven fertile male of the same strain. Putative zygotes with polar bodies were collected from the oviducts of mated mice, 25-28 h after hCG injection, and were cultured in vitro. Embryo development was evaluated at either 96 h and 120 h or every 24 h for 120 h. The results obtained showed that the CZB medium was better than the KSOM and HCO3HTF media, and the use of 1 mL of paraffin oil was better than the use of 0.5 mL of paraffin oil. The effect of paraffin oil and mineral oil on embryo development was examined and the results indicated that the use of paraffin oil was better than the use of mineral oil. Repeated observations did not influence the proportion of embryos developing to blastocysts.

The objective of this study was to evaluate the effects of equine chorionic gonadotropin (eCG) treatment on the number of induced accessory corpora lutea (CL), plasma progesterone concentrations and pregnancy rate in cross-bred heifers after transfer of frozen-thawed (1.5M ethylene glycol) embryos. All recipients received 500 microg PGF2alpha (dl-cloprostenol, i.m.) at random stages of the estrous cycle (Day 0) and were observed for estrus for 7 days. On Day 14, heifers detected in estrus between 2 and 7 days after PGF2alpha treatment were randomly allocated to four groups ( n=83 per group) and given 0 (control), 200, 400, or 600 IU of eCG. Two days later (Day 16), these recipients were given PGF2alpha and observed for estrus. Six to eight days after detection of estrus, plasma samples were collected to determine progesterone concentration and ultrasonography was performed to observe ovarian structures. Heifers with multiple CL or a single CL >15 mm in diameter received an embryo by direct transfer. Embryos of excellent and good quality were thawed and transferred to the recipients by the same veterinarian. Pregnancy was diagnosed by ultrasonography and confirmed by transrectal palpation 21 and 83 days after embryo transfer (ET), respectively. Plasma progesterone concentrations on the day of transfer (Day 7 of the estrous cycle) were 3.9+/-0.7, 4.2+/-0.4,6.0+/-0.4 and 7.8+/-0.6 ng/ml for groups Control, 200, 400, and 600, respectively (Control versus treated groups P=0.009; 200 versus 400 and 600 groups P=0.0001; and 400 versus 600 P=0.012 ). Conception rates 83 days after ET were 41.9, 50.0, 25.0, and 20.9% for groups Control, 200, 400, and 600, respectively (200 versus 400 and 600 groups P=0.0036 ). In conclusion, an increase in progesterone concentration, induced by eCG treatment, did not improve pregnancy rates in ET recipients. Conversely, there was a decline in conception rates in the animals with the highest plasma progesterone concentrations.

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.

A standard dose of 500 IU of eCG is commonly given to progestogen pre-treated anestrous ewes for induction of estrus. Twelve seasonally anestrous and 12 cyclic Western White Face ewes were treated for 12 days with intravaginal sponges impregnated with medroxyprogesterone acetate (MAP). In trials in both the breeding and nonbreeding seasons, six randomly selected ewes were given 500 IU of eCG at sponge removal to determine the effects of low dose of eCG on ovarian antral follicular dynamics and ovulation. Ultrasound scanning and blood sampling were done daily. Treatment with eCG did not have marked effects on antral follicular growth. All ewes ovulated, except for five of six control anestrous ewes. Luteal structures and progesterone secretion were confirmed in all but the control anestrous ewes. In the breeding season, peak progesterone concentrations were greater (P<0.05) in eCG-treated compared to control ewes. Daily serum estradiol concentrations were greater in the periovulatory period in eCG-treated compared to control ewes (treatment-by-day interaction; P<0.05), particularly in anestrus. Progestogen-treated ewes ovulated follicles from several follicular waves, in contrast to ovulations of follicles from the final wave of the cycle in untreated, cyclic ewes. Anestrous ewes exhibited more frequent follicular waves and FSH peaks compared to cyclic ewes after a progestogen/eCG treatment. In conclusion, 500 IU of eCG given after 12 days of progestogen treatment had limited effects on the dynamics of ovarian follicular waves. However, eCG treatment increased serum concentrations of estradiol during the periovulatory period, particularly in anestrous ewes; this probably resulted in the synchronous estrus and ovulation in anestrous ewes.

An unpredictability of ovarian response still remains the major problem concerning ovine reproductive programs. The influence of several environmental, genetic, and ovarian cycle effects on oocyte/embryo yield from donor females has been previously reported. The present research has been designed to exclude aforementioned causes of variability, thus to verify embryogenic competence in homogenous groups of animals. For this purpose we used prepubertal ewes kept under identical conditions. Initially, we stimulated three groups of prepubertal ewes at various ages and used a number of gonadotropin treatments to assess differences in oocyte competence between individuals. The results revealed the repeatability of response within individual donor lambs throughout the study. Moreover, once the variability in both oocyte and embryo yield between homogenous groups of donors was revealed alongside the influence of age and type of gonadotropin treatment (P < 0.001), we investigated whether the individual donor effect persisted among genetically similar animals. Therefore, we compared oocyte and subsequent embryo output of sibling lambs derived from the most efficient donor. Here the genetic homogeneity of sisters kept under identical conditions substantially improved the uniformity of either follicular response or embryo production, suggesting that the genotype plays a primary role in establishing follicular recruitment and developmental capability of oocytes. This observation consents to predict the ovarian performance from a single ewe already in early prepuberty (i.e., to qualify the female to breeding programs).

Two experiments were performed to examine sow fertility following the pre-weaning injection of gonadotrophin. In experiment 1, sows received PG600 2-days pre-weaning (PW2), on the day of weaning (W) or received no injection and served as controls (CT1). Compared to controls, the injection of PG600 resulted in shorter (P<0.02) wean-estrus intervals and more (P<0.05) sows exhibiting estrus by 7 days after weaning (4.5+/-0.2, 5.1+/-0.3, 6.1+/-0.3 days, and 81.3, 82.9, 68.8%, for PW2, W, and CT1, respectively). No effect of treatment was apparent for farrowing rate or subsequent litter size. In experiment 2, PG600 was injected at 4 days pre-weaning (PW4) and then either GnRH or hCG was injected at the time of weaning. Control (CT2) sows received no injections. Injection of PG600 was associated with shorter (P<0.0001) wean-estrus intervals and a higher (P<0.003) farrowing rate (1.1+/-0.2, 5.1+/-0.2 days, and 94.8, 78.5% for PW4 and CT2, respectively). There was no effect of treatment on subsequent litter size and results were the same for GnRH and hCG treated sows. We conclude that injection of gonadotrophins prior to weaning will result in very short wean-estrus intervals and that, when combined with a hormonally controlled ovulation, sow fertility may be enhanced.

The aim of the present study was to investigate the endocrinology of the periestrus period and that of the subsequent estrous cycle in ewes synchronized during the breeding season. Animals were treated for 14 days with either MAP intravaginal sponges or subcutaneous progesterone implants, followed by administration of 500 IU PMSG at the time of withdrawal. The time to estrus occurrence following progestagen withdrawal differed significantly between groups (45.3+/-2.7h for the MAP and 21.5+/-1.2h for the implant group, P<0.001). Estradiol levels around estrus did not differ between groups, but a significant difference was detected for the interval from peak estradiol to estrus, with a shorter interval for the implant group (26.7+/-0.7 and 2.7+/-0.9h, P<0.001). Progesterone implants shortened the interval from removal to LH surge, compared to the MAP group (31.2+/-4.4 and 56.5+/-3.6h, respectively, P<0.05). An earlier response was also observed for the interval from estradiol peak to LH peak in the implant group (12.1+/-3.3 and 37+/-2h, respectively, P<0.005), but no difference was observed for the interval from estrus to LH surge. Progesterone levels, particularly during the Days 6 to 10 of the subsequent estrous cycle were significantly higher (P<0.05) in the implant group. It is concluded that the kind of progesterone treatment may affect the time of estrus and the LH peak as well as the progesterone levels of the subsequent cycle.

Adult female mice were superovulated with PMSG followed by HCG and 140 blastocysts and 69 morulae were recovered from 24 mice. On the basis of the response, mice were divided into six groups; non responders, 1-5, 6-10, 11-20, 21-30 and >30 embryos. The ovaries of the animals were pooled group wise, homogenized in PBS (pH 7.4) and after centrifugation for 10-15 minutes, the supernatant was analyzed for the enzymes, guanine oxaloacetate transaminase (GOT), guanine pymvate transaminase (GPT), acid phosphatases (ACP) and alkaline phosphatases (AKP). Acid and alkaline phosphatase activities did not show any variation in relation to response to superovulation but GOT and GPT showed significantly increased activity in response to induction of superovulation. A statistically significant positive correlation was found between GOT and GPT activities and the superovulatory response in mice.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in the 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 the murine endometrium during this period is TIMP-3. Quantitative, competitive-reverse transcription-PCR was performed to evaluate the expression of TIMP-3 mRNA during the implantation period in the hyperstimulated murine endometrium from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of 5 and 10 IU pregnant mare serum gonadotropin (PMSG). In PMSG groups, the expression of TIMP-3 mRNA, normalized by MMP-9 mRNA, showed a significant increase compared to the control group (natural coitus without PMSG injection) on g.d. 6.0 (p = 0.039) and an increasing tendency on g.d. 7.0. This study suggests that ovarian hyperstimulation by gonadotropin may have a detrimental effect on embryonic implantation possibly via an altered endometrial remodeling process by the increase in expression of TIMP-3 in the murine endometrium., (Copyright 2003 S. Karger AG, Basel)

The existence of a vasoactive molecule released in response to hCG is believed to be the main feature in the development of ovarian hyperstimulation syndrome (OHSS) in women, and vascular endothelial growth factor (VEGF) is the main candidate as the human chorionic gonadotropin (hCG) mediator. This study was conducted to investigate the role of VEGF in increasing vascular permeability (VP) in vivo, a characteristic of OHSS. We analyzed the source and specific isoforms of VEGF involved and developed strategies to reverse increased VP in hyperstimulated rats targeting the VEGF system. Ovarian hyperstimulation was induced with pregnant mare's serum gonadotropin, or pregnant mare's serum gonadotropin plus hCG. Time-course experiments analyzed VP and the expression of whole VEGF mRNA in the mesentery and the ovaries. VP and ovarian mRNA VEGF expression increased to peak values after 48 h. No significant change in expression was observed in the mesentery. To further prove the ovarian origin of VEGF, we showed that VP was not altered when ovariectomized rats were treated with gonadotropins. The ovary expressed VEGF(120) and VEGF(164) isoforms. Immunohistochemistry showed VEGF in granulosa and zona pellucida of preovulatory and atretic follicles and in granulosa-lutein and endothelial cells of whole corpus luteum. A specific VEGF receptor-2 inhibitor (SU5416) was administered in three different protocols: on a daily basis, every 48 h, or two injections after hCG. Increased VP was reversed when SU5416 was administered every 48 h or two injections after hCG. These results show that the ovary is the main source of VEGF(120) and VEGF(164), which act through the VEGF receptor-2 to increase VP, and provide new insights into the prevention of OHSS.

Background: The aim of this study was to characterize the roles of nitric oxide (NO) on the rat ovarian blood flow (OBF) during the preovulatory period., Methods and Results: Immature Sprague-Dawley rats were primed with pregnant mares' serum gonadotrophin (PMSG, 15 IU) and given hCG (15 IU) 48 h later. The ovary was exposed 48-56 h after PMSG, a laser Doppler probe was attached to the ovarian surface and OBF was measured at two time periods: preovulatory (PO) 48 h after PMSG and ovulatory (OV) 6-8 h after hCG. A non-selective NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), was injected i.v. (4 and 10 mg/kg) or intrabursally (1 mg/kg). Intravenous administration of L-NAME to OV rats rapidly increased blood pressure and reduced OBF by 30%, which returned to the pretreatment level within 30 min. L-NAME given into the ovarian bursa of both PO and OV rats did not affect blood pressure and reduced OBF by nearly 40%, which remained low throughout the experiment. Intravenous injection of hCG to PO rats increased OBF to 116.1% at 5 min and 133.5% at 30 min in relation to the pretreatment level. When L-NAME was given intrabursally, subsequent hCG injection was without effect., Conclusions: These results indicate that locally produced NO is important for the maintenance and increase of rat OBF during the preovulatory period.

A plasma progesterone profile obtained from three consecutive blood samples with an interval of 7 days was evaluated for usefulness as the basis for the diagnosis and treatment of anestrous gilts and sows. Four reproductive statuses were categorized based on the plasma progesterone levels and pathological examination of the reproductive organs from 25 gilts and 12 sows with anestrus. Category 1: fluctuating (at least one sample <2.5 ng/ml and one >10 ng/ml) with normal ovary; Category 2: sustained low (<2.5 ng/ml) with inactive ovary; Category 3: persistent high (>5 ng/ml) with normal sized or cystic corpora lutea; and Category 4: animals not included in the categories mentioned, such as pigs with luteinized cysts and follicular cysts. Using the plasma progesterone profiles and this categorization, the reproductive status of 54 gilts and 38 sows with anestrus was predicted. Hormonal treatments were performed with moderate to high success. Results from this study indicate that plasma progesterone profiles can be useful for the determination of estrus status, for the diagnosis of the causes of anestrus, and for the prediction of the next estrus for an appropriate hormonal treatment in anestrous gilts and sows.

Prepubertal gilts were treated with 1,500 iu equine chorionic gonadotrophin, followed 72 hours later by 500 iu human chorionic gonadotrophin (hCG), and inseminated 36 and 48 hours later. Embryos were collected at slaughter 168 hours after the hCG treatment. Blastocysts classified as 'good' or 'fair' were transferred to synchronised recipients, either by conventional surgical means or by a 'semi-endoscopic' approach, and the recipients were slaughtered four weeks later. Of 238 donor gilts, 98.4 per cent had responded with a mean (se) 23.5 (1.0) ovulations and 19.1 (1.0) ova or embryos, of which 47 per cent were considered morphologically intact and transferable. The large proportion of non-transferable embryos was not associated with the age or weight of the gilts, the season or with their housing conditions. Conventional surgical transfer of 15 to 20 (mean 17.4) blastocysts to synchronised recipients yielded 88 percent (14 of 16) pregnancies with between seven and 14 (mean 8.2) viable fetuses, and an embryo survival rate of 47 per cent in the pregnant recipients and 41 per cent in all the recipients. The corresponding data for the semi-endoscopic transfers were 16 to 20 (mean 17.7) blastocysts transferred, 47 per cent (eight of 17) pregnancies, four to 12 (mean 7.3) viable fetuses per pregnant recipient and an embryo survival rate of 41 per cent in the pregnant recipients and 19 per cent in all the recipients. Significantly fewer of these recipients became pregnant and a significantly smaller proportion of the embryos survived (P<0.05).

In the present studies atrial natriuretic factor (ANF) was characterized immunocytochemically in the reproductive tract of immature female rats, and changes of ANF levels in response to different hormonal conditions were demonstrated. Administration of pregnant mare serum gonadotropin (PMSG) to immature animals has shown to be a useful method to synchronize growth, differentiation and atresia of ovarian follicles. ANF immunoreactivity was investigated in rat uterus and oviduct during follicular growth and estrogenic dominance (48 h after PMSG treatment) and during follicular atresia and progesterone dominance (96 h after PMSG treatment). Our immunocytochemical results showed that in rat uterus ANF was localized in endometrial mucosal and glandular epithelium and smooth muscle cells of the myometrium. In the oviduct ANF immunoreactivity was observed in mucosal cells and muscle layers. Immunocytochemical staining patterns and Western blot analysis revealed that ANF levels in rat uterus and oviduct are modulated by the hormonal status. ANF immunoreactivity was elevated during estrogenic dominance (48 h after PMSG) in uterus and oviduct. However, during progesterone dominance (96 h after PMSG) elevation of ANF immunoreactivity was observed in the uterus only. These results raise the possibility that ANF expression in rat oviduct is positively controlled by estrogen and negatively by progesterone. ANF staining in uterus during progesterone phase provides evidence that both estrogen and progesterone regulate ANF levels in uterus. The observed staining patterns indicate that ANF may have intracellular functions as well as a role in priming the extracellular environment. Accordingly, the possibility that ANF might be an important regulatory molecule for autocrine/paracrine communication within the female reproductive tract should be considered.