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This study evaluated the use of flunixin meglumine to prevent the occurrence of premature corpus luteum (CL) regression in superovulated ewes, improving embryo recovery and viability. Ewes (n=23) submitted to conventional superovulatory protocol and laparoscopic artificial insemination were treated with 2.2 mg/kg/day of flunixin meglumine (FLU, n=12) or 1.5 mL saline solution (CONT, n=11) on Days 2, 3, and 4 (Day 0 = 48 h after device removal). Serum progesterone (P 4 ) concentrations were measured (Day 1-6). Ultrasound (US, Days 3 and 6) and laparoscopic evaluation (Day 6) were performed to identify luteinized structures. In the US, laparoscopy, and P 4 assessments, the percentage of ewes with premature CL regression differed (P<0.05) between CONT (54.5; 63.6; and 54.5 %) and FLU (0.0; 0.0; and 0.0 %), respectively. The US exams revealed the effect (P<0.05) of treatment on the number of regressing CL between CONT (1.4 ± 0.6) and FLU (0.0 ± 0.0). Greater (P<0.05) number of normal CLs (10.5 ± 1.8 vs. 4.4 ± 1.5), ova/embryos (9.1 ± 2.1 vs. 3.7 ± 1.3), viable embryos (5.1 ± 1.1 vs. 2.6 ± 1.2), and recovery rate (79.5 ± 9.6 vs. 41.3 ± 15.0 %) were observed in FLU compared to CONT, respectively. The embryo viability rate did not differ (P>0.05) between FLU (60.7 ± 10.5 %) and CONT (45.5 ± 16.1 %). In conclusion, the flunixin meglumine protocol was able to prevent the occurrence of premature CL regression in superovulated ewes, increasing the recovery rate and embryo production., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study evaluated the effect of prostaglandin F2α (PGF2α) associated with gonadotropin-releasing hormone (GnRH) for ovulation induction in precocious indicus heifers submitted to a fixed-time superovulation (SOV) programme. Precocious Nellore heifers (n = 35), aged 13 months, were subjected to the SOV protocol. On day 0 (D0), all animals received intravaginal insertion of a progesterone (P4) device along with intramuscular administration of 2 mg of oestradiol benzoate, plus 200 IU of follicle-stimulating hormone in decreasing doses, with 12-h intervals between D4 and D7, in addition to 150 μg of D-cloprostenol on D6 and device removal on D7. On D8, the donors received 10.5 μg of buserelin acetate and the treatment group received 300 μg of D-cloprostenol/PGF 2α . Artificial insemination was performed 12 h and 24 h after GnRH administration using frozen semen. On D15 of the protocol (i.e., D7 after insemination), the embryos were collected and evaluated. All animals passed through the control and treatment groups. Results were evaluated by analysis of variance using an adjusted mixed-effects model (p < 0.05). There was no difference in the total number of embryos between the control and treatment groups (10.40 ± 1.52 vs. 9.60 ± 1.36; p = 0.63) or viable embryos (6.30 ± 1.22 vs. 4.30 ± 0.71). For precocious indicus heifers, treatment with PGF 2α in association with GnRH did not affect embryo production in the fixed-time SOV protocol., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Purpose: To compare fertility outcomes of obese patients (body mass index [BMI] ≥ 30 kg/m 2 ) undergoing superovulation and intrauterine insemination (SO-IUI) using gonadotropins versus letrozole., Methods: A single centre retrospective cohort study of obese patients undergoing SO-IUI using gonadotropins or letrozole between January/2019 and June/2022. Primary outcome was clinical pregnancy rate (intrauterine pregnancy with positive fetal heart rate). Secondary outcomes included rates of multifollicular development, multiple pregnancy, spontaneous abortion and cycle cancellation. Subgroup analysis was done stratifying by obesity class. A multivariate logistic regression model was used for primary/secondary outcomes, adjusting for clinically determined covariates., Results: Out of 802 total identified SO-IUI cycles, 715 cycles were completed (518-gonadotropins and 197-letrozole cycles). The clinical pregnancy rates were not significantly different in obese patients undergoing SO-IUI with gonadotropins versus letrozole when adjusted for age, gravidity, parity, cause of infertility, IUI cycle number, endometrial thickness, sperm source and post-wash motile sperm count (adjusted odds ratio [aOR] 1.37, 95% confidence interval [CI] 0.72-2.59). Similarly, no significant associations were found in spontaneous abortion (aOR1.46, 95%CI 0.42-5.08), multiple pregnancy (aOR1.33, 95%CI 0.20-8.88) or cancellation rates (OR0.89, 95%CI 0.55-1.45) between the two groups. The rates of multifollicular development were also comparable between the two groups (aOR0.51, 95% CI 0.19-1.38). For cycles involving gonadotropins, higher BMI classes required higher total gonadotropin dose (p < 0.001)., Conclusion: After adjusting for patient and cycle factors, gonadotropins and letrozole led to comparable odds of achieving pregnancy in obese patients undergoing SO-IUI. Future research in the obese population will help to better understand how to optimize fertility treatments for this growing population., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring., Results: Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed., Conclusions: These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health., (© 2024. The Author(s).)

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 μg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8-12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study explored the alteration in kisspeptin and reproductive hormones during different superovulation protocols (SOP) in dromedary camel. The kisspeptin and reproductive hormonal profile, ovarian response, and the quality and quantity of embryos in dromedary camel donors were evaluated. A total of thirty donor camels were divided into two groups: the 5dSOP group, which received diluent containing 400 mg pFSH dissolved in 20 ml and administered two times daily for 5 days at decreasing doses (2.5, 2, 1.5, and 1 ml); and the 3dSOP group, which received diluent containing 400 mg pFSH dissolved in 12 ml and administered two times daily for 3 days at decreasing doses (3 ml, 2 ml, and 1 ml). Ultrasonography was used to monitor the ovarian environment, recording daily follicle count and dimensions and the time taken for follicles to mature. On the sixth day after mating, a corpus luteum (CL) count was conducted. On the 8th day after mating, records of the quantity and quality of embryos collected were kept. Blood samples from the jugular vein were collected at the commencement of the superovulation protocol and at 8:00 a.m. for the following 48 h to measure the concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), kisspeptin (KP), and progesterone (P4). The findings indicated that the 3dSOP yielded superior results compared to the 5dSOP in terms of follicle quantity and size, as well as the quantity of CL and embryos. This improvement was attributed to significantly higher concentrations of reproductive hormones, including FSH, LH, E2, kisspeptin, and P4 (P ≤ 0.05), in the 3dSOP than in the 5dSOP. In conclusion, reducing the duration of superovulation protocols contributed to the proliferation of follicles with improved dimensions and counts, ultimately resulting in a greater quantity of embryos of superior quality. The levels of FSH, LH, E2, KP, and P4 were affected significantly by SOP and time of evaluation., Competing Interests: Declaration of competing interest No competing interests exist., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 μg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P<0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P<0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P>0.05) between groups 22.5 and 30.0 μg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 μg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P>0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 μg rhFSH. The number of follicles >7 mm on day 4 did not differ (P=0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P=0.0005), ovulation rate (P<0.0001), and number of CL (P=0.0155), as well as a trend towards a greater number of ova (P=0.07) and viable embryos (P=0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 μg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P>0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha)., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study evaluated the use of the GnRH agonist hormone, deslorelin, to control the follicular population before initiating multiple ovulation and embryo transfer (MOET) treatment. Twenty-four cross-bred Santa Inês ewes, aged between 2 and 4 years, were randomly assigned to either a control group (n = 11) or a treated group (n = 13). All ewes received an intravaginal device containing 60 mg of medroxyprogesterone acetate on day 0, and a new device on day 7, which remained in place until day 14. Additionally, the ewes were administered 125 μg of cloprostenol on day 7. The superovulatory treatment involved administering 200 mg of pFSH, divided into eight decreasing doses at 12-h intervals starting on day 12. On day 14, 300 IU of eCG was administered. In the deslorelin group, three doses of 100 μg of deslorelin were administered starting on day 3 after the insertion of the vaginal device, with subsequent doses given at 72-h and 144-h intervals. Natural mating was performed 36 h after the removal of the progesterone implant using males with proven fertility. Embryo collection took place on the 6th day after mating, and the recovered structures were quantified and evaluated for quality and developmental stage. Transrectal ultrasonography was conducted on days 12, 16 and 21 to evaluate the ovaries, specifically to assess the ovarian follicular population and the presence of the corpus luteum. Ewes in the control group had higher embryo recovery rates (p < .01) compared to the treated group (5.2 ± 0.8 vs. 1.1 ± 0.8), with differences observed primarily in the number of morulae. The number of corpus luteum observed during the laparotomy on day 21 was significantly higher (p < .01) in the control group (10.44 vs. 4.5 corpus luteum per ewe). Yet, the treated group had a significantly higher number of follicles (p < .05) on the first day of pFSH application (5.5 vs. 3.0 follicles per ewe). In conclusion, although the inclusion of deslorelin in the superovulation protocol resulted in increased synchronization of oestrus and follicle number, it did not lead to an increase in the number of corpus luteum or harvested embryos., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Anti-Müllerian Hormone (AMH) serves as a crucial parameter in assessing the reproductive herd life and ovarian reserve in cattle. Consequently, extensive research is conducted on AMH levels. Various measurement methods can be employed to determine AMH levels. However, to our knowledge, no study has been conducted on Holstein donors using the Elecsys ® AMH kit., Objective: This study was designed to determine AMH levels in donors utilising the Elecsys® AMH kit and to evaluate the relationship between superovulation response parameters and AMH levels., Methods: In this study, we measured the serum AMH levels of 36 cows using the Elecsys ® AMH automated assay before the superovulation protocol (1st sample) and FSH injections (2nd sample). The cows were categorised into three groups based on their AMH levels: low, medium, and high AMH., Results: Positive correlations were identified between AMH and parameters associated with superovulation response. The high AMH level group exhibited significantly greater numbers of corpus luteum, total embryos, transferable embryos, and grade 1 embryos compared to the medium and low AMH groups (p < 0.05) There was no significant difference between AMH levels before the superovulation protocol and FSH injections(p > 0.05). Body condition score and parity did not significantly affect AMH levels in cows (p > 0.05). Also, AMH cut-off values for the number of corpus luteum, total embryo, and transferable embryos were detected as 234, 227, and 210 pg/mL, respectively., Conclusion: These findings demonstrate that a high serum AMH level has a positive influence on the superovulation response. AMH can be used as a reliable marker for the selection of donors in Holstein cows., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The aim of this study was to determine the effect of exogenous melatonin administration on transferable embryos by increasing total antioxidant status before superovulation in Assaf ewes. Selected ewes were randomly divided into two equal groups: melatonin (n = 9) and control (n = 9). In the melatonin group, a melatonin implant (18 mg melatonin, Regulin®, Ceva, Turkey) was placed under the skin of the ear 7 days prior to insertion of the progesterone-containing sponge. In the control group, a physiological saline solution was injected under the skin of the ear on the same day. The same superovulation protocol was used in both groups. In addition, blood samples for determination of Glutathione peroxidase, superoxide dismutase, total antioxidant status and total oxidant status concentrations were collected on five different days, including the day of melatonin implant placement (Day-7), vaginal sponge insertion (Day 0), vaginal sponge removal (Day 11), mating (Day 12-13) and uterine flushing (Day 19). Embryos were collected by laparotomy on the 7th day after mating. Uterine flushing taken into petri dishes were scanned under a stereomicroscope, and the quality and developmental stages of the embryos were recorded. In the study, total corpus luteum count and total cell count were found to be higher in the control group than in the melatonin group (p < .05). When the results were evaluated in terms of oxidative stress index, a negative correlation was found between the total number of corpus luteum, number of cells obtained, count of transferable embryos and number of Grade 1 embryos on Day 0. There was also a positive correlation oxidative stress index and the number of unfertilized oocytes on Day-7. As a result, exogenous melatonin administration prior to superovulation during the breeding season is thought to have a negative effect on embryo yield and quality. Therefore, the use of exogenous melatonin in MOET studies during the breeding season is recommended to be investigated in new studies., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Background: Anti-Müllerian hormone (AMH) holds potential as a biomarker for assessing the superovulation (SO) response in cattle. Nonetheless, there exists scant information regarding this aspect in the literature concerning dairy heifers. Given this gap, our objective is to explore the viability of AMH as an indicator for gauging the SO response specifically in Holstein heifers. Furthermore, our aim encompasses examining the variations in AMH levels within the same individuals before and after undergoing SO., Methods: The study included 41 Holstein heifers. All heifers were superovulated and blood samples were taken both before and after the SO protocol., Results: The findings revealed that the mean values of serum AMH concentrations before and after SO were 0.122 ng/mL (0.093-0.248 ng/mL) and 0.119 ng/mL (0.084-0.170 ng/mL), respectively. AMH concentrations in heifers were stratified into low (<0.106 ng/mL), medium (0.107-0.126 ng/mL) and high (>0.127 ng/mL) categories both before and after SO., Conclusions: There was no significant correlation between AMH levels in the heifers both before and after SO treatment with the number of follicles, corpora lutea, total embryos collected or embryos transferred (p > 0.05). Furthermore, this study showed that serum AMH concentrations in Holstein heifers did not change after SO treatment. In this study, as AMH levels in Holstein heifers were in a narrow range, a relationship between AMH and SO response could not be determined. In future studies, we believe that it would be more useful to plan more studies in Holstein donor heifers, taking into account the number of animals and AMH levels., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

This study compared the follicular growth, superovulatory response, and in vivo embryo production after administering two doses of porcine follicle-stimulating hormone (pFSH) in Santa Inês ewes. The estrous cycle of 36 multiparous ewes was synchronized with the Day 0 protocol and superovulated with 133 mg (G133, n=18) or 200 mg (G200, n=18) of pFSH. Ultrasonographic evaluations of the ovaries were performed, ewes were mated and submitted to non-surgical embryo recovery. Viable blastocysts were stained with Nile Red and Hoechst. The G200 had a greater number of medium and large follicles, as well as a larger size of the third largest follicle. A total of 97.2% (35/36) of the ewes came into estrus and it was possible to transpose cervix in 80.6% (29/36). There were no effects of treatments in the response to superovulation, the proportion of ewes in which was possible to transpose the cervix, the number of corpora lutea, the number of anovulatory follicles, the proportion of ewes flushed with at least one recovered structure, number of recovered structures, number of viable embryos, viability rate, and recovery rate. The G200 ewes were in estrus for a longer period of time than the G133 ewes (54.0 ± 4.5 h vs. 40.3 ± 3.6 h) and produced more freezable embryos (6.5 ± 1.6 vs. 2.3 ± 0.7) than G133. Both doses promoted an efficient superovulatory response and did not affect embryonic lipid accumulation. The dose of 200 mg of pFSH showed greater potential to increase the superovulatory response, as it increased follicular recruitment and the recovery of freezable embryos., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rats are multiparous rodents that have been used extensively in research; however, the low reproductive performance of some rat strains hampers the broader use of rats as a biomedical model. In this study, the possibility of increasing the litter size after natural mating in rats through superovulation using an anti-inhibin monoclonal antibody (AIMA) was examined. In outbred Wistar rats, AIMA increased the number of ovulated oocytes by 1.3-fold. AIMA did not affect fertilization and subsequent embryonic development, resulting in a 1.4-fold increase in litter size and a high pregnancy rate (86%). In contrast, conventional superovulation by eCG/hCG administration decreased the pregnancy rate to 6-40% and did not increase the litter size. In inbred Brown Norway rats, AIMA increased the litter size by 1.2-fold, and the pregnancy rate increased more than twice (86% versus 38% in controls). AIMA also increased the litter size by 1.5-fold in inbred Tokai High Avoiders and Fischer 344 rats. AIMA increased the efficiency of offspring production by 1.5-, 2.7-, 1.4-, and 1.4-fold, respectively, in the four rat strains. Thus, AIMA may consistently improve the reproductive performance through natural mating in rats, which could promote the use of AIMA in biomedical research., (© 2024. The Author(s).)

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Oocyte quality is one of the key factors affecting the outcome of ART. Therefore, how to improve oocyte quality has become an urgent problem in the field of ART. In this study we evaluated the effect of resveratrol (RSV), added during the process of superovulation, on embryonic development in mice. The results showed that the blastocyst rate was significantly higher in the RSV treated group than in the control group when oocytes were parthenogenetically activated in vitro (61.67 vs 41.51%, P = 0.032). In the naturally fertilized oocytes group, the rates of cleavage and blastocyst were significantly higher in the RSV treatment group than in the control group (74.47% vs 60.98%, P = 0.035; 96.19% vs 70.00%, P = 0.000, respectively). For the aged mice, the average number of oocytes, the rates of cleavage and blastocyst were also significantly higher in RSV treated groups than in the control group (19.47 ± 5.98 vs 10.30 ± 4.82, P = 0.028; 69.03 vs 50.75%, P = 0.014; 64.10% vs 44.12%, P = 0.049, respectively). Mitochondrial membrane potential and mtDNA copy number in oocytes were significantly increased after RSV treatment in both the young and aged populations. The expression of mitochondrial biogenesis related genes was significantly upregulated in cumulus cells of young and aged mice following RSV treatment. Our data suggest that supplementation of RSV during superovulation improves oocytes quality in young and aged mice, increases the number of oocytes retrieved from aged mice, and improves oocytes mitochondrial function., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Contamination of animal feed with Fusarium spp results in accumulation of mycotoxins including deoxynivalenol. In animals, deoxynivalenol is metabolized to de-epoxy deoxynivalenol (DOM-1), which is generally considered to be a non-toxic metabolite; however, recent studies demonstrated that DOM-1 can reduce steroid production and induce apoptosis in the bovine ovary. The objectives of this study were to assess the effects of DOM-1 on applied aspects of reproductive function in cattle, specifically sperm function and embryo development in vitro and follicle growth and superovulatory responses in vivo. The effect of naturally contaminated feed on superovulatory responses was assessed; a dose of 6 ppm deoxynivalenol increased blood DOM-1 concentrations to 20 ng/ml, but this did not alter the number of viable embryos recovered on day 7. However, intrafollicular injection of DOM-1 (100 ng/ml) directly into the growing dominant follicle resulted in cessation of follicular growth over the subsequent 3 days. Treatment with DOM-1 reduced motility of bull spermatozoa over a 10-h period in vitro. Addition of DOM-1 to oocytes in vitro during IVM did not alter rates of cumulus expansion and nuclear maturation, but treatment during IVF reduced the rate of blastocyst formation. These data illustrate that DOM-1 is more biologically active than previously thought and negatively impacted reproductive outcomes in cattle., (© 2020 John Wiley & Sons, Ltd.)

The purpose of this study was to determine effects of various sources of omega-3 and omega-6 fatty acids on ovarian response and embryo quality in Boer does when there was a superovulation treatment regimen imposed. Pluriparous does were randomly assigned to be treated with 300 g of one of four experimental supplements containing linseed oil (LO), soybean oil (SO), palm oil (PO), or a control supplement without fatty acids (CO), for 15 days. Does were fitted with a controlled internal drug release (CIDR) device containing 0.3 g progesterone for 7 days. At 48 h before CIDR withdrawal, does were treated with 80 mg follicle-stimulating hormone (FSH) administered at 12 h intervals. Embryos were collected 7 days after the last natural mating. Estrous response and interval between CIDR withdrawals to estrous onset were similar between treatments (P > 0.05). Number of ovulations was similar for does in the different groups (10.0, 9.2, 7.0, and 7.0, in LO, SO, PO, and CO, respectively; P > 0.05). There was premature luteal regression in does of the SO, PO, and CO groups, except in LO group. The LO-treated does had a larger (P < 0.05) mean number of ova/embryos recovered than does of SO, PO, and CO groups (7.2, 2.0, 0.2, 0.2, respectively) and transferable embryos (5.1, 1.4, 0.2, 0.2, respectively). These results indicate that including LO in supplements may be a feasible strategy for preventing premature luteal regression and improving embryo quality in goats treated to induce follicular super-stimulation for induction of superovulation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

When women with small ovarian reserves are subjected to assisted reproductive technologies, high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive follicle-stimulating hormone (FSH) doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for 4 days, beginning on Day 1 of the estrous cycle with 35, 70, 140, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F2α injections were given at 12-h interval, starting at the seventh FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine the number and size of follicles and corpora lutea, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, number of ovulatory-size follicles, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In mice, ovarian stimulation via hormone administration is an effective method for obtaining many ova simultaneously, but its effect is reduced by the influence of aging. In this study, we demonstrate that this problem can be improved by administering the gonadotropin-releasing hormone antagonist Cetrorelix prior to ovarian stimulation. Before 12-month-old female mice were injected with 5 IU pregnant mare serum gonadotropin and 5 IU human chorionic gonadotropin, we administered 5 µg/kg Cetrorelix for 7 consecutive days (7 times) or 3 times once every 3 days. As a result, 8.7 ± 1.9 (mean ± SEM, n=10) and 9.8 ± 1.3 (n=10) oocytes were obtained, respectively, as opposed to 4.7 ± 1.2 oocytes (n=9) in the case of no administration. Collagen staining of ovarian tissue showed that Cetrorelix administration reduced the degree of fibrosis, which improved ovarian function. In addition, equivalent fertilization and fetal development rates between control and Cetrorelix-treated aged mouse-derived oocytes were confirmed by in vitro fertilization and embryo transfer (Fertilization rate; control: 92.2% vs. 3 times: 96.9%/7 times: 88.5%, Birth rate; control: 56.4% vs. 3 times: 58.3%/7 times: 51.8%), indicating the normality of the obtained oocytes. It is concluded that Cetrorelix improved the effect of superovulation in aged mice without reducing oocyte quality. This procedure will contribute to animal welfare by extending the effective utilization of aged female breeding mice.

The goal was to investigate the relationship among mRNA expressions of anti-Mullérian hormone (AMH), follicle-stimulating hormone receptor (FSHR) and responses to superovulation (SO) in embryo donor dairy cows. Holstein cows (n = 19) were submitted to a standard SO protocol, with twice daily FSH treatments, and artificially inseminated. Prior to SO (Day 0), relative mRNA expressions of AMH and FSHR in blood were determined for all cows. Day 7 embryos were collected and were graded to determine superovulatory response for each donor. Results showed that relative mRNA expressions of AMH and FSHR were positively correlated (R 2  = 0.94). Relative mRNA expressions of both AMH and FSHR were positively correlated with total embryos (R 2  = 0.68 and 0.69, respectively), total transferable embryos (R 2  = 0.92 and 0.97, respectively) and total grade 1 embryos (R 2  = 0.54 and 0.59, respectively). Further, transcript abundances of AMH and FSHR positively associated with milk production of donor cows, and meanwhile, they were negatively associated with days in milk (DIM) at submission of cows to SO (p < .05) protocol. The relative mRNA expression of AMH was higher (p < .05) in donor cows <5 years of age. However, age of donor at superovulation did not influence mRNA expression of FSHR. Collectively, we infer that the mRNA expressions of AMH and FSHR prior to superovulation can predict donor cows' positive response to superovulation., (© 2020 Wiley-VCH GmbH.)

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6-8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-β (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG., (© 2020 Wiley-VCH GmbH.)

This study was conducted to assess effects of different doses of pFSH on follicular recruitment, superovulatory response, ova/embryo recovery, and embryo yield in lactating ewes. Ewes (n = 24) had a superovulation treatment regimen imposed. All ewes were implanted with a progesterone intravaginal device for 9 d, and administered either 100 (G-100) or 200 (G-200) mg pFSH, proportioned into six doses administered at 12-h intervals, starting 60 h before device removal. At 7 days subsequent to progesterone device removal, there were non-surgical embryo recoveries (NSER) from ewes having three or more corpora lutea. At the time of the first pFSH injection, number of antral follicles were similar (P < 0.05) between ewes in the G-100 and G-200 group, however, there were more 3.1-4.0 mm follicles in ewes of the G-200 than G-100 group at the time of the second pFSH administration. Estrous response and CL number were less (P < 0.05) in ewes of the G-100 (66.7 % and 2.6 ± 0.7) than G-200 (91.7 % and 11.6 ± 1.2) group. There were embryo collections from 100 % and 90.9 % of ewes in the G-100 and G-200 groups, respectively (P > 0.05). Viable embryo numbers and ova/embryo recovery rate were greater (P < 0.05) in ewes of the G-200 (6.9 ± 1.1 and 67.8 %) than G-100 (1.0 ± 0.5 and 27.6 %) group. A dose of 200 mg pFSH was more effective in inducing a superovulatory response and embryo yield after NSER in ewes, however, the 100 mg dose was insufficient for these purposes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the potential negative effects of superovulation on subsequent fertility of dairy heifers. Holstein heifers (n = 1,783), 312 to 387 d of age, and 273 to 307 kg of body weight (BW) from 2 commercial farms were enrolled. These animals were first selected to be donors (446) or nondonors (CON, n = 1,327) according to their genomic values. Nondonor heifers (CON) were artificially inseminated (AI) according to standard procedures of each farm after reaching 320 kg of BW. Donor heifers were superovulated using a fixed FSH dose (180 mg NIH-FSH-P1; Folltropin-V, Vetoquinol Brasil, Mairiporã, SP, Brazil) and embryos were collected following standard procedures. Heifers that produced fewer than 8 viable embryos after first superovulation (SOV1, n = 337) were no longer used as donors, whereas the remaining heifers (SOV2, n = 109) were superovulated a second time within an interval of 48 to 54 d. Donor heifers (SOV1 and SOV2) were AI once they reached 320 kg of BW, at least 15 d after the last embryo collection. Data on age at first AI, at conception, and at parturition, as well as the number of services per conception, were analyzed by ANOVA, using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC) procedure. Binomial variables (pregnancy per AI, overall pregnancy rate, open heifers at 500 d age, and late pregnancy loss) were analyzed using the GLIMMIX procedure of SAS. The heifers selected to undergo superovulation twice (SOV2) yielded more total (12.6 ± 5.3 vs. 6.8 ± 4.6; respectively) and viable embryos (8.5 ± 3.8 vs. 3.9 ± 2.8; respectively) than those superovulated only once (SOV1). Age at first AI, conception, and at parturition was greater in SOV2, but not in SOV1 compared with nondonor controls. In addition, pregnancy per first AI, overall pregnancy rate, services per conception, open heifers at 500 d of age, and occurrence of pregnancy losses after 60 d of gestation were similar among CON, SOV1, and SOV2 heifers. In summary, a single superovulation performed before heifers reach a minimum weight for breeding did not affect age at conception, calving or other indicators of reproductive efficiency. On the other hand, heifers superovulated twice were first inseminated at a later age than their birth cohorts, but had similar reproductive performance., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: With the medical advancement some studies put forward that letrozole (LE), a specific aromatase inhibitor with the function of reducing oestrogen synthesis, has recently been applied as a potentially better alternative compared with clomiphene citrate (CC), owing to that it has a superior efficacy as compared with CC in patients of unexplained infertility undergoing intrauterine insemination (IUI). However, there is no one study can clear and definite whether LE can replace the CC as first line drug., Objective: Our objective is to compare the LE with CC in the induction of ovulation in patients with unexplained infertility IUI., Method: Searching databases consist of all kinds of searching tools, such as Medline, The Cochrane Library, Embase, PubMed, etc. All the include studies should meet our demand of this meta-analysis: RESULT:: Based on the current meta-analysis, we rigorously consider that LE has a likelihood to improve dominant follicles (MD= -0.56, I= 100%, P= .04; MD= -0.39, I= 73%, P = .0003, respectively) and reduces the miscarriage rate (RR= 0.61, I= 0%, P = .03). There is no significant differences between the 2 groups in The total rate of pregnancy, pregnancy rate per cycle, multiple pregnancy and endometrial thickness. (RR= 1.06, I= 11%, P = .38; RR= 1.09, I= 7%, P = .32; RR= 0.79, I= 0%, P = .46; respectively) CONCLUSION:: Combined with the results of current systematic review and meta-analysis through subgroup analysis and sensitivity analysis, we can be cautious: in general, compared with CC, LE is an effective treatment in the IUI cycle, has a likelihood to improve dominant follicles and reduces the miscarriage rate.

This study was conducted to develop simple superovulation protocols for dromedary camels using eCG. In experiment 1, camels received either 1000, 2000, 3000, 4000, 5000 or 6000 IU eCG. In experiment 2, camels received either 400 mg FSH (Folltropin-V) twice-daily over 5 days or 3000 IU eCG. In experiment 3, camels received 3000 IU eCG either at 2, 3, 4 or 5 days after ovulation induction. Ovarian response and embryo yield were evaluated in all experiments and embryos collected from camels treated with FSH and eCG were transferred to recipients to examine pregnancy rates. The mean number of ovulations (12.6 ± 1.5 and 13.3 ± 1.2 vs 3.4 ± 0.3, 6.2 ± 0.6 and 9.3 ± 1.0, respectively) and transferable embryos (4.6 ± 1.3 and 4.8 ± 1.0 vs 1.6 ± 0.2, 2.2 ± 0.4 and 1.1 ± 0.4, respectively) with 3000 and 4000 IU eCG doses were higher compared to 1000, 2000 and 6000 IU eCG doses (P < 0.05). Doses of 5000 and 6000 IU eCG resulted in a higher number of unovulatory follicles than other doses (P < 0.05). The FSH treatment resulted in higher number of ovulatory follicles (21.8 ± 1.3 vs 14.8 ± 1.7) and ovulations (18.5 ± 1.1 vs 13.9 ± 1.4) compared to eCG (P < 0.05). However, the number of transferable embryos and pregnancy rates were similar in these treatments. The timing of eCG treatment after ovulation induction did not affect the number of ovulatory follicles and transferable embryos but eCG treatment at 5 days after ovulation induction reduced the number of ovulations (P < 0.05). In conclusion, the optimal dose of eCG to induce superovulation is 3000-4000 IU and it produces a comparable embryo yield to FSH, and can be administered at 2-4 days after ovulation induction., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nutrition is an important source of exogenous AGEs and thermally processed foods present in western-style diets contain a large amount of these pro-inflammatory AGEs. Additionally, the intake of dietary AGEs could upregulate ovarian gene expression of inflammatory macrophage markers. The objective of this study was to investigate the effect of diet rich in AGEs on estrous cyclicity and ovarian function in a mouse model. Six-week old C57BL/6 J female mice were randomly subjected to either a diet low in AGEs (L-AGE) or a diet high in AGEs (H-AGE) for a total of 13 weeks. Experiments performed included daily vaginal smears to assess estrous cyclicity, ovarian superovulation with gonadotropins to assess the number of oocytes released, whole ovarian tissue mRNA quantification by RT-PCR to quantify genes involved in folliculogenesis, steroidogenesis, and macrophage markers, and ovarian morphology for follicle count. Outcome measures included estrous cyclicity, number of oocytes following superovulation, expression of genes involved in folliculogenesis, steroidogenesis, and macrophage infiltration as well as the number of primordial, primary, secondary, antral follicles and corpora lutea. Compared to mice on L-AGE diet, mice on H-AGE spent significantly longer time in the diestrus phase, had similar number of oocytes released following ovarian superovulation, and showed significant alterations in genes involved in steroidogenesis (increase in Star mRNA expression levels) and folliculogenesis (increase in Gdf-9 and Fshr mRNA expression levels). Mouse macrophage marker F4/80 mRNA expression was upregulated in mice on H-AGE diet compared to mice on L-AGE diet. Finally, mice on H-AGE diet had significantly fewer corpora lutea in their ovaries. These results indicate that the ingestion of high amounts of dietary AGEs could disrupt folliculogenesis and steroidogenesis that might lead to abnormal estrous cyclicity. Intake of dietary AGEs could also upregulate ovarian gene expression of inflammatory macrophage markers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Conventional methods for determining the reproductive performance of sheep bred either after estrus synchronization during the breeding season or after induction of estrus/ovulation during the non-breeding season take a long time and may give misleading results due to the effect of environmental factors. Laparoscopic observations allow real-time monitoring of ovarian activity around estrus or ovulation. This study was aimed at assessing the superovulatory effects of follicle-stimulating hormone (FSH) and equine chorionic gonadotropin (eCG) treatments by laparoscopy during breeding (September-November, n=12) and non-breeding (April-June, n=12) seasons in Akkaraman sheep. In both seasons, after CIDR withdrawal, the ewes were injected either with 600 IU eCG or 300 μl (20 mg/ml) FSH twice at 12 hour intervals. Plasma P4, E2 and LH concentrations were determined at the time of intra-vaginal CIDR insertion (day 0) and then at its withdrawal (day 12), followed by 3 and 6 days of eCG or FSH injections. After 3 (first observation) and 6 (second observation) days of hormone injections, laparoscopy was performed to record ovarian activity in both seasons. The eCG increased (p⟨0.05) the numbers of large follicles (first observation) and CL (first and second observations) in the breeding season compared to FSH treatment. CL, small-moderate and large follicle numbers of eCG treated ewes were higher (p⟨0.05) than those of FSH at both observations in the non-breeding season. In the breeding season, eCG treated ewes had higher (p⟨0.05) plasma P4 (3 and 6 days after hormones injections) and E2 (3 days after hormones injections) concentrations than those of FSH. In conclusion, the results of the present study indicate that treatment with eCG during the non-breeding season can support ovarian activity, and thus increase ovulation rate and plasma hormone concentrations around induced estrus/ovulation in Akkaraman ewes., (Copyright© by the Polish Academy of Sciences.)

Outcomes of short- (6.5 days) and long-term (14.5 days) estrous synchronization for 6.5 d (G-6.5d) or 14.5 d (G-14.5d) and followed by the 4-day or 3-day declining-dose follicle-stimulating hormone superovulatory regimen, respectively, were compared using 16 estrous-cycling Santa Inês ewes. Non-surgical embryo recovery (NSER) procedures were performed 60 d apart starting 6 or 7 d after the onset of estrus; an i.m. injection of estradiol benzoate and of d-cloprostenol at 16 h was followed by an i.v. oxytocin injection administered 20 min before NSER. There was a longer (P < 0.05) period before estrous onset in ewes during the second (September) compared with the first study replicate (July) by approximately 14 h. The NSER could be performed in 11 of 15 ewes that were in estrus, with an average of three viable-embryos/donor and the mean duration of the procedure being 29 min. There were no differences in superovulatory responses between the two groups of ewes, but there were only degenerated embryos in ewes of the G-6.5d group. In summary: i. the duration of progestin-priming and of multiple-dose pFSH treatment had a limited effect on superovulatory responses in estrous-cycling Santa Inês ewes; ii. NSER is a safe and repeatable method of embryo collection in ewes subsequent to superovulation; and iii. duration of the superovulatory treatment regimen may alter the effects of endogenous steroids on oocyte/embryo quality in ewes., Competing Interests: Declaration of Competing Interest Nothing to declare., (Copyright © 2020. Published by Elsevier B.V.)

Background: Epigenetic abnormalities caused by superovulation have recently attracted increasing attention. Superovulation with exogenous hormones may prevent oocytes from establishing an appropriate epigenetic state, and this effect may extend to the methylation programming in preimplantation embryos, as de novo DNA methylation is a function of developmental stage of follicles and oocyte size. Follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation, and appropriate concentrations of these gonadotropins might be necessary. However, no systematic study on the effects of DNA methylation alterations in oocytes associated with superovulation with different dosages of FSH/hMG at the single-cell level has yet been reported. In the current study, different dosages of FSH/hMG combined with human chorionic gonadotropin (hCG) were used in female mice to generate experimental groups, while naturally matured oocytes and oocytes superovulated with only hCG were respectively used as controls. Single-cell level DNA methylation sequencing was carried out on all these matured oocytes., Results: In this study, we revealed that the genome-wide methylation pattern and CG methylation level of the maternal imprinting control regions of all mature oocytes were globally conserved and stable. However, methylation alterations associated with superovulation were found at a specific set of loci, and the differentially methylated regions (DMRs) mainly occurred in regions other than promoters. Furthermore, some of the annotated genes in the DMRs were involved in biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation and were altered in all dosages of FSH/hMG group (for example, Gfod2 and SYF2). Other genes were impaired only after high gonadotropin dosages (for instance, Sox17 and Phactr4)., Conclusions: In conclusion, the current study addressed the effects of superovulation on DNA methylation from the perspective of different dosages of gonadotropins at the single-cell level. We found that the genome-wide DNA methylation landscape was globally preserved irrespective of superovulation or of the kind and dosage of gonadotropins used, whereas the methylation alterations associated with superovulation occurred at a specific set of loci. These observed effects reflect that superovulation recruits oocytes that would not normally be ovulated or that have not undergone complete epigenetic maturation. Our results provide an important reference for the safety assessment of superovulation with different dosages of gonadotropins. However, it should be noted that this study has some limitations, as the sample number and library coverage of analyzed oocytes were relatively low. Future studies with larger sample sizes and high-coverage libraries that examine the effects of superovulation on embryo development and offspring health as well as the underlying mechanisms are still needed.

Purpose: Tyrosine kinase inhibitors (TKIs) such as imatinib are commonly used chemotherapeutics, but the effects of long-term treatments on reproductive outlook for cancer survivors are unknown. The purpose of this study was to examine the effects of long-term imatinib treatments on follicle development and embryo quality. Since prospective studies are not possible in healthy humans, we have incorporated a commonly used mouse model., Methods: Adult female mice were treated with daily IP injections of imatinib for 4-6 weeks. Liquid chromatography-mass spectrometry was used to measure imatinib in serum and ovarian tissues. At the end of treatments, females were superovulated and mated to yield fertilized embryos. Oocytes and embryos were collected from oviducts, assessed for development by microscopy, and fertilized embryos were cultured in vitro. Blastocysts were fixed and stained for differential cell counts., Results: Long-term imatinib treatments caused a shift in follicle development, with imatinib-treated females having fewer primordial follicles, but an increase in primary and secondary follicles (P < 0.05). There was no effect on ovulation or fertilization rates. However, blastocysts from imatinib-treated females had fewer total cells (P < 0.05) and a significant shift from inner cell mass to increased trophectoderm cells., Conclusion: This pilot study indicates that long-term TKI treatments may have significant impact on ovarian reserve and embryo developmental capacity. More studies are needed in other model systems to determine the long-term impact of TKIs in patients. Knowing the potential effects of chemotherapeutics on reproductive outlook is critical for quality of life and more research is needed.

The objective was to determine associations between response to superovulation and body condition, subclinical endometritis and circulating metabolic biomarkers [adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL6, and urea] in lactating dairy cows. Ten multiparous lactating Holstein cows in each body condition score (1-5; 1 emaciated; 5 obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00 (BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) groups (total n = 50) were randomly selected and superovulated, timed artificially inseminated with frozen-thawed semen from three sires and embryos collected (n = 50 collections). At embryo collection, blood samples and embryo recovery fluid were collected for determination of metabolic markers and presence of subclinical endometritis (lavage technique; > 6% PMN). In total, 379 embryos were collected (average of 7.6 embryos per superovulation). Mean numbers of total ova and embryos was greater for cows in BCSC2, BCSC3 and BCSC4 groups compared with cows in BCSC1 and BCSC5 groups (P < 0.01). Total number of transferrable embryos were greater for cows in BCSC 2 and BCSC3 groups compared with cows in BCSC1, BCSC4 and BCSC5 groups (P < 0.01). Mean number of total ova and embryos and of transferrable embryos was higher for cows with 0 or 1-6% PMN compared to cows with >6% PMN (P < 0.01). In addition, there was a quadratic association between blood urea nitrogen concentrations and % transferrable embryos (r 2  = 0.85; P < 0.05) and between BCS and % transferrable embryos (r 2  = 0.73; P < 0.05). Circulating adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body condition compared to thin or obese cows (P < 0.05). Circulating adiponectin, leptin, IGF1 and insulin were greater in normal cows (≤6% PMNs), whereas, TNFα and IL1β and IL6 were greater in cows with subclinical endometritis (P < 0.05). In conclusion, BCS and subclinical endometrial inflammation were associated with superovulatory response and embryo quality. Further, circulating metabolic biomarkers were associated with superovulatory response and embryo quality, likely due to donor's metabolic status and uterine environment. Optimizing superovulatory responses and embryo quality in lactating dairy cows requires management of nutrition and uterine health., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Published by Elsevier Inc.)

We aimed to evaluate the relationship of anti-Müllerian hormone (AMH) and progesterone concentrations with superovulation response in goats and to determine donors exhibiting better superovulation response by measuring AMH concentrations. For this, blood samples were collected from multiparous Angora goats (n = 24) for measuring the progesterone and AMH concentrations on the day the synchronization protocol was initiated (Day 0), on the day of the first FSH administration (Day 9), on the day the progesterone source was removed (Day 11), and on the day of uterine flushing. Descriptive statistics (mean, standard deviation, median, minimum value, maximum value, and percentile) were given for superovulation response and embryo yield. To compare the differences between the two groups, the Student's t-test was used. The relationship between two continuous variables was assessed by the Pearson Correlation Coefficient. The AMH cutoff values in superovulation responses were evaluated by ROC analysis on the day the synchronization protocol was initiated. A strong positive correlation was found between the AMH concentrations measured on the day the synchronization protocol was initiated (Day 0), on the day of the first FSH administration (Day 9), and on the day of removal of the progesterone source (Day 11) and the count of total corpus luteum (CL), total oocyte/embryo, transferable embryo, and Code I quality embryo (P < 0.05). Furthermore, AMH concentration increased on the day the synchronization protocol was initiated, the donor's superovulation response increased as well. The cutoff value was 4.74 ng/ml, as assessed by the ROC curve analysis conducted for selecting donors exhibiting better superovulation responses. The sensitivity and specificity of the selected cutoff value were found to be quite high (P < 0.01). However, a positive correlation was noted between the progesterone concentrations measured on the day of uterine flushing and total CL count, total oocyte/embryo count, transferable embryo count, and Code I quality embryo count (P < 0.01). In conclusion, it was determined that an increase in AMH concentrations in goats led to an increase in the total CL count, embryo count, and embryo quality and that AMH measurement could be used to identify donors that responded better to superovulation. Additionally, a positive correlation was found between the progesterone concentration measured on the day of uterine flushing and the total CL count, transferable embryo count, and embryo quality., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim was to define the pattern and physiological concentrations of FSH and LH required for the selection of a single dominant follicle in mono-ovulatory species. A series of five experiments was carried out using gonadotrophin-releasing hormone agonist-induced hypogonadal heifers. Animals were infused with different patterns of either FSH and/or LH followed by an ovulatory dose of human chorionic gonadotrophin. Follicular response was monitored by ultrasound scanning and blood samples were collected to measure concentrations of FSH, LH, oestradiol and progesterone. The main findings were: (1) physiological concentrations of FSH given as a continuous infusion and for an adequate duration, in the presence of basal LH, with or without LH pulses, are capable of inducing a superovulatory response, (2) initial exposure to FSH followed by LH pulses alone stimulate the development of multiple preovulatory follicles, confirming that ovarian follicles are capable of transferring dependence on gonadotrophins from FSH to LH, (3) while LH pulses appear not to have a major effect on the pattern of preovulatory follicle development, adequate LH pulsatile support is required for full oestradiol synthesis and (4) the duration of initial exposure to FSH and the ability to transfer the dependence from FSH to LH are critical for the selection of a single dominant follicle. In conclusion, this experimental series confirms that the duration of initial exposure to FSH and the ability of the selected follicle to transfer its gonadotrophic dependence from FSH to LH are critical for the selection of a single dominant follicle in cattle.

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July-August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n = 10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH, Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline (control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10 days. When the largest follicle reached ≥35 mm in diameter, treatment was discontinued and 2500 IU hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen every 48 h until ovulation(s) were detected or one dose of frozen semen (250 × 10 6 motile sperm) after the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: The present study aims to define maturation, yield, health, and ease of collection of murine immature oocytes recovered using the conventional method or from mice treated with cilostazol., Main Methods: The conventional method included the superovulation of mice and the recovery of germinal vesicle (GV) or metaphase (MI) oocytes from preovulatory follicles. The cilostazol method included the oral treatment of superovulated mice with 7.5 mg cilostazol once or twice to result in the ovulation of MI or GV oocytes, respectively., Key Findings: The cilostazol method resulted in >95% of GV or MI oocytes with a diameter range of 60-90 μm or 50.1-70 μm in comparison to <60.0% of GV or MI oocytes resulting from the conventional method, respectively (P < 0.0001). The cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules (CG) and chromatin configuration of surrounded nucleolus and MI oocytes having higher levels of co-occurrence of normally organized spindles/chromosomes and peripheral CG with free domains than did the conventional method (P < 0.001). The cilostazol method was more time and labor efficient and resulted in higher oocyte yields of normal morphology than did the conventional method (P < 0.01)., Significance: The presented method provides not only oocytes with uniform size and synchronized developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all immature oocytes resulting from the conventional method are from preovulatory follicles nor have been developed adequately and consequently ovulated as opposed to the presented method., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study evaluated embryo production after superovulation (SO) with a reduced number of FSH applications and increased eCG dose in 26 Bos taurus × Bos indicus donors. On Day 0, donors received an intravaginal device (CIDR) with 1.9 g of progesterone plus 2.5 mg of estradiol benzoate and 50 mg of progesterone via IM. On Day 4, donors were randomly allotted to one of three SO treatments: 1) 455 IU of Folltropin +400 IU of eCG (n = 9), 2) 350 IU of Folltropin +600 IU of eCG (n = 9), and 3) 500 IU of Pluset + 600 IU of eCG (n = 8). In treatment 455 IU of Folltropin +400 IU of eCG, donors received eight IM Folltropin injections in decreasing dose 12 h apart from Day 4 to Day 7. On Day 6, at the same time as the Folltropin, donors received via IM 25 mg of dinoprost tromethamine (PGF 2a ). On Day 7, the CIDR was removed, and together with the Folltropin, donors received 200 IU of eCG via IM. In treatment 350 IU of Folltropin +600 IU of eCG, donors received four IM Folltropin injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF 2a 12 h apart. On Day 7, the CIDR was removed. Donors from treatment 500 IU of Pluset +600 IU of eCG received four IM Pluset injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF 2a 12 h apart. On Day 7, the CIDR was removed. In the morning of Day 8, donors from the three treatments received 0.25 mg of GnRH via IM. Artificial insemination was performed on Day 8 (pm) and Day 9 (am). Embryos were collected on Day 15. Variables evaluated were number of CL before embryo collection, number of structures recovered, transferable embryos, degenerate embryos and unfertilized oocytes, recovery rate, and viability rate. There was no difference in any variable among treatments (P > 0.05). In conclusion, replacement of four Folltropin or Pluset injections from a conventional eight FSH-injection SO protocol, by a single injection of 600 IU of eCG, is a good alternative to reduce donor handling without decreasing yield of transferable embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two experiments were done using a two-by-two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo-derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every-other-day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus-oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p < .05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p < .05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo-derived embryos is significantly lower during the anovulatory season., (© 2019 Blackwell Verlag GmbH.)

An efficient system to collect large numbers of vital zygotes is a pre-requisite for application of zygote genome-editing technology, including development of efficient models for xenotransplantation using pigs. Owing to the sub-optimal in vitro production of zygotes in pigs, efficient collection of in vivo developed zygotes is required. Timing of ovulation is a key factor to sustain efficiency since the interval between pronuclear formation and the first division is very short in pigs. The weaning-to-estrus interval can, due to its inverse relation with length of estrus and time of ovulation, interfere with ovulation and make it asynchronous, which reduces the probability of obtaining zygotes. This retrospective study compared the effects of three weaning-to-estrus intervals of 3, 4 or 5 days on zygote collection efficiency in a total of 17 trials over a 3-year period including 223 sows. Donor sows in groups of 10-15 animals were super-ovulated with eCG 24 h after weaning and those in estrus at 48-72 h post-eCG were immediately treated with hCG, followed by insemination 6 and 24 h thereafter. Collected structures during laparotomy on Day 2 (Day 0: onset of estrus) were morphologically evaluated and only those with a single cell and two visible polar bodies were considered as zygotes. Zygotes were injected with CRISPR-Cas9 editor mixture and cultured for 6 days to evaluate their developmental ability against non-injected control zygotes. Of all recovered structures (N = 5,468), 67.4%, 30.8% and 1.8% were zygotes, 2-cell embryos and oocytes-degenerated embryos, respectively. The different weaning-to-estrus intervals did not affect either the percentages of collected zygotes (range: 64.1%-70.0%) or the percentages of sows with zygotes at collection time (range: 69.0%-73.3%). The weaning-to-estrus intervals did not affect the in vitro developmental ability of zygotes. After 24 h of culture, 78.1 ± 2.0% and 95.1 ± 0.6 (P < 0.05) of injected (N = 2,345) and non-injected (N = 335) zygotes, respectively, developed to 2-to-4-cell embryo stage. The total efficiency of the system was 64.1 ± 2.2% and 85.8 ± 1.5% (P < 0.05) for injected and non-injected zygotes, respectively. In conclusion, the results indicate that neither the efficiency of collecting in vivo derived porcine zygotes from superovulated sows nor the zygote ability to develop to blastocyst after cytoplasmic genome-editing injection were affected by a weaning-to-estrus interval between 3-to-5 days., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF-IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos., (© 2019 Blackwell Verlag GmbH.)

Optimal results in cattle embryo transfer are limited by the variation in ova recovery, fertilization rate and embryo quality experienced with superovulation. Inflammation and immune dysregulation may be contributing factors. This study, evaluated feeding OmniGen-AF® (OG), a nutritional supplement that reduces inflammation and supports immune health, on superovulatory response and serum progesterone and cortisol concentrations in embryo donors treated with two different doses of Folltropin®-V (FSH). Angus cross-bred beef cows (n = 24) were assigned to four groups, fed OG at 0 or 56 g/animal/day for 49 days and were treated with 200 or 400 mg FSH to induce superovulation. Treatments for superovulation started after feeding OG for 28 days and ova were non-surgically recovered 7 days after estrus and graded for quality. More transferrable embryos (P < 0.05) were recovered from cows fed 56 g OG and treated with 400 compared with 200 mg FSH. Percent degenerate embryos recovered from cows treated with the 400 mg FSH dose was threefold greater (P < 0.05) when fed no OG compared with 56 g OG. Serum progesterone on day of embryo collection was greater (P < 0.05) in OG-supplemented cows and cows treated with 200 mg FSH. Serum cortisol was not affected (P > 0.10) by FSH dose or OG-feeding, but was greatest (P <  0.05) on Days 0 and 42 of the feeding period. In summary, the improvement in embryo quality with OG-feeding may relate to a greater serum progesterone concentration., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The most significant focal points of the embryo transfer technology are as follows: the selection of donors, the response of the selected donor to the superovulation protocol and the obtained number of the transferable embryos. For this purpose, it is suggested that donor selection can be done by anti-Müllerian hormone (AMH) levels, and embryo production is evaluated. AMH is secreted by the granulosa cells of primordial, pre-antral and antral follicles below 4 mm in the ovary, independent of FSH. Therefore, the aim of this study was to investigate the relationship between serum AMH levels and the number of corpus luteum (CL), total embryos and transferable embryos that were shaped after a uniform superovulation protocol. For this reason, 48 Simmental cows, which were located at General Directory of Agricultural Enterprises (region, province, etc. instead of the general directorate), were used as donors for the embryo transfer. Blood samples were taken at random, regardless of the stage of animal's sexual cycle. AMH levels were measured by enzyme-linked fluorescent assay (ELFA) method of the miniVIDAS ® (bioMérieux SA) using AMH Bovine Test Kit. According to the statistical analyses of the obtained data, AMH levels were positively correlated with CL and total embryos (p < .05). No significant correlations between AMH and transferable embryos were approved (p > .05). It was also determined that each 200 pg/ml increase in serum AMH level resulted in one increase in CL number. Overall, considering the positive correlation between AMH level and the obtained number of CL and total embryos after a superovulation treatment, it was concluded that measuring blood AMH level prior to any further costly implementation may be an effective method in donor selection., (© 2019 Blackwell Verlag GmbH.)

Superovulation technique is important to improve the efficiency of oocyte and animal production and reduce the number of oocyte donors. Previously, we have reported that the coadministration of inhibin antiserum (IAS) and equine chorionic gonadotropin (eCG) results in the production of >100 oocytes in a 4-week-old female C57BL/6 mice. It is well established that superovulation depends on the age of the female mice. However, detailed data regarding the ovulation of juvenile, mature, and aged female mice following the administration of IAS and eCG as well as the performance of reproductive technologies using oocytes have not yet been investigated. In the present study, we examined the effect of the age of female mice (3-50 weeks old) on the number of ovulated oocytes via the coadministration of IAS and eCG or eCG alone. Treatment with IAS plus eCG produced the maximum number of oocytes at 4 weeks of age. Moreover, IAS plus eCG produced more oocytes than eCG alone in mice aged between 3 and 5 weeks or 7 and 30 weeks. The fertilization and birth rates were similar between the two treatments at any age. Moreover, after vitrifying and warming the embryos, the survival and birth rates of two-cell embryos were similar between the two treatments. Subsequently, we examined the optimal ages of female mice (between 24 and 34 days) to obtain a high and stable number of oocytes. In mice aged between 24 and 32 days, IAS plus eCG induced the production of more eggs than eCG alone. Notably, the coadministration of IAS and eCG in mice aged between 25 and 31 days resulted in stable ovulation and high number of oocytes. Using the tip of the optimal female aged between 25 and 31 days old, we demonstrated an efficient production of embryos and offspring between homozygous knockout males and few females aged 26-28 days via in-vitro fertilization and embryo transfer. In summary, the coadministration of IAS and eCG resulted in a higher number of oocytes in juvenile, mature, and aged female mice. This treatment may be useful for the efficient production of homozygous mutant mice from a limited number of female mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effects of plasma progesterone (P 4 ) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day -2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P 4 (P 4 group) and (2) no ID (Control group). On Day 5, females were administered PGF 2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P =  0.04) in the control (9.7 ± 4.2) than P 4 -treated (6.7 ± 4.9) group; number of corpora lutea did not differ (5.5 ± 3.1 and 5.2 ± 3.4 respectively). Ovulation rate was greater (P <  0.01) in the P 4 -group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P =  0.07) in the P 4 -group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P 4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ppm1b, a metal‑dependent serine/threonine protein phosphatase, catalyzes the dephosphorylation of a variety of phosphorylated proteins. Ppm1b‑/‑ mouse embryos die at the fertilized oocyte stage, whereas Ppm1b+/‑ mice with a C57BL/6 background exhibit no phenotypic abnormalities. Because the C57BL/6 strain produces a limited number of pups, in an attempt to produce Ppm1b‑/‑ mice, congenic Ppm1b+/‑ mice with an ICR background were established, which are more fertile and gave birth to more pups. As a result, however, no Ppm1b‑/‑ offspring were obtained when pairs of Ppm1b+/‑ ICR mice were bred again. Ppm1b+/‑ male and female ICR mice were analyzed from the viewpoint of fecundity. The Ppm1b haploinsufficiency had no effect on testicular weight or the number of sperm in male mice. Despite the fact that the levels of Ppm1b protein in the ovaries of sexually mature Ppm1b+/‑ mice were decreased compared with those of Ppm1b+/+ mice, there appeared to be no significant difference in the histological appearance of the ovaries, litter sizes or plasma progesterone levels at the estrous stage. When superovulation was induced by stimulation using a hormone treatment, the number of ovulated oocytes were the same for Ppm1b+/‑ and Ppm1b+/+ mice at 4 weeks of age when the estrous cycle did not proceed, however, the number of ovulated oocytes was lower in sexually mature Ppm1b+/‑ mice at 11 weeks of age compared with Ppm1b+/+ mice in the first and the second superovulation cycles. These collective results suggest that follicle development is excessive in Ppm1b+/‑ mice, and that this leads to a partial depletion of matured follicles and a corresponding decrease in the number of ovulated oocytes.

The aim of this study was to evaluate the effect of a co-treatment of follicle-stimulating hormone (FSH) plus equine chorionic gonadotrophin (eCG) on serum insulin and insulin-like growth factor 1 (IGF-1) concentrations, superovulatory response, ovulatory rate, and number and production of embryos in Katahdin breed ewes during the non-breeding season. Twenty Katahdin ewes were synchronized with progestagens (CIDR) and assigned to two superovulation treatments (n = 10): (1): ewes treated with 200 mg ewe -1 of FSH from day 5 to 8 after CIDR insertion at decreasing doses every 12 h (FSH group) and (2) ewes treated as FSH group plus 300 IU of eCG on day 5 after CIDR insertion (FSH + eCG group). Estrous behavior was monitored and direct mating was performed. On days - 7 (CIDR insertion), 0 (CIDR withdrawal), and 7 (embryo recovery), blood samples were collected to determine serum hormone concentrations. Co-treatment with eCG (FSH group) did not affect (P > 0.05) serum hormone levels. Superovulation response, ovulation rate, recovery rate, fertilization, and number of embryos were also similar (P > 0.05) between treatments. Compared with FSH group, FSH + eCG ewes had lower (P < 0.05) number of transferable embryos and higher (P < 0.05) number of oocyte and a tendency to increase the number of degenerated embryos (P = 0.07). Overall results suggest that the administration of eCG is not beneficial either to improve the ovulatory response or the amount of transferable embryos in Katahdin ewes superovulated with a protocol using progesterone and FSH at decreasing doses.

Superovulation protocols are designed to achieve maximum embryo yields. Nevertheless, ovarian response control and the quality of obtained embryos are still a challenge. On the other hand, to save the superovulated embryos until their subsequent use, it is usual to cryopreserve them, so it is also crucial to assess their cryotolerance. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (FSH-CTP) alone or supplemented with human chorionic gonadotropin (hCG) and to determine the impact of this stimulation on in vitro and in vivo development of fresh or devitrified embryos. Our outcomes showed that ovulation rate and recovered embryos were significantly increased when hCG was used. In vitro development of fresh and devitrified embryos and survival at birth were not significantly affected by superstimulation treatment. Results of this study suggest that a single injection of long-acting FSH-CTP supplemented with hCG can be effectively used in rabbits to elicit an increase in ovulation rate and number of recovered embryos. Furthermore, we demonstrated that hCG supplementation had no negative effects in embryo cryosurvival and development, showing similar survival rate at birth than FSH-CTP alone group., (© 2019 Blackwell Verlag GmbH.)

This study was conducted to develop simple superovulation protocols in dromedary camels. Using two commercial FSH products, a series of experiments was conducted to evaluate the effect of different superovulation protocols on ovarian response and embryo production. In experiment 1, camels in control group (n = 15) received 400 mg Folltropin-V in a traditional protocol (FSH diluted in saline and given twice daily in decreasing doses over 5 days) and camels in split-injection (2 doses 48 h apart) groups received either 400 (n = 16, first dose: 320 mg, second dose: 80 mg) or 200 mg (n = 16, first dose: 120 mg, second dose: 80 mg) of slow-release (SR) preparation of Folltropin-V [Folltropin-V diluted in hyaluronan (5 mg/mL) solution]. In experiment 2, camels in control group (n = 13) received 2000 IU Pluset in a traditional protocol and camels in split-injection groups received either 2000 (n = 14, first dose: 1600 IU, second dose: 400 IU) or 1000 IU (n = 16, first dose: 600 IU, second dose: 400 IU) of SR preparation of Pluset (Pluset diluted in hyaluronan solution). In experiment 3, camels received SR preparation of 200 mg Folltropin-V (n = 45, first dose: 120 mg, second dose: 80 mg) or 1000 IU Pluset (n = 42, first dose: 600 IU, second dose: 400 IU) in a split-injection protocol. In experiments 1 and 2, the mean number of ovulations, corpora lutea, transferable embryos and unfertilized ova were similar (P > 0.05) between groups. In experiment 3, there was no difference (P > 0.05) between SR preparations of Folltropin and Pluset on the number of ovulatory sized (≥9 mm) follicles (15.8 ± 0.9 vs 16.7 ± 0.9) and transferable embryos (5.0 ± 0.4 vs 5.2 ± 0.5). In conclusion, split-injection of SR preparation of FSH resulted in a similar superovulatory response compared to a traditional protocol and a similar superovulatory response can be achieved with SR preparations of Folltropin-V and Pluset in a split-injection protocol., (Copyright © 2018 Elsevier Inc. All rights reserved.)