Optimizing ReBreed21 I: Evaluation of endocrine and ovarian dynamics in non-bred Bos indicus heifers.

The present study evaluated follicular and endocrine dynamics during ReBreed21, a reproductive strategy that allows resynchronization of ovulation every 21 days in Bos indicus (Nelore) heifers. A synchronized estrous cycle was induced using a standard timed ovulation protocol (d -10: P4 implant inserted + 2 mg estradiol benzoate; d -2: P4 removed+ 0.5 mg cloprostenol + 0.6 mg estradiol cypionate + 200 IU equine chorionic gonadotropin (eCG); d0: 8.4 µg buserelin) without AI to ensure nonpregnancy in heifers. Day of GnRH was designated d0 of estrous cycle. On d12, heifers (n = 80) were randomized into three experimental groups: (1) ReBreed21 (n = 28) d12 P4 device inserted, d19 P4 device withdrawal plus 200 IU eCG, and d21 8.4 µg buserelin (GnRH); (2) ReBreed21+G (n = 26) same as ReBreed21 plus GnRH (16.8 µg) treatment on d12; and (3) Control (n = 26) no treatment. ReBreed21+G increased two-fold (62.9%; 18/26) percentage of heifers with synchronized follicular wave emergence compared to Control (34.6%; 9/26) whereas ReBreed21 (53.6%; 15/28) was intermediate. The ReBreeed21 groups (eCG on d19) increased (P < 0.01) follicular growth between d19 and d21 in ReBreed21 (2.3 ± 0.2 mm) and ReBreed21+G (3.4 ± 0.2 mm) compared with Control (1.2 ± 0.3 mm), resulting in greater (P < 0.01) follicle diameter on d21 for ReBreed21 (10.7 ± 0.4 mm) and ReBreed21+G (10.8 ± 0.4 mm) compared with Control (9.1 ± 0.5 mm). Structural luteolysis was similar among groups (P = 0.51), although the average day when P4 was <1 ng/mL was later (P < 0.01) for ReBreed21 (20.5 ± 0.2) and ReBreed21+G (20.7 ± 0.2) compared to Control (19.2 ± 0.4). Overall ovulation at the end of the estrous cycle was increased (P = 0.03) for ReBreed21 groups (83.3%; 45/54) compared with Control (57.7%; 15/26). Synchronized ovulation on day 22-23 was greater (P < 0.01) for ReBreed21 (78.6%; 22/28) and ReBreed21+G (76.9%; 20/26) compared with Control (30.8%; 8/26). Thus, the ReBreed21 resynchronization program produced acceptable endocrine and follicular dynamics, including synchronized ovulation at the end of the protocol in nonpregnant heifers providing good rationale for testing the fertility and practical implementation of this protocol under field conditions.

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

A combined treatment with progesterone, anti-inhibin serum, and equine chorionic gonadotropin improves number of ovulated oocytes in young C57BL/6J mice.

Superovulation procedures are routinely and widely used in mouse reproductive technology. Previous studies have shown that a large number of oocytes can be obtained from adult mice (> 10 weeks old) using a combined treatment with progesterone (P4) and anti-inhibin serum (AIS). However, these effects have not been fully investigated in young (4 weeks) C57BL/6J mice. Here, we found that a modified superovulation protocol (combined treatment with P4, AIS, eCG (equine chorionic gonadotropin), and hCG (human chorionic gonadotropin); P4D2-Ae-h) improved the number of oocytes compared to the control (eCG and hCG) (39.7 vs. 21.3 oocytes/mouse). After in vitro fertilization, pronuclear formation rates were 69.3% (P4D2-Ae-h group) and 66.2% (control group). After embryo transfer, 46.4% (116/250) of the embryos in the P4D2-Ae-h group successfully developed to term, which was comparable to the control group (42.9%; 123/287 embryos). In conclusion, our protocol (P4D2-Ae-h) was effective for superovulation in young C57BL/6J mice.

Low-Dose Sodium Salicylate Promotes Ovulation by Regulating Steroids via CYP17A1.

To meet the current demand of assisted reproduction and animal breeding via superovulation and reduce the impact of hormone drugs, it is necessary to develop new superovulation drugs. This study examined the role of inflammation and steroids in ovulation. Sodium salicylate can regulate inflammation and steroids. However, the effect of sodium salicylate on ovulation has not been studied. In this study, mice were intraperitoneally injected with different concentrations of sodium salicylate for four consecutive days. The effects of sodium salicylate on oocyte quality and on the number of ovulations were examined, and these effects were compared with those of pregnant horse serum gonadotropin (PMSG)/follicle-stimulating hormone (FSH) treatment. We found that low-dose sodium salicylate increased the levels of ovulation hormones and inflammation by promoting the expression of CYP17A1. Sodium salicylate had the same effect as the commonly used superovulation drug PMSG/FSH and reduced the histone methylation level. Sodium salicylate can promote ovulation in mice and Awang sheep. It can greatly decrease the use of hormone drugs, reduce breeding costs and physical impacts, and can thus be used for livestock breeding.

Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.

Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.

Evaluation of hCG as gonadotropic support to timed embryo transfer protocol in beef cattle.

This study compared the reproductive performance of embryo recipients treated with a timed embryo transfer (TET) protocol using human chorionic gonadotropin (hCG) or equine chorionic gonadotropin (eCG). On a random day of the estrous cycle (Day -10) indicus-taurus recipients (n = 341; 194 nulliparous and 147 multiparous cows) with a body condition score between 3.0 and 4.0, were submitted to the TET protocol consisting of an intramuscular (i.m.) injection of 2.0 mg estradiol benzoate (EB) and the insertion of intravaginal progesterone (P4) device that remained until Day -2.5. On the same day (-2.5), the recipients received i.m. 150 mg D-cloprostenol and 1 mg estradiol cypionate and were randomly divided into two groups: the eCG group (n = 179), in which females received i.m. 300 IU eCG and the hCG group (n = 162), in which females received 150 IU hCG. Then, estrus intensity and the diameter of the dominant follicle (DF) were monitored on D0 and the quality of the corpus luteum (CL) (B mode and color Doppler) was assessed on D7 to select recipients eligible for receiving the transfer of an embryo produced in vitro. Pregnancy diagnosis was assessed 23 days after the transfer. Continuous data were analyzed by ANOVA using a mixed-effects model and Tukey's test. The rates were analyzed using a logistic regression model. The diameter of the DF on day 0 of the TET protocol was influenced by the interaction between gonadotropic treatment and category (P = 0.01), and nulliparous recipients treated with hCG had the smallest diameter. Treatment with hCG and eCG resulted in a high rate of estrus expression; however, the proportion of females with a high-intensity of estrus was higher in the hCG group (79.84 vs. 68.61%, respectively; P = 0.03). The utilization rate (recipients with CL) showed a tendency (P = 0.06) to be influenced by the interaction between gonadotropic treatment and category, wherein nulliparous recipients treated with hCG exhibited a lower utilization rate than the other groups. The diameter, perimeter, and area of the CL were similar (P > 0.1) in all groups. However, the hCG group resulted in CL with a better Doppler evaluation score (P = 0.04), central blood flow (P = 0.03), and tendency towards greater peripheral blood flow (P = 0.08). The rates of conception (32.00% hCG vs. 35.10% eCG; P = 0.46) and pregnancy (24.69% hCG vs. 29.61% eCG; P = 0.20) were similar between the hCG and eCG groups. However, an interaction between the gonadotropic treatment and category revealed lower conception (P = 0.01) and pregnancy rates (P = 0.001) in nulliparous recipients treated with hCG. Treatment with hCG resulted in a greater intensity of estrus expression and CL with a higher Doppler score, which determined rates of utilization, conception, and pregnancy similar to conventional protocols using eCG. However, nulliparous recipients treated with hCG exhibited a lower overall reproductive rate.

Equine chorionic gonadotropin administered on day 5 of a 7-days fixed-time artificial insemination program improves ovulation synchrony and corpus luteum function in anestrous beef cows.

In order to assess the effect of equine chorionic gonadotropin (eCG) administered on Day 5 or 7 of a fixed-time artificial insemination protocol (FTAI) in anestrous suckled beef cows, two experiments were performed to determine the following endpoints: Experiment 1 (n = 22), preovulatory follicle (POF) diameter, ovulation time, corpus luteum (CL) area, estradiol (E2) and progesterone (P4) concentrations; and Experiment 2 (n = 676), a field trial to evaluate conception rate using the same experimental design. In both experiments, a synchronization protocol using estradiol benzoate (EB) (Day 0), intravaginal progestin device (IVD) (Days 0 through 7), prostaglandin (PGF) (Day 7), eCG (Day 5 or 7), and GnRH (Day 9). Treatment consisted of administering 400 IU of eCG on Day 5 (T5) or Day 7 (T7 or control) concomitant with treatment with PGF2α. In experiment 1, all cows of T5 ovulated by 16 h after GnRH administration. The POF tended (P = 0.06; P = 0.07) to be larger at 1 and 2 d before ovulation in T5. The day before ovulation, E2 tended to be lower (P = 0.06) in T5 compared with T7. The CL area and the P4 concentrations were greater (P = 0.04) on day 9 in T5 compared with T7. In experiment 2, the conception rate was greater (P = 0.04) in T5 (72.2%) compared with T7 (61.0%) group. Therefore, administration of eCG on Day 5 of the designed protocol hastened ovulation of a greater follicle, which produced a larger CL and greater concentrations of progesterone by Day 9 after ovulation, resulting in 11.2% increase in cows pregnant.

The Corticosterone-Glucocorticoid Receptor-AP1/CREB Axis Inhibits the Luteinizing Hormone Receptor Expression in Mouse Granulosa Cells.

Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to a disorder of the body's endocrine system and damage to the body. Our previous work showed that CORT can block follicular development in mice. Since LH acts through binding with the luteinizing hormone receptor (Lhcgr), the present study aimed to investigate whether and how corticosterone (CORT) influences Lhcgr expression in mouse ovarian granulosa cells (GCs). For this purpose, three-week-old ICR female mice were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG). In addition, the treatment group was injected with CORT (1 mg/mouse) at intervals of 8 h and the control group was injected with the same volume of methyl sulfoxide (DMSO). GCs were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, the mouse GCs obtained from healthy follicles were treated with CORT alone, or together with inhibitors against the glucocorticoid receptor (Nr3c1). The results showed that the CORT caused a downregulation of Lhcgr expression in GCs, which was accompanied by impaired cell viability. Moreover, the effect of the CORT was mediated by binding to its receptor (Nr3c1) in GCs. Further investigation revealed that Nr3c1 might regulate the transcription of Lhcgr through inhibiting the expression of Lhcgr transcription factors, including AP1 and Creb. Taken together, our findings suggested a possible mechanism of CORT-induced anovulation involving the inhibition of Lhcgr expression in GCs by the CORT-Nr3c1-AP1/Creb axis.

Combined synchronization and superovulation treatments negatively impact embryo viability possibly by the downregulation of WNT/ß-catenin and Notch signaling genes in the porcine endometrium.

The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes ≥2 or ≤2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 ± 10.2 and 38.3 ± 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 ± 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% ± 15.2%) compared to SC and NC groups (93.8 ± 7.6% and 91.8 ± 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/ß-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation of ß-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk.

Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/ß-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.

Establishment of a useful in vitro decidual induction model using eCG-primed nonpregnant mouse endometrial stromal cells†.

Uterine endometrial differentiation is essential for developmental continuity and female health. A convenient in vitro model mimicking the physiological status is needed to effectively evaluate implantation and uterine response mechanisms. Thus, we developed a promising in vitro model, the FSS (FSH mimic-stimulated synchronized) model, by using primary mouse uterine stromal cells (mUSCs) obtained from equine chorionic gonadotropin (eCG)-primed mice. These mUSCs could be differentiated into decidualized cells with 17 beta-estradiol (E2) and progesterone (P4). The pregnancy day 4 (PD4) model, in which mUSCs are obtained at day 4 of pregnancy, was used as a control. The cell shape index and polyploidy rates were similar between the two models. The staining intensities of lipids and glycogen were significantly higher in the induced groups in both models but stronger in the FSS model than in the PD4 model. The expression levels of AP-TNAP, cathepsin L, Prl8a2, Gja1, Cebpb, and Igfbp1 were increased at 24 h after decidual induction. PR-alpha and PR-beta levels were also increased at 24 h after decidual induction in both models. These results indicate that the FSS model provides a convenient method for obtaining USCs that are usable for various experimental approaches due to their physiological competence and flexibility for triggering induction. This may serve as a model system for the study of pathogeneses originating from the endometrium or communication with other tissues and lead to a better understanding of embryo implantation mechanisms. Furthermore, the results of this study will be integral for further refinements of 3D uterine culture manipulation techniques.

Effect of oestrogen on mouse follicle growth and meiotic resumption.

Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus-oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 µm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 µM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 µM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

Induction of right open reading frame kinase 3 (RIOK3) during ovulation and luteinisation in rat ovary.

Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were treated with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development, followed 48h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.

Approaches of estrous synchronization in sheep: developments during the last two decades: a review.

The objective of the current review was to summarize the protocols used for estrous synchronization in ewes during the last two decades. Progesterone (P4) is a major hormone used in most protocols. P4 in the form of a controlled internal drug releasing (CIDR) device, medroxyprogesterone acetate (MAP), and fluorogestone acetates (FGA) has been used for estrous synchronization. Also, gonadotropins such as equine chorionic gonadotropin (eCG) and gonadotropin-releasing hormone (GnRH) are often administered at the end of P4-based protocols to improve fertility. Moreover, the administration of prostaglandins (PG) and ram effects have been used for estrus induction and synchronization of ewes. The findings of previous studies indicate that the outcome of administering various synthetics P4 analogues (CIDR, MAP, and FGA) in ewes is comparable in terms of estrous synchronization/induction. The supplementation of P4-based protocols with eCG, however, improves the estrus response and pregnancy rate during breeding and non-breeding season. On the other hand, PG is effective for successful estrous synchronization during the breeding season only. Often, two injections of PG are administered either 11 or 14 days apart along with P4-based protocols to lyse ovine corpus luteum (CL) when it is receptive to PG i.e., 3 days post-ovulation. Alternatively, the "ram effect" has been shown to improve the efficacy of P4-based protocols and can be used as an alternative to eCG in ewes. The current review describes the methods of synchronization and their outcomes during breeding and a non-breeding season in ewes.

Anticentromere antibody induced by immunization with centromere protein and Freund's complete adjuvant may interfere with mouse early-stage embryo.

BACKGROUND: Anticentromere antibody (ACA) is a member of the antinuclear antibody spectrum (ANAs) which has been speculated to be associated with subfertility. Thus, the present study aimed to investigate the induction of ACA production and its potential interference with early-stage embryos. METHODS: Recombinant centromere protein-A (CENP-A) or centromere protein-B (CENP-B) and complete Freund's adjuvant (CFA) were used to immunize mice. Serum ACA level was then evaluated by using an indirect immunofluorescence test. Immunofluorescence assay was performed to detect IgG in follicles in ovarian tissues and early-stage embryos. RESULTS: Following treatment, serum positive ACA was observed in mice treated with CENP and CFA. Furthermore, IgG were detected in follicular fluid and early-stage embryos from mice treated with CENP and CFA. CONCLUSIONS: This study preliminarily indicated that ACA induced by CENP and CFA may penetrate into the living embryos of early-stage in mice.

Induction of oestrus by administering Inhibin antiserum along with equine chorionic gonadotropin in anoestrous bitches.

As dogs experience oestrus only once or twice a year, it is necessary to establish an effective method of oestrous induction for efficient breeding. In the present study, we evaluated inhibin antiserum (IAS) on oestrous induction in anoestrous females. Bitches were administered 0.5 ml/kg IAS or a mixture of 50 IU/kg equine chorionic gonadotropin (eCG) and 0.5 ml/kg IAS and 500 IU human chorionic gonadotropin (hCG) administered 7 days after the mixture injection. As a control, bitches received 50 IU/kg eCG, with 500 IU hCG administered 7 days after eCG injection. Blood-tinged vaginal discharge, vulvar swelling, plasma progesterone concentrations and ovarian follicular development were assessed from day 0 to day 14. IAS alone injection did not induce oestrus in bitches at the anoestrous stage. Conversely, vulvar swelling, blood-tinged vaginal discharge and an estimated luteinizing hormone (LH) surge appeared on days 3-7, days 3-6 and days 7-9 after the IAS+eCG mixture injection, respectively, in all five bitches at the anoestrous stage. The average number of developing and ovulated follicles in bitches administered IAS+eCG was 8.8 and 9.6 respectively. A single eCG injection followed by hCG induced oestrous signs, with an average of 8.3 developing follicles and 4.5 ovulated follicles. This study revealed that IAS alone did not induce oestrus, but when IAS was used in combination with eCG, it induced oestrus and promoted a considerable number of ovulations in anoestrous dogs.

Improvement in early antral follicle development and gene expression modulation prior to follicle aspiration in bovine cumulus-oocyte complexes by equine chorionic gonadotropin.

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at -80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0-5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 µg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Vaginal stimulation enhances ovulation of queen ovaries treated using a combination of eCG and hCG.

Follicular changes throughout the oestrous phase have been poorly documented in queens because of the location and the small size of ovaries. We investigated follicular development in queens treated with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and evaluated the effects of vaginal stimulation by a tomcat on ovulation induction. A hormonal treatment was administered using a simple crossover design. Four queens were administered 150 IU of eCG (day 1) and 250 IU of hCG on day 5 and 6. Half of the queens were mated with a vasectomised tomcat for 3 days after hCG injection. Ultrasound imaging of the ovaries clamped at a subcutaneous site was performed once a day from day 1 to 7, and on day 13, and the serum concentrations of oestradiol and progesterone were examined on day 1, 5, 7 and 13. The mean number of follicles gradually increased with the eCG treatment and decreased after hCG injection. The ovulation rate of follicles was significantly higher in the vaginal stimulation group (70.0%) than in the control group (42.6%). During the hormonal treatments, the serum concentration of oestradiol and progesterone did not differ between the two groups. Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that eCG and hCG treatment promoted the follicular growth and corpus luteum formation, respectively. The combination of hCG injection with vaginal stimulation by a vasectomised tomcat enhanced the ovulation rate of follicles.

Morphological changes in mouse ovary due to hormonal hypersecretion and matrix metalloproteinase -2 activity.

We analyzed whether aberrant gonadotropin secretion affects the morphological remodeling of murine ovarian tissues facilitated by activated matrix metalloproteinase (MMP) enzymes. Six mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG) or human chorionic gonadotropin (HCG) every two days after estrus synchronization. Morphology and expression of various MMPs were assessed following the successful induction of hormonal secretion in these tissues. HCG treatment, but not PMSG treatment, resulted in the expanded production of granulose second follicular cells. In addition, the number of developing follicular cells in the HCG group increased compared with that in the PMSG group. Ovarian diameters were also very small in the PMSG group. Immunohistochemistry revealed decreased MMP-2 protein activity in the HCG group and increased MMP-2 activity in the PMSG group. Activity was particularly high in theca and granulose cells of the PMSG group, but only partial activity was observed in the theca cells of the HCG group. Vascular endothelial growth factor activity was increased in both the external and internal theca cell walls in the PMSG group while the HCG group showed high overall expression of this protein in the internal theca cells. These data indicate that follicular cell activity and remodeling of the ovaries differ based on the type of secretory hormone signals they receive. Inappropriate gonadotropin secretion may induce functional changes in the ovaries, and follicular remodeling may be facilitated by the activity of various MMPs.