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The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty-nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B-mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11-15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose., (© 2018 Blackwell Verlag GmbH.)

Physical damage and oxidative stress may occur in prepubertal cumulus cells, due to insufficient glutathione synthesis. To determine potential epigenetic mechanisms related to antioxidant effects of melatonin on ovine prepubertal cumulus cells, 30 lambs, 4-wk-old were randomly allocated into two groups: a control (C, n = 20) group and a melatonin (M, n = 10) group given a subcutaneous implant containing 18 mg melatonin. All lambs were superovulated (250 IU FSH and 250 IU eCG). Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected by ovarian follicular aspiration and dissociated with hyaluronidase. Compared to the C group, the M group had greater superovulation, better antioxidant capacity, a higher proportion of fully expanded COCs and a lower proportion of apoptotic cumulus cells (P < 0.05). Melatonin up-regulated mRNA expression of genes for melatonin receptors MT1 and nuclear binding site RORα, antioxidants (SOD1, GPx4 and CAT) and cumulus cell expansion (PTX3, HAS2 and PTGS2), as well as Bcl2, but down-regulated expression of Bax (P < 0.05). Regarding epigenetics, there were less methylation at five CpG sites of SOD1, three CpG sites of GPx4 and two CpG sites of CAT in M versus C groups (P <  0.05), leading to lower total methylation of SOD1, GPx4 and CAT promoters region on M group (P < 0.05). In a mechanistic study, addition of MT1 or RORα antagonist increased ROS and MDA concentrations, but decreased T-AOC, GPx, CAT and T-SOD concentrations (P < 0.05), whereas there were no significant difference between the melatonin and MT2 antagonist treatment groups for T-AOC, GPx, CAT and T-SOD concentrations. Furthermore, addition of RORα agonist decreased total DNA methylation of SOD1, GPx4 and CAT, with no significant difference after MT1 agonist treatment. These studies provided new information regarding epigenetic mechanisms by which melatonin promoted ovine prepubertal cumulus cells antioxidant through RORα, both in vitro and in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of the PI3K/Akt-cyclin D1 pathway against nicotine-induced DNA damage is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as a reproductive toxicant. 48 female balb/c mice (6⁻8 weeks) (23⁻25 g) were randomly divided into eight groups (Grp.1⁻Grp.8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for seven consecutive days. On Day 8, the females were superovulated and mated before euthanization for embryo collection (46 h post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (Grp.2) significantly decreased ( p < 0.05) the number of produced 2-cell embryos compared to the control (Grp.1). Intervention with mixed annatto δ-TCT (Grp.3) and pure annatto δ-TCT (Grp.4) significantly increased the number of produced 2-cell embryos by 127% and 79%, respectively compared to Grp.2, but these were lower than Grp.1. Concurrent treatment with soy α-TOC (Grp.5) decreased embryo production by 7%. Supplementations with δ-TCT and α-TOC alone (Grp.6-Grp.8) significantly increased ( p < 0.05) the number of produced 2-cell embryos by 50%, 36%, and 41%, respectively, compared to control (Grp.1). These results were found to be associated with alterations in the PI3K/Akt-Cyclin D1 genes expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against nicotinic embryonic damage. To our knowledge, this is the first attempt in studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.

Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.7 ± 3.5 and 9.0 ± 6.0, respectively). Next, we compared in vitro embryo production by ovum pick-up and in vitro fertilization (OPU-IVF) between control (n = 12) and epidural groups (n = 12). The rate of development to transferable blastocysts was higher in the epidural group than in the control (23.3 vs. 10.5%, P < 0.001). In conclusion, a single epidural administration of FSH can induce follicular development comparable to that of the conventional superovulation protocol and may improve the productivity of OPU-IVF.

The aim of the present study was to evaluate the effect of serum progesterone concentrations during the superstimulatory treatment of the first follicular wave on fertilization rate and embryo development in sheep. A total of 71 Merino ewes received a superstimulatory FSH treatment during Wave 1 of ovarian follicular development (Day 0 Protocol), which was administrated under low progesterone concentrations typical of the early luteal phase (control group, n = 33) or under high progesterone concentrations induced by the administration of an intravaginal device from Day 0 to Day 3 containing 0.3 g progesterone (n = 38). Intrauterine insemination after FSH superstimulation was followed by uterine flushing 6 days later. Serum progesterone concentrations from Day 0 to 3 were greater in those ewes treated with progesterone (P < 0.05), while serum estradiol-17β concentrations were not affected by the treatment. Although the mean number of corpora lutea per donor was not affected by the progesterone treatment, the number of collected ova and embryos was greater in progesterone treated than untreated ewes (6.6 ± 0.7 compared with 4.6 ± 0.9, respectively; P < 0.05). Furthermore, progesterone treatment increased fertilization rate (93.3% compared with 83.3%; P < 0.05) and the proportion of Grade 1 embryos (67.7% compared with 52.7%; P < 0.05) compared with the control group. In conclusion, oocyte fertilization rate and embryo quality are improved by high progesterone concentrations during FSH superstimulation, which suggests an important role of progesterone during preovulatory follicular development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although melatonin has some of the broadest ranges of actions on the physiology of vertebrates, especially on their reproductive processes, the mechanism by which melatonin regulates animal reproduction is still incompletely understood. This study was designed to determine the effect of oral melatonin on the reproductive performance of female mice. Female ICR mice (7 weeks old) were given melatonin-containing water (3, 30 and 300 μg/mL; melatonin) or water only (control) until 10 weeks of age. Then, some of the mice were successfully mated (confirmed by vaginal plugs), and the number of live births and their weights were recorded. Some mice were used for a histological analysis of the number of follicles in the ovaries. Others were used for oocyte collection after superovulation, and in vitro fertilization (IVF) was performed. The mRNA expression of the apopotosis-related genes ( BAX , BCL 2) in the IVF embryos were analyzed. After melatonin administration, the mice showed similar serum melatonin levels to that of the control. The number of antral follicles per mm² unit area in the 30 μg/mL melatonin-treated group (14.60) was significantly higher than that of the control (7.78), which was lower than that of the 3 μg/mL melatonin-treated group (12.29). The litter size was significantly higher in the 3 μg/mL melatonin-treated group (15.5) than in the control (14.3). After IVF, the hatched blastocyst formation rate in the 30 μg/mL melatonin-treated group (85.70%) was significantly higher than that of the control (72.10%), and it was the same for the BCL 2/ BAX expression ratio. Although oral melatonin did not appear to have an effect on the serum melatonin rhythm in the mouse, melatonin did increase litter size at the 3 μg/mL dose level, and improved the developmental competency of IVF embryos at the 30 μg/mL level.

We determined the effect of monogamous or polygamous mating with 2 females on vaginal plug (VP) rate, embryo donors (ED), 2-cell embryo production, and male performance after superovulation of females aging 24d or 45-48d. C57BL/6NCrl (B6N), BALB/cAnCrl (BALB/cN), FVB/NCrl (FVB/N), and Crl:CD1(ICR) (CD-1) females received 5 IU eCG and 5 IU hCG (24d) or 7.5 IU eCG and 7.5 IU hCG (45-48d) 48 h apart. After the hCG injection, females were paired with males, which alternated weekly in monogamous or polygamous mating. Significant differences in the percentage of VP-positive females between monogamous and polygamous mating were observed for B6N (71% vs. 49%), FVB/N (77% vs. 51%), and CD-1 (90% vs. 67%) at 45-48d. BALB/cN and CD-1 showed higher VP rates than B6N and FVB/N. A significantly higher percentage of ED was found for monogamous than for polygamous mating for FVB/N (87% vs. 61%) at 24d and for B6N (91% vs. 53%) and CD-1 (90% vs. 68%) at 45-48d. In all strains of mice and in both age groups, no significant differences were observed in the number of intact 2-cells per VP-positive female, ED or treated female between monogamous and polygamous mating except in the B6N strain where monogamous mating resulted in a significantly higher number of intact 2-cell embryos per treated female than polygamous mating at both ages. The present results imply that polygamous mating can be implemented for 2-cell embryo production in all strains studied except for B6N when all females are euthanized. However, when only VP+ females are sacrificed polygamous mating can be employed for all 4 strains studied., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study aims to compare four different methods for selecting high responding sheep donors for in vivo embryo production. These methods include a pre-selection eCG test (eCG), antral follicle count (AFC), plasma anti-Müllerian hormone measurement (AMH) and genotyping for the presence of the FecG E mutation (a polymorphism in the GDF9 gene associated with increased ovulation rate). Santa Ines ewe lambs (n = 25) underwent superovulation (SOV) with 800 IU equine chorionic gonadotropin (eCG) and the corpus luteum (CL) count was recorded by laparoscopy after eight days. At the D0 eCG , blood samples for AMH and genotyping analysis were collected. Twenty-one days after the end of the eCG test, the same animals underwent SOV with 200 mg of FSH, administered in six decreasing doses, and then naturally mated. Immediately before the beginning of the FSH protocol (D0 FSH ), and at the moment of the first FSH dose (D9 FSH ), the AFC was assessed. Plasma AMH was again determined at the D9 FSH . After each screening process, animals were classified as having a high (HR), or low (LR), potential of response (using specific thresholds for each method). Then, the ewes' response to SOV and embryo yield for each screening method, classified as HR or LR, were compared. Animals classified as HR by AFC (HR AFC ) and by AMH concentration (HR AMH ) at the D9 FSH , produced more viable embryos than those classified as LR AFC and LR AMH (HR AFC 6.2 ± 3.2 vs LR AFC 2.8 ± 3.0 and HR AMH 6.6 ± 3.6 vs LR AMH 3.0 ± 2.9). Pre-selection tests with eCG and different FecG E genotypes, either heterozygous (+/E) or wild type (+/+), were unable to discriminate HR or LR animals. A tendency (P = 0.06) to have lower plasma AMH was observed in heterozygous FecG E (+/E) ewes. In conclusion, both AFC and plasma AMH can be used to select donor ewes with a higher potential of response for in vivo embryo production., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Multidrug resistance protein 1 (MDR1), a phase III drug transporter that exports substrates out of cells, has been discovered in both cancerous and normal tissues. The over expression of MDR1 in cancer cells contributes to multiple drug resistance, whereas the MDR1 in normal tissues protects them from chemical-induced toxicity. Currently, the role of MDR1 in the ovary has not been entirely understood. Our objective is to determine the function of MDR1 in protecting against chemotherapy-induced ovarian toxicity. Using both the in vivo transgenic mouse model and in vitro follicle culture model, we investigated the expression of MDR1 in the ovary, the effect of MDR1 deficiency on doxorubicin (DOX)-induced ovarian toxicity, and the ovarian steroid hormonal regulation of MDR1. Results showed that the MDR1 was expressed in the ovarian epithelial cells, stroma cells, theca cell layers, endothelial cells, and luteal cells. The lack of MDR1 did not affect female ovarian function and fertility; however, its deficiency significantly exacerbated the DOX-induced ovarian toxicity in both in vivo and in vitro models. The MDR1 showed significantly higher expression levels in the ovaries at estrus and metestrus stages than those at proestrus and diestrus stages. However, this dynamic expression pattern was not regulated by the ovarian steroid hormones of estrogen (E2) and progesterone (P4) but correlated to the number and status of corpus luteum. In conclusion, our study demonstrates that the lack of MDR1 promotes DOX-induced ovarian toxicity, suggesting the critical role of MDR1 in protecting female ovarian functions during chemotherapy.

The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study evaluated colour-Doppler ultrasound imaging (UI) as a substitute for laparoscopy to count the corpora lutea (CL) in superovulated sheep. Twenty-five Santa Ines ewes were superovulated three times at 21-day intervals. Corpora lutea were counted by colour-Doppler UI (CL DOPPLER ) 6 days after each superovulation and confirmed by laparoscopy (CL LAP ) 12 hr later. The mean number of CL was similar for both techniques (2.1 ± 2.5 vs. 2.1 ± 2.7 for CL DOPPLER and CL LAP , respectively) with a significant positive correlation (r = .94; r 2 =.89). Colour-Doppler UI effectively evaluated the ovarian response in superovulated ewes and efficiently identified animals that did not respond to superovulation., (© 2017 Blackwell Verlag GmbH.)

Purpose: The purpose of the study is to determine whether continued stimulation of mature follicles to allow "catch up" growth of medium-sized follicles in assisted reproductive technology compromises the clinical pregnancy (CPR) and live birth (LBR) rates in IVF/ICSI cycles., Methods: This retrospective cohort study reviewed 200 first IVF ± ICSI cycles out of a total of 340 cycles with complete data. Women underwent stimulation protocols with gonadotropins (Gn) and GnRH antagonist. Treatment cycles were divided into two groups (Gp): hCG administration delayed despite the presence of two mature follicles, defined as ≥ 18 mm [Gp1, n = 79] and hCG administration given when there were two mature follicles [Gp2, n = 121]., Results: The patients in Gp1 were significantly younger than those in Gp2 [32.9 (4.5) vs. 34.3 (4.8), p = 0.04] and needed a median of one more day of superovulation before ovulation was triggered with hCG. The extra days was associated with the use of 450 [75-2025] more Gn, such that at the time the hCG was administered, patient's in group 1 had developed significantly greater number of follicles ≥ 18 mm [mean (SD), 4.9 (1.8) vs. 3.4 (1.7), p < 0.0001]. The clinical pregnancy (48.1 vs. 38.0%, [OR (95% CI)] [1.6 (1.0-2.5), p = 0.09]) and live birth (43.0 vs. 35.5%, [1.4 (0.9-2.3), p = 0.21]) rates per cycle started were not significantly different between the two groups. Forward stepwise logistic regression showed that only maternal age (p = 0.04) influenced clinical pregnancy rates (OR = 0.88, CI 0.78-0.99) and only the number of days for superovulation influenced live birth rates (OR = 0.65, CI 0.486-0.869)., Conclusion: This study demonstrated that delaying hCG administration to allow further growth of the medium-sized follicles added further days of superovulation and cost without improvement in CPR and LBR.

The relationship between circulating concentration of anti-mullerian hormone (AMH) and the LH content of follicle stimulating hormone (FSH) preparation on follicular growth and ovulatory response in water buffaloes was evaluated. A single blood sample was taken from cows (N=31; age: 9.06±0.98years) to determine systemic AMH. Animals with concentrations higher or lower than 194±30pg/ml were placed into LOW and HIGH AMH groups and were assigned randomly to be superovulated FSH containing either a high (FSHp, HLH) or low (FolltropinV, LLH) LH content. Follicular growth and ovulation were monitored using transrectal ultrasonography. In animals with HIGH systemic AMH, treatment with FSH with a high LH content was associated with more small follicles (AMH X FSH; P=0.02). AMH had no effect on small follicles in animals treated with LLH. Females with a HIGH AMH had greater numbers of small follicles (P=0.01) and total follicles (P=0.005) than LOW AMH cows. Animals treated with HLH had more small follicles (P=0.001) but fewer large (P<0.001) and total follicles (P=0.0005) than those treated with LLH. Among animals with HIGH AMH, those treated with LLH, ovulated more follicles than those treated with HLH. (AMH X FSH; P=0.03). In conclusion, selecting animals with high AMH concentration and the use of FSH preparations with a lower LH content may improve the superovulatory response in water buffaloes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

The aim of the present study was to compare hormonal treatments to induce and synchronize follicular wave emergence to improve the results of superovulatory (SOV) treatments in ewes. In Experiment 1 (n = 66), ewes were treated with a progesterone intravaginal implant plus a PGF 2α analogue (group G P4 ), or with the same treatment plus estradiol benzoate (G P4+EB ), a GnRH agonist (G P4+GnRH ), or both, estradiol benzoate and a GnRH agonist (G P4+EB+GnRH ) in a 2 × 2 factorial arrangement. Follicular wave emergence was determined by ultrasound. Follicular wave did not emerge during the studied period in 10 females (one from G P4 , six from G P4+EB and three from G P4+EB+GnRH ). Follicular emergence was less synchronized (P = 0.007) when estradiol was administered (G P4+EB : 103.6 ± 22.0 h), without any interaction with GnRH treatment (G P4+EB+GnRH : 80.1 ± 21.4 h, G P4+GnRH : 52.5 ± 8.7 h, G P4 : 56.6 ± 10.4 h). Estradiol administration delayed the moment of follicular emergence (P = 0.007) and the follicular wave emergence moment in which follicular dominance was achieved (P = 0.009), without interactions between estradiol and GnRH in the moment of follicular wave emergence or dominance. In Experiment 2 (n = 22), two SOV protocols were compared: the best treatment of Experiment 1 (G P4 ) was used to synchronize follicular wave emergence, initiating the SOV treatment 2.5 days later; in the control treatment, SOV treatment started 80 h after a short-term protocol to synchronize ovulation (G control ). The number of corpora lutea (CL) and the evaluation of the collected embryos were performed six days after estrus. Blood samples were collected daily for plasma progesterone determination. Although the number of CL was similar in G control (7.1 ± 1.0) and G P4 (6.9 ± 5.1), the number of structures and viable embryos recovered were greater in G control (P < 0.05). The occurrence of luteal premature regression was significantly greater in G P4 (60%) than in G control (8.3%). The use of GnRH agonist alone did not improve synchronization of follicular wave emergence. When EB was used (alone or associated) follicular wave emergence was less synchronized. The SOV protocol proposed had a similar ovarian response; however, it resulted in less transferable embryos., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Antagonistic relationship between milk yield and reproduction is reported in several livestock species. This study aimed to investigate whether genetic merit for milk production in dairy sheep affects responses to superovulation, embryo yield and quality. A total of 21 cross-bred Sarda x Lacaune ewes homogeneous for age, parity and stage of lactation were included. The ewes were stratified as high-producing or low-producing based on their genetic merit for milk production estimated by a pentatrait repeatability animal model. Oestrus was synchronized using an intravaginal progesterone pessary inserted on Day 0 and removed on Day 14. Superovulatory treatment consisted of 350 I.U. of porcine FSH administered in eight decreasing intramuscular doses every 12 hr with a total dose of 10 ml of solution starting 12 days after insertion of sponges. Laparoscopic artificial insemination (AI) was performed 48 hr after pessary removal. Surgical embryo recovery was performed at Day 8 after pessary removal. Correlation between breeding value for milk production and the number of corpora lutea (CL) was significantly different from zero (-0.49). High-producing ewes had a lower number of CL than low-producing counterparts (7.6 ± 2.50 vs 12.1 ± 5.16 respectively; p < .02). Furthermore, there was a tendency for high-producing ewes to yield fewer embryos than low-producing females (5.3 ± 3.46 vs 9.18 ± 5.11; p = .09). No differences were observed between ewes in both genetic groups with regard to the number of embryos of grades 1, 2 and 3. To our knowledge, this is the first report highlighting an antagonism between genetic merit for milk production and the ability to produce embryos in sheep. These results deserve to be considered in sheep breeding programmes., (© 2017 Blackwell Verlag GmbH.)

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced., (Published by Elsevier Inc.)

There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, posology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or supplemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to superovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP + LH showed significantly higher implantation rates and survival at birth among the different ovarian stimulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplementation allows us to obtain similar in vivo embryo development results as in the control group., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P<0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P<0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P<0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

The aim of this study was to examine the association between antral follicular blood flow velocity and the response of ewes to hormonal ovarian superstimulation. Ten Santa Inês ewes were subjected to a short- (7days; Group 1) or long-term (13days; Group 2) progesterone (CIDR ® ; InterAg, Hamilton, New Zealand) priming, and a superovulatory treatment with porcine follicle-stimulating hormone (pFSH; Folltropin ® -V; Bioniche Animal Health, Belleville, ON, Canada), given twice daily for four consecutive days in decreasing doses and initiated four or ten days after CIDR insertion, respectively. Embryos were recovered surgically seven days after the last pFSH dose. From one day prior to until the end of the pFSH regimen (Days -1 to 3), all ewes underwent daily transrectal ultrasonography of ovaries. The number of high-velocity pixels (HVPs; 0.055-0.11m/s or upper 50% of recordable velocities) on Day 1 correlated directly with the number of corpora lutea (CL; r=0.92, P=0.0002) and of viable embryos (r=0.77, P=0.01). Correlations were also recorded between the number of HVPs on Day 3 and the recovery rate (r=-0.69, P=0.03), viability rate (r=-0.64, P=0.05), and percentage of degenerated embryos (r=0.65, P=0.04). The percentage of HVPs relative to the total area of ovarian cross section on Day 1 was correlated with the number of CL (r=0.95, P<0.001) and of viable embryos (r=0.85, P=0.002). This parameter on Day 3 was also correlated with the recovery rate (r=-0.69, P=0.03). The percentage of HVPs relative to the total Doppler area on Day 0 was correlated with the recovery rate (r=0.72, P=0.02). It can be concluded that sonographic assessment of high-velocity antral follicular blood flow has the makings of a useful non-invasive method to predict the outcome of the superovulatory treatment in ewes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate the effect of two sources of commercial porcine pituitary-derived follicle-stimulating hormone (pFSH) and pFSH-porcine Luteinizing Hormone (pLH), including equine chorionic gonadotropin (eCG), in ovulatory and embryonic response in Pelibuey sheep. Twenty-four Pelibuey sheep were used and were assigned randomly to four treatments (n = 6): (T1; 200 mg pFSH-Folltropin ® ); (T2; 200 mg pFSH + 300 UI eCG-Folligon ® ); (T3; 250 UI pFSH/pLH-Pluset ® ) and (T4; 250 UI pFSH/pLH + 300 UI eCG). The interval of hours from withdrawal of the device to the beginning of oestrus (BO) was lower (p < .05) in sheep treated with eCG (T2 = 8.0 ± 1.4 and T4 = 10.0 ± 2.8) than in those without eCG (T1 = 12.6 ± 0.6 and T3 = 20.6 ± 2.4). The ovulatory rate (OR) was higher (p < .05) in T1 = 15.5 ± 2.8 and T2 = 15.6 ± 1.4, compared to T3 = 8.1 ± 3.2 and T4 = 11.8 ± 2.8; a significant difference was not shown between them (T1 vs. T2 and T3 vs. T4) when including eCG. The number of non-fertilized oocytes (NFO) was lower (p ˂ .05) in T1 = 0.8 ± 0.4 and T3 = 1.8 ± 1.8, compared to those that included eCG (T2 = 6.3 ± 2.4 and T4 = 2.1 ± 1.2). The number of transferable embryos (TE) was higher (p < .05) when FSH was applied (T1 = 5.8 ± 1.1), compared with (T2 = 2.6 ± 1.1, T3 = 2.3 ± 1.4 and T4 = 2.8 ± 1.5). The commercial treatments (pFSH or pFSH-pLH) in combination with eCG did not improve OR, NFO and TE. However, the exclusive pFSH (Folltropin) treatment presented a higher OR, lower number of NFO and higher number of TE., (© 2017 Blackwell Verlag GmbH.)

The effect of extending the length of the FSH treatment protocol on superovulatory response and embryo production was investigated in wood bison during the anovulatory and ovulatory seasons. In Experiment 1 (anovulatory season), follicular wave emergence was synchronized by follicular ablation (Day -1) and bison were assigned randomly to two groups (n = 14/group) and given 200 mg FSH on Day 0 and Day 2 (non-extended group), or 133 mg FSH on Days 0, 2, and 4 (extended group). Human chorionic gonadotropin (hCG; 3000 IU) was given on Day 5 and Day 6 in the non-extended and extended groups, respectively, and bison were inseminated 12 and 24 h later. Ova/embryos were collected 8 days after hCG treatment. In Experiment 2 (ovulatory season), bison were synchronized and superstimulated as in Experiment 1 (n = 12/group), but prostaglandin was given to control CL development. Data were compared by t-test and Chi-square test. In Experiment 1, no differences in ovarian response or embryo production between groups were detected. In Experiment 2, there was no difference in the ovarian response between groups, however, a greater number of ova/embryos (4.3 ± 0.8 vs. 2.3 ± 0.4; P ≤ 0.05) and freezable embryos (2.5 ± 0.6 vs. 1.2 ± 0.4; P ≤ 0.05) were obtained in the extended group. The number of freezable embryos was greater during the ovulatory vs anovulatory season (1.8 ± 0.4 vs. 0.3 ± 0.2; P ≤ 0.05). In conclusion, extending the FSH treatment in wood bison did not improve the superovulatory response during the anovulatory season, but resulted in twice as many freezable embryos during the ovulatory season. The number of freezable embryos collected during the anovulatory season was <20% that of the ovulatory season., (Copyright © 2017. Published by Elsevier Inc.)

Experiments were done to determine if inclusion of eCG and progesterone in the superstimulation protocol will increase the ovarian response and embryo production in wood bison, and to provide preliminary information regarding the effect of season. In Experiment 1 (anovulatory season), bison (n=26) were synchronized by follicular ablation (Day -1) and given FSH on Days 0 and 2, and assigned to 3 groups: Progesterone (Days 0-4), eCG (Day 3), or progesterone+eCG. On Day 5, bison were given hCG and inseminated 12 and 24h later. Ova/embryos were collected 8days after hCG. In Experiment 2 (ovulatory season), bison (n=24) were synchronized and assigned randomly to two groups in which superstimulation was induced with FSH, either with or without eCG, as in Experiment 1. No differences among groups were found in ovarian response or embryo production in either experiment. The follicular count at wave emergence was positively correlated with the number of large follicles at the end of superstimulation in all groups. A significantly greater number of follicles present at wave emergence in the anovulatory vs. ovulatory season was associated with a greater number of CL at the time of embryo collection, but only half the number of freezable embryos. In conclusion, the number of transferable embryos collected (1-2/bison) was higher than in any previous report, but was not attributable to the inclusion of eCG or progesterone in the superovulatory protocol. The apparent effect of season on oocyte competence, and not superovulatory response, is worthy of further investigation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effect of varying intervals between successive gonadotropin injections on the superovulatory outcomes in anestrous Rideau Arcott ewes superstimulated for ovarian follicular development with multiple doses of porcine FSH (pFSH) was evaluated in a single study. Twenty-five animals received six (1×2.5ml and 5×1.25ml) injections of Folltropin ® -V given at 0800 and 1600h or at 0800 and 2000h in Group 1 (n=9) or Group 2 (n=16), respectively. An i.m. injection of 500 IU of equine chorionic gonadotropin (eCG; Folligon ® ) was given concurrently with the first pFSH dose. Time of estrus was synchronized among ewes with intravaginal sponges containing 60mg of medroxyprogesterone acetate (Veramix ® ) that were left in place for 14days; sponges were removed at the time of the 5th pFSH injection. Six days after insertion of MAP sponges, all ewes received an i.m. injection of estradiol-17β dissolved in 1ml of sesame oil (350μg/ewe) to synchronize follicular wave emergence. Following the last pFSH dose, all animals were given a single i.m. injection of 50μg of gonadotropin-releasing hormone (GnRH; Cystorelin ® ) to induce ovulations before placing in a pen with four fertile rams for 36h. The ovarian responses were assessed and embryos recovered surgically 7days after GnRH injections. The mean number of corpora lutea was greater (P<0.05) in Group 1 compared with Group 2 ewes (21.0±2.9 compared with 10.4±1.6, respectively; mean±SEM) but there was no difference (P>0.05) in the number of transferable embryos (5.4±2.4 compared with 5.4±1.3/ewe, respectively), and Group 1 animals had significantly more degenerated embryos than Group 2 ewes (2.6±1.2 compared with 0.6±0.3/ewe, respectively). A superovulatory protocol wherein pFSH injections were given at 0800 and 1600h was more effective in terms of inducing multiple ovulations than the protocol with 12-h intervals between consecutive pFSH doses, but it was not associated with an increased production of transferable quality embryos by anestrous ewes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The current study was performed to investigate the effect of oocyte donor status, including age and body weight, on metaphase II (MII) oocyte recovery using two superovulation methods in cynomolgus monkeys. The use of Method A [recombinant gonadotrophin (75 IU/kg, 3 ×, 3-day intervals) and human chorionic gonadotropin (hCG)] led to great increases in ovary size and the mean number of MII oocytes retrieved in age- and body-weight-dependent manner; in contrast, both the parameters were similar in Method B [recombinant gonadotrophin (60 IU, twice daily, 6 days), recombinant gonadotropin and recombinant human luteinizing hormone (rhLH) (60 IU, twice daily, 3 days), and hCG]. Importantly, Method A showed maximal MII oocyte recovery rate in > 60-month-old or 4.5-5.0-kg female monkeys, whereas Method B was equally effective regardless of the donor age and body weight. These results indicate that superovulatory responses depend on the interaction between oocyte donor status and the superovulation method used in cynomolgus monkeys.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

To optimize the superovulation protocol in Thai native cattle, the present research was designed to (1) compare three different protocols designed to induce superstimulation and (2) study the effect of gonadotropin-releasing hormone (GnRH) administration at insemination time (to induce ovulation) on ovarian follicular activities in terms of the number of large follicles, corpora lutea (CLs) and unovulated follicles, and the number and quality of ova/embryos recovered in Thai native heifers. Initially, the estrous cycles of animals (n = 36) at unknown stages were synchronized by two prostaglandin F 2α (PGF 2α ) injections at an interval of 12 days. Follicular development of heifers was randomly superstimulated with one of three different treatment protocols: treatment A-a total of 100 mg of pituitary-derived FSH (pFSH; Folltropin®-V) administered in eight decreasing doses; treatment B-a single dose of 100 mg pFSH dissolved in 30% (w/v) polyvinylpyrrolidone; or treatment C-ablation of all follicles ≥5 mm with a single dose of pFSH. All heifers received PGF 2α 48 h after the initiation of FSH treatment to induce luteolysis from the previous cycle, and they were twice inseminated at 12 and 24 h after the onset of estrus. Heifers in each treatment were assigned to be injected or not with GnRH at the time of first insemination with frozen/thawed semen to induce ovulation. About 7 days after artificial insemination (AI), ova/embryos were collected and classified. The numbers of large follicles at the onset of estrus were not statistically significantly different; meanwhile, the maximum diameters of follicles at the time of first insemination in treatment C were smaller compared with the other treatment groups (p < 0.001). The administration of GnRH at the first insemination time resulted in a greater number of CLs and fewer unovulated follicles at the time of ova/embryo collection (p = 0.001), which subsequently resulted in a higher number of total ova/embryos recovered (p = 0.030). Among heifers treated with different superstimulation protocols, the ablation of all follicles ≥5 mm in diameter before superstimulation (treatment C) resulted in significantly higher quality of fertilized ova and transferable embryos (p = 0.001). In summary, it could be inferred that GnRH treatment improved ovarian function rather than embryo quality. Dominant follicle ablation prior to superstimulation is preferable for collecting a greater number of transferable embryos.

The goal of this study is to estimate the effects of low-level laser irradiation (LLLI) on the superovulatory response according to the number of corpora lutea (CL), follicles (F) and the embryo yield. In recent years, while searching for new, more efficient and organic methods to improve superovulatory response and embryo yield with respect to the conventional methods, low-level laser irradiation (LLLI) is a more sensitive and less costly technology that can be used to improve animal reproduction, namely, artificial insemination and the embryo production system. The dairy-cow donors were treated for superovulation with Pluset®, at any time during the oestrus cycle, and the total dose per donor was 700 IU. The first group of the donors (n=25), test group (TG), was irradiated on the sacroiliac area for 180 seconds per day, from the 1st to 11th superovulatory treatment (ST) days in a row, with LLLI in the 870-970-nm wavelength, 65.93 J/cm dose, frequencies in the 20-2000 Hz range and pulse durations commonly in the range of about 1 second. For the second control group (CG) (n=25), the ST was performed without LLLI. After the ST, The mean number of CL in the right side ovaries in the TG was 25.43% (p<0.05) greater than in those of the CG. The number of total recovered and transferable embryos was greater in the TG compared with the CG by 28.97% (p<0.05) and 15.8% (p>0.05), respectively. With respect to conventional methods, LLLI can be used to improve the superovulatory response and embryo yield as a supplementary environment and animal-friendly method of treatment.

Two commercial FSH products were compared in a retrospective study on 3990 commercial superovulations and embryo recoveries in dairy heifers and cows. In addition, the 56-day nonreturn rate of 19,400 embryos produced with these two preparations was analyzed. Embryo collections were performed during a 16-year period from donors of Holstein and Ayrshire breeds. Folltropin (Vetoquinol S.A., Lure cedex, France) group (Group F) consisted of 2592 superovulations, of which 80% were performed on heifers and 20% on cows, and Pluset (Laboratorios Calier, S.A., Barcelona, Spain) group (Group P) of 1398 treatments, of which 66% and 34% were on heifers and cows, respectively. Total number of recovered structures, number of transferable embryos, and the proportion of unfertilized ova (UFO) and degenerated embryos were analyzed. Distribution of embryos into quality grades (1-3) and developmental stages (4-9) according to the IETS classification guidelines and means for each collection were evaluated. The proportion of low-responders having fewer than five corpora lutea and yielding fewer than five embryos or ova was investigated for each treatment. Group P yielded 1.1 recovered structures more than Group F (P < 0.001). Consequently, however, the number of transferable embryos did not differ among the groups, being 7.0 and 7.1 in Groups F and P, respectively. Instead, there was an increase in the number of UFO from 2.0 in Group F to 3.0 in Group P (P < 0.001). The quality of embryos and the developmental stages were similar between the groups and there was no difference in the proportion of low-responding donors in Group F and Group P. Also, there was no difference in the nonreturn rate after transfer of embryos originating from donors superovulated with Folltropin or Pluset. It was concluded that equal numbers of transferable embryos and pregnancies can be achieved with Folltropin and Pluset., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study investigated the effect of sex-sorted semen compared with conventional semen on the outcome of embryo recovery, placing special emphasis on the quality, and developmental stage of embryos. Data were analyzed for 443 embryo collections with sex-sorted semen (SEX group) and 1528 with conventional semen (CONV group) in superovulated dairy heifers and cows. The insemination protocol for conventional semen included two inseminations, comprising a total dose of 30 million sperm passing into the uterine body. For sex-sorted semen, two (30%) to three (70%) deep uterine inseminations were performed, the total dose ranging from eight to 12 million sperm. The data were analyzed separately for heifers and cows. The total number of recovered structures was similar among the groups. The number of viable embryos decreased in the SEX groups compared with the CONV (with 1.4 and 3.2 fewer embryos in heifers and cows, correspondingly, P < 0.001), and correspondingly the proportions of unfertilized ova and degenerated embryos increased in the SEX groups (P < 0.001). The proportion of unsuccessful collections, yielding no transferable embryos, increased in the SEX groups for both heifers (from 7.2% to 11.2%, P = 0.025) and cows (from 9.0% to 20.7%, P < 0.001). Regarding the quality of viable embryos, the quality grades were superior in the CONV group compared with the SEX group for heifers (P < 0.001) and cows (P < 0.001). The proportion of grade 1 embryos decreased by 6.5 percentage points in heifers and 11.9 percentage points in cows when sex-sorted semen was used. Correspondingly, the proportions of grade 2 and 3 embryos increased in heifers and cows when sexed semen was used. The mean developmental stages of embryo collections were numerically slightly lower in the SEX group. In heifers, the delay in developmental stage was statistically significant (P = 0.001), but in cows, there was only a tendency toward that (P = 0.067). In conclusion, sex-sorted sperm decreased the transferable embryo yield and increased the risk of a recovery yielding no transferable embryos. Furthermore, use of sex-sorted semen decreased the proportion of grade 1 embryos. In addition, it also seemed to delay embryonic development, although the delay in embryonic development was minimal and its biological relevance remains undefined. Despite the compromised embryo production, taken into account the optimization of recipient resources, the use of sex-sorted semen is advantageous, especially in superovulated heifers, which are of most importance in the modern breeding strategies using genomic selection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Superovulation (SOV) is a necessary technique to produce large numbers of embryos for embryo transfer. In the conventional methods, follicular stimulating hormone (FSH) is administered to donor cattle twice daily for 3 to 4 days. As this method is labor intensive and stresses cattle, improving this method has been desired. We previously developed a novel and simple SOV method, in which the intramuscular injection of a single dose of FSH in aluminum hydroxide gel (AH-gel) induced the growth of multiple follicles, ovulation and the production of multiple embryos. Here we show that AH-gel can efficiently adsorb FSH and release it effectively in the presence of BSA, a major interstitial protein. When a single intramuscular administration of the FSH and AH-gel mixture was performed to cattle, multiple follicular growth, ovulation and embryo production were induced. However, the treatments caused indurations at the administration sites in the muscle. To reduce the muscle damage, we investigated alternative administration routes and different amounts of aluminum in the gel. By administering the FSH in AH-gel subcutaneously rather than intramuscularly, the amount of aluminum in the gel could be reduced, thus reducing the size of the induration. Moreover, repeated administrations of FSH with AH-gel did not affect the superovulatory response. These results indicate that a single administration of FSH with AH-gel is an effective, novel and practical method for SOV treatment.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Improvement of the superovulation technique will help to enhance the efficiency of embryo and animal production. Blocking inhibin using inhibin antiserum (IAS) is known to promote follicular development by increasing the level of FSH. Previously, we reported that coadministration of IAS and eCG produced more than 100 oocytes from a single female C57BL/6 mouse at 4 weeks old. The oocytes derived from the IAS + eCG (IASe) treatment were able to fertilize and develop normally into offspring. In this study, we examined the effect of IASe treatment on the numbers of ovulated oocytes in major inbred (A/J, BALB/cByJ, C3HeJ, DBA/2J, and FVB/NJ) and outbred (CD1) mice strains at 4 weeks old. We confirmed the fertilization and developmental ability of the IASe-derived oocytes. IASe treatment ovulated 1.5 to 3.2 times higher numbers of oocytes than eCG treatment alone. The fertilization rate of IASe-derived oocytes was similar to that of eCG-derived oocytes. In vitro and in vivo developmental rates of the embryos derived from IASe were similar to the rates of embryos derived from eCG. We have shown that superovulation by IASe is very effective in obtaining high numbers of ovulated oocytes from small numbers of oocyte donor in a number of mice strains. The superovulation technique will contribute to the archiving of cryopreserved embryos of genetically engineered mice using small numbers of donors and has the potential to produce more live animals for rederivation of the archived mouse lines in mouse repositories., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF-induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Controlled breeding programmes utilising exogenous hormones are common in the Australian sheep industry, however the effects of such programmes on cervicovaginal mucus properties are lacking. As such, the aim of this study was to investigate cervicovaginal (CV) mucus from naturally cycling (NAT), progesterone synchronised (P4), prostaglandin synchronised (PGF2α), and superovulated (SOV) Merino ewes. Experiment 1; volume, colour, spinnbarkeit, chemical profile and protein concentration of mucus (NAT, P4, PGF2α and SOV; n=5 ewes/treatment) during the follicular (5 d) and luteal phases (8 d) was investigated. Experiment 2; in vivo mucus pH and in vitro mucus penetration by frozen-thawed spermatozoa (NAT, P4 and SOV; n=11 ewes/treatment) was investigated over oestrus (2 d) and the mid-luteal phase (pH only, 2 d). Oestrus mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus (p<0.05). SOV increased mucus production and protein concentration (p<0.05) while PGF2α reduced mucus volume (p<0.05). Mucus pH (oestrus 6.2-6.5), chemical profile and mucus penetration by sperm were unchanged (p>0.05). Results indicate that exogenous hormones used for controlled breeding affect cervicovaginal mucus production, but few other tested characteristics. Further research is required to explain fertility differences between synchronised and naturally cycling animals following cervical AI., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Japanese field vole (Microtus montebelli) is a wild-derived rodent and have unique characteristic. Thus, these species have been expected as model animal. This study was performed to develop novel superovulation procedure for Japanese field vole. First, when 30 IU pregnant mare's serum gonadotropin (PMSG) and 30 IU human chorionic gonadotropin (hCG) were administrated 48 hours apart, females showed higher response to hCG compared with three concentrations of PMSG. Second, to effectively induce ovulation on females after vaginal opening, they were mated with vasectomized male instead of hCG administration. Average number of ovulated oocytes using PMSG mating (13.9 ± 1.9 oocytes) was higher than PMSG-hCG (control; 6.9 ± 2.3 oocytes) or PMSG-hCG mating (6.8 ± 0.8 oocytes). Finally, we attempted superovulation using GnRH agonist (GnRHa). With this treatment, we speculated that GnRHa might induce endogenous luteinizing hormone releasing to cause ovulation. Such superovulation was performed with 30 IU PMSG and different concentration of 20% polyvinylpyrrolidone-GnRHa (15, 30, 45, and 60 μg/kg). As results, average number of ovulated oocytes was highest with 30 μg/kg GnRHa (14.5 ± 4.1 oocytes). The numbers of ovulated oocytes of other concentrations were 5.0 ± 1.4 (15 μg/kg), 12.8 ± 2.7 (45 μg/kg), and 8.8 ± 3.7 oocytes (60 μg/kg). Nuclear status of most collected oocytes was the second meiotic division (range, 94.3%-100%). These superovulation procedures will be useful for development of in vitro culture systems and assisted reproductive technologies for not only Japanese field vole but also other voles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Traditional superovulation protocols that include multiple gonadotropin treatments are time-consuming and labor intensive. These protocols require multiple handling and restraining of embryo donors. This will likely increase the risks of injuries in both animals and humans and induce stress that may lead to a reduced superovulatory response. These are more evident when working with cattle that are rarely handled or raised on extensive grazing. The objectives of this experiment were to compare the efficacy of a split-injection protocol of porcine pituitary-derived porcine FSH (pFSH) preparation (slow release [SR] group) to the traditional 4-day treatment with pFSH administered twice daily (C group) and to determine the concentrations of cortisol in the hair as a marker of activation of the hypothalamic-pituitary-adrenal (HPA) axis during the two superovulatory treatments. Thirty-two heifers were stimulated twice in a 2 × 2 crossover design and compared for ovarian response and numbers and characteristics of recovered ova-embryo among treatments. No differences between SR and C groups were found in terms of percentage of responsive animals (100% vs. 93.8%) and ovulation rate (83.7 ± 1.1 vs. 79.5 ± 1.0%). A positive correlation was found between the number of follicles responsive to pFSH (2-8 mm) at the beginning of treatments and the superovulatory response, and no differences were found in these follicular populations between the two treatment groups. The numbers of CLs, ova-embryos, fertilized ova, transferable and freezable embryos recovered per cow were found to be significantly higher in SR compared with C group (14.0 ± 1.6 vs. 10.6 ± 1.0, 12.1 ± 1.6 vs. 7.6 ± 1.0, 11.1 ± 1.1 vs. 7.3 ± 1.0, 9.6 ± 1.4 vs. 6.6 ± 1.0, and 9.4 ± 1.4 vs. 6.0 ± 1.0 for SR and C group, respectively). The SR group produced also a significantly greater number of excellent- and/or good-quality embryos compared with the C group. The concentrations of cortisol in the hair at Days 14 and 21 were significantly greater in the C compared with the SR group (P < 0.05). In conclusion, these results indicate that the dilution of gonadotropin in a 0.5% hyaluronan solution, reducing the administration frequency, improves the quantitative and qualitative superovulatory response of Marchigiana heifers [corrected]. Further studies using other breeds of cattle are needed to verify the results herein obtained and to confirm the lower activation of the hypothalamic-pituitary-adrenal axis caused in the donor by the split-injection protocol., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.

Two experiments were carried out in goats to evaluate the effects of the FSH/LH ratio during treatment on ovarian response and embryo production (experiment 1) and the efficiency of progestin supplementation on pregnancy and the survival of vitrified embryos (experiment 2). In experiment 1, 30 goats were synchronized and allocated to 2 groups (n = 15) corresponding to the following superovulatory treatments with p-FSH (250 IU, over 3 days) having different doses of purified FSH and LH: (group A) control, FSH/LH ratio of 1, kept constant during treatment; (group B) FSH/LH ratio of 2 and daily FSH/LH ratio of 5.0:1.0:0.3 for the first, second, and third days of treatment, respectively. Ovarian response and embryo production were assessed 7.5 days after estrus. In experiment 2, 46 vitrified blastocysts from p-FSH-superovulated donors were transferred to 26 recipients (2 blastocysts per goat) 7.5 days after estrus. The recipients were synchronized with donors and allocated to 2 experimental groups (n = 13). Group C received progestin supplement as fluorgestone acetate (FGA) inserted into the vagina at the time of embryo transfer, replaced with a new one 16 days later, and maintained until the 45th day of pregnancy; group D, no treatment (control). Pregnancy was diagnosed by transrectal ultrasound scanning on Days 30 and 45 after estrus and followed to term. The results indicated that the increase in FSH/LH ratio from 1 to 2 with decreasing daily FSH/LH (treatment B) did not improve the superovulatory response. Superovulatory treatment A (control) advanced (P < 0.05) the onset of estrus and showed a higher ovulation rate compared to group B (14.9 vs. 10.9; P < 0.05). Fertilization rate, embryo yield, and mean number of transferable embryos in group A (7.5) were higher (P < 0.05) than those in group B (3.2). Recipient goats receiving progestin supplementation (group C) showed a higher (P < 0.05) pregnancy rate and embryo survival (kids born per embryos transferred; 69.3% and 73.1%) than the controls (group D; 23.3% and 19.2%). In conclusion, regimen A with FSH/LH ratio of 1 kept constant during the treatment gave the best ovarian response and embryo production. The progestin supplementation as FGA-pessary administered at embryo transfer time to the 45th day of pregnancy improved the pregnancy rate, kidding rate, and embryo survival of transferred vitrified embryos. Intravaginal progestin supplement has the potential to reduce the incidence of pregnancy losses during early pregnancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Producing many mature oocytes is of great importance for assisted reproductive technologies. In mice, superovulation by consecutive injections of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) has been the gold standard for oocyte collection. However, the yield of mature oocytes by this regimen can fluctuate according to the stage of the estrous cycle, strain, and age. Therefore, our objective was to develop a high-yield superovulation protocol to collect higher numbers of oocytes from adult female mice of different strains and ages. First, we aimed to synchronize the estrous cycle using C57BL/6 (B6) female mice. Most (93%) were synchronized to metestrus after two daily injections of progesterone. Second, we found that with the injection of anti-inhibin serum (AIS) instead of eCG, the mean number of ovulated oocytes almost doubled (21 vs. 41 per mouse). Third, by combining estrous cycle synchronization with two AIS injections, we obtained 62 oocytes per mouse, about three times that with the eCG-hCG protocol. Importantly, this approach increased the proportion of mice that ovulated >25 oocytes from about 40% (eCG-hCG) to 90%. The same protocol was also effective in other inbred (BALB/cA), outbred (ICR), and hybrid (B6D2F1) strains. In addition, B6 female mice aged over 1 yr ovulated 1.8-fold more oocytes by this protocol. Thus, estrous cycle synchronization followed by AIS-hCG yielded a broadly applicable, highly efficient superovulation. This protocol should promote the effective use of invaluable female mouse strains and decrease the numbers of animals euthanized., (© 2016 by the Society for the Study of Reproduction, Inc.)

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.

The effects of equine chorionic gonadotropin (eCG) on follicular development and ovulation in cyclic guinea pigs were investigated by histological and immunohistochemical analyses. Three groups of guinea pigs (n=12) were administrated subcutaneously with saline, 20 or 50 IU of eCG, respectively, on cyclic Day 12 (Day 1=vaginal openings). Ovaries were collected at 4 and 8 d after administration (6 animals per group each time). The eCG administration induced significant and distinct morphological changes in the ovaries, as it promoted the luteinization of granulosa cells, but not follicular development. In addition, proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) were immunolocalized specifically in luteinized follicles. Our experiments together indicate that eCG administration can induce follicular luteinization but not superovulation in guinea pigs. The eCG in cyclic guinea pigs functions similar to that of luteinizing hormone (LH), but not follicle-stimulating hormone (FSH).

Many studies identified new components of the renin–angiotensin system (RAS), such as Angiotensin-(1-7) [Ang-(1–7)] and Angiotensin-converting enzyme type 2 (ACE2), in mammalian ovaries.We previously showed Angiotensin-Converting Enzyme (ACE) inhibition, which increases the level of Ang-(1–7), stimulated ovarian estradiol output in ewe after estrous synchronization. Considering that Ang-(1–7) stimulates ovarian function and elevated estradiol before ovulation is associated with increased chance of achieving pregnancy, the present study investigated whether ACE inhibition throughout a superovulation protocol in ewe might improve ovulation outcome. At first, immunohistochemistry in ovaries of nonpregnant ewes revealed localization of Angiotensin II (Ang II), Ang-(1–7) and ACE2 in theca cells of antral follicles and in corpus luteum. Ang II and Ang-(1–7)were also detected in follicular fluid (FF) by Radioimmunoassay (RIA). Enalapril treatment throughout the superovulation protocol decreased 17β-estradiol (E2) output and raised progesterone:estradiol (P4:E2) ratio without a direct influence on ovulation and quality of embryos.

In this study, the performance of 300 Changbaishan Black cattle treated for superovulation from June to September was evaluated to determine the optimal conditions and herds for bovine embryo production. Data analysis revealed that cattle treated in July and August had higher numbers of available embryos (NAE), M1 embryos (NM1), and total embryos (NTE), as well as a higher percentage of M1 embryos (PM1). The temperature and precipitation observed during July and August were greater than those seen in the other two months; strong correlations were observed between these traits and the choice of month of treatment. In addition, multiparous cattle showed a better performance, higher NTE, NAE, NM1, and PM1 values, higher percentages of available embryos, and a lower percentage of degenerated embryos. The co-efficient correlation analysis showed that the month chosen for the treatment did not affect the superovulation traits of nulliparous cattle; however, the choice of the month affected multiparous cattle. Multiparous and nulliparous cattle exhibited many significant differences when treated in July and in August. In addition, the superovulatory traits of multiparous cattle, and not the nulliparous cattle, were strongly correlated to the choice of month of treatment. The results suggested that superovulation is more effective during a period with appropriate environmental temperature and humidity, and that multiparous cattle are more suitable for morula production.

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n=4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24h after induced luteolysis (penultimate day of FSH) and cumulus-oocyte complexes were collected 24h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P<0.01). On average across all groups, oocytes contained 22pL of lipids (3.3% of ooplasm volume) distributed as 3000 droplets. Average volume of individual lipid droplets was higher in the FSH Starvation (11.5±1.5 10(-3) pL, P=0.03) compared with the Short and Long FSH groups (7.2±0.6 10(-3) and 8.0±0.8 10(-3) pL, respectively). In conclusion, both FSH Starvation and Short FSH treatments yielded a lower proportion of mature oocytes compared with the Long FSH treatment. Furthermore, FSH starvation led to an accumulation of larger lipid droplets in the ooplasm, indicating atresia. Our results indicate that a longer superstimulation period in beef cattle yields higher numbers and better-quality oocytes.

Follicular wave status at the beginning of exogenous FSH administration is an important contributor to variability in superovulatory responses in ruminants. Studies in ewes have shown a decrease in the number of ovulations when superovulation is initiated in the presence of ostensibly ovulatory-sized ovarian follicles. Hormonal ablation of large antral follicles with the progestin-estradiol (E2-17β) treatment significantly reduces this variability in superovulated anestrous ewes, but the effects of the treatment in cycling ewes have not yet been assessed. Sixteen Rideau Arcott × Polled Dorset ewes (November-December) received either medroxyprogesterone acetate (MAP)-releasing intravaginal sponges (60 mg) or controlled internal drug release (CIDR) devices (containing 300 mg of natural progesterone) for 14 days (Days 0-14), with a single intramuscular injection of 350 μg of E2-17β on Day 6. The superovulatory treatment consisted of six injections of porcine FSH (Folltropin-V) given twice daily, followed by a bolus GnRH injection (50 μg intramuscular) on Day 15. There were no differences (P < 0.05) in the ovulatory responses and embryo yields between the two groups of ewes. In both subsets of animals, the next follicular wave emerged ∼2.5 days after an E2-17β injection (P > 0.05). A decline in maximum follicle size after an E2-17β injection was more abrupt in CIDR- compared with MAP-treated animals, and the ewes pretreated with exogenous progesterone had significantly more 3-mm follicles at the start of the superovulatory treatment. The metabolic clearance rate of exogenous E2-17β appeared to be greater in MAP-treated ewes, but circulating concentrations of porcine FSH failed to increase significantly after each Folltropin-V injection in CIDR-treated animals. The CIDR-treated ewes exceeded (P < 0.05) their MAP-treated counterparts in serum E2-17β concentrations during superovulation. In spite of differences in antral follicle numbers and endocrine profiles between MAP- and CIDR-treated cyclic ewes receiving E2-17β before ovarian superstimulation, there were no differences in superovulatory responses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A study was designed to compare the effects of a conventional (4 days) versus a lengthened (7 days) superstimulation protocol on follicle dynamics and to test the hypothesis that superstimulatory treatment only rescues small follicles within the wave. Nonlactating beef cows received a progesterone-releasing intravaginal device [PRID] and were superstimulated with 400-mg FSH on the day of follicle ablation-induced wave emergence (Day 0). The control group (n = 5) received FSH over 4 days, whereas the long group (n = 5) received FSH over 7 days. PGF was administered twice on Day 2 (control group) or 5 (long group), and PRIDs were removed 24 hours after the first PGF. Cows received 25-mg LH 24 hours after PRID removal. The cows chosen for the present study represented a subset from a larger group of 24 cows in which superovulation results were obtained and published. Cows in the present study were those with the lowest antral follicle counts at the time of wave emergence in order to facilitate tracking of individual follicles. Daily ultrasonographic examinations monitored follicle diameters and numbers. A reduction (P < 0.01) in the number of 1- to 2-mm follicles from Day 0 to the end of the superstimulatory treatment was associated with a progressive shift to the next size category in both groups. On the day of LH treatment, the number of follicles in any size category did not differ between groups. The number of follicles of diameter 5 mm or less decreased during superstimulation (i.e., no continuous recruitment of small follicles; P < 0.001), and the number of follicles of diameter 1 mm or greater at the end of superstimulation did not differ from that at the beginning of superstimulation (P = 0.739). However, the total number of follicles of diameter 3 mm or greater at the end was greater than the number of follicles of diameter 3 mm or greater at the beginning of superstimulation because of growth of the 1- to 2-mm population during treatment (P < 0.001). Follicle growth was slower in the long group than in the control group. Results support the hypothesis that both 4-day and 7-day superstimulatory protocols rescue small antral follicles present at the time of wave emergence; there was no evidence for continuous recruitment of new follicles. Results also provide rationale for the hypothesis that a lengthened treatment protocol is associated with greater follicle maturation and capacity to ovulate., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To evaluate multiple follicular development synchronization after estrogen stimulation in prepubertal mice, follicular responsiveness to gonadotropin superovulation, the prospective reproductive potential and ovarian polycystic ovary syndrome (PCOS)-like symptoms at adulthood, prepubertal mice were intraperitoneally injected with estrogen to establish an animal model with solvent as control. When synchronized tertiary follicles in ovaries, in vitro oocyte maturation and fertilization rates, blastocyst formation rate, developmental potential into offspring by embryo transfer, adult fertility and PCOS-like symptoms, and involved molecular mechanisms were focused, it was found that estrogen stimulation (10 μg/gBW) leads to follicular development synchronization at the early tertiary stage in prepubertal mice; reproduction from oocytes to offspring could be realized by means of the artificial reproductive technology though the model mice lost their natural fertility when they were reared to adulthood; and typical symptoms of PCOS, except changes in inflammatory pathways, were not remained up to adulthood. So in conclusion, estrogen can lead to synchronization in follicular development in prepubertal mice, but does not affect reproductive outcome of oocytes, and no typical symptoms of PCOS remained at adulthood despite changes related to inflammation.