Roles of kisspeptin in IVF/ICSI-treated infertile women and in human granulosa cells

Kisspeptin, a crucial central regulator of reproduction, has been used as a trigger in in vitro fertilization (IVF) treatment. This study aimed to investigate the roles of kisspeptin in IVF treatment in infertile females (n = 30); and in steroidogenesis in human granulosa-like tumor cell line (KGN). In the human study, blood was collected at three time points including (1) the beginning of gonadotropin stimulation (Phase I), (2) around eight days after gonadotropin stimulation (Phase II), and (3) on the day of ovum pick-up (Phase III). Follicular fluid (FF) was collected at Phase III. Serum human chorionic gonadotropin (hCG) was measured 15 days after embryo transfer and fetal heart beats were determined around 42 days of menstrual cycle to classify the subjects into successful and unsuccessful groups. FF kisspeptin levels were higher in successful compared with unsuccessful subjects (P < 0.01). Kisspeptin levels were significantly higher in FF than in serum in successful subjects (P < 0.05) but were comparable in unsuccessful subjects. Serum kisspeptin was comparable among three phases in the successful group but its levels in Phase III were significantly lower compared with Phase I in the unsuccessful group (P < 0.01). Serum kisspeptin in Phase II/III had positive correlations with serum E2 in Phases II and III and the outcomes of IVF/intracytoplasmic sperm injection (ICSI) treatment including serum hCG levels. For the cell experiment (n = 3), kisspeptin treatment in the presence of FSH together with IGF-1 enhanced CYP19A1 (aromatase) mRNA expression compared with control. FSH alone increased aromatase concentrations in the supernatant compared with control and kisspeptin at the dose of 10^-2 mmol/L with FSH enhanced aromatase concentrations in the supernatant compared with FSH alone (P < 0.001 all). In conclusion, kisspeptin enhanced aromatase expression and secretion and was associated with positive outcomes of IVF/ICSI treatment. Further studies regarding supplementation of kisspeptin could reveal its beneficial effects on IVF/ICSI treatment.     

Coagulansin-A has beneficial effects on the development of bovine embryos in?vitro via HSP70 induction

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.     

Renin and ovarian vascularization in cows with follicular cysts after epidural administration of a GnRH analogue

The ovarian renin–angiotensin system may play an important role in follicular growth and maturation, as well as in the process of ovulation. The aim of this study was to investigate the effects of administration of a GnRH analogue to cows with ovarian follicular cysts on plasma renin concentrations and ovarian vascularization. This study was performed with 60 Friesian cows, which were diagnosed with follicular cysts, and randomly allocated into two groups: group A (treatment; n = 30) received 2 ml of lecirelin (Dalmarelin^® – Fatro), per head via sacro-coccygeal epidural, and group B (control; n = 30) received 2 ml saline solution (0.9% NaCl) per head by the same route. Blood samples were immediately collected prior to administration (T0) and then 24 h (T1), 48 h (T2) and 8 days (T3) after administration of the treatment, for both groups. Ovarian vascularization was evaluated utilizing Power Doppler on these same days in 10 animals from each group. The number of pixels detected by Power Doppler was used as an indicator of the degree of vascularization. Plasma renin concentrations remained relatively constant for the control (group B) animals, but increased as the sampling period progressed (NS) for the treated cows (group A). Similarly, there were no changes in ovarian vascularization (number of pixels) for the control cows, but vascularization increased throughout the sampling period in the treated animals. The number of pixels associated with cysts was significantly higher for treated compared to control cows at 24 h after treatment (P < 0.001). The epidural administration of a GnRH analogue was determined to be a highly effective therapy for follicular cysts (regression occurred in 82% of treated cows within 8 ± 2 days after treatment, but in none of the control cows), which also enhanced ovarian vascularization.     

Correlations between ovarian follicular blood flow and superovulatory responses in ewes

The primary goal of this study was to employ ultrasonography to examine the ovaries of ewes undergoing superovulatory treatment for correlations between antral follicular blood flow and ovarian responses/embryo yields. Five Santa Inês ewes were subjected to a short- (Days 0–6, Group 1) and five to a long-term progesterone-based protocol (Days 0–12, Group 2) to synchronize estrus and ovulations after the superovulatory treatment. Porcine FSH (pFSH, 200 mg) was administered in 8 decreasing doses over 4 days, starting on Days 4 and 10 in Groups 1 and 2, respectively. After CIDR removal, all ewes were bred by a ram and embryos were recovered surgically 7 days later. Transrectal ovarian ultrasonography was performed the day before and on all 4 days of the superovulatory treatment. Both an arbitrary-scale [(0) non-detectable; (1) small; (2) moderate; (3) intense blood flow] and quantitative analysis of the blood flow area were used to assess the follicular blood flow in color Doppler images. There were no significant correlations between the arbitrary blood flow scores and superovulatory responses in the ewes of the present study. However, there was a positive correlation between the quantitative estimates of follicular blood flow on the final day of the superovulatory treatment, and the number (DA: r = 0.68, P < 0.05; DA/TA × 100%: r = 0.85, P < 0.05) and percentage (DA: r = 0.65, P < 0.05; DA/TA × 100%: r = 0.91, P < 0.001) of unfertilized eggs (DA: Doppler area, TA: total area of the largest ovarian cross section). This experiment presents a commercially practical tool for predicting superovulatory outcomes in ewes and evidence for the existence of follicular blood flow threshold that may impinge negatively on oocyte quality when surpassed during hormonal ovarian superstimulation.     

Suitability of antral follicle counts and computer-assisted analysis of ultrasonographic and magnetic resonance images for estimating follicular reserve in porcine,ovine and bovine ovaries ex situ

This study was conducted to determine if correlations exist between the numbers of microscopic follicles comprising ovarian follicular reserve (OFR) and antral follicle counts (AFCs), and to assess the usefulness of computerized analyses of ovarian ultrasonograms and magnetic resonance (MR) images for estimating OFR in excised porcine, ovine and bovine ovaries. As a pre-requisite to these analyses, we characterized and compared ovarian cortical histomorhpology and follicle populations in the three species varying in prolificacy and overall reproductive longevity, and hence the total number of microscopic and antral follicles. Ultrasonographic and MR images were obtained at the scanner settings optimized to provide opposing contrasts between antral follicles and the ovarian stroma. Commercially available ImageProPlus® analytical software was used to calculate numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of the pixel values) along the computer-generated lines (4–6) placed in the area corresponding to the ovarian cortex. The numbers of primordial (r = 0.38, P < 0.01) and intermediate follicles (r = 0.37, P < 0.01) were correlated with the numbers of antral follicles in bovine ovarian sections. The numbers of primordial (r = 0.28, P < 0.05), intermediate (r = 0.31, P < 0.01) and primary follicles (r = 0.27, P < 0.05) correlated directly with mean NPVs of the ultrasonographic ovarian images in cattle. There was a negative correlation between primary follicle numbers and NPVs of MR images (3D FAST-SPOILED GRADIENT ECHO) of the porcine ovarian cortex (r = −0.31, P < 0.05). To summarize, the numbers of primordial and intermediate follicles could only be estimated from AFCs in cows. Using ultrasound NPVs, the numbers of primordial, intermediate and primary follicles could be directly estimated in bovine ovaries and the quantitative image attributes of MR images were useful for quantifying porcine primary follicles. The bovine ovarian model is compatible with human situation and hence future studies should be undertaken to ascertain the usefulness of AFCs and ultrasonographic image analyses for estimating OFR in women.     

The Local Effects of Ovarian Diathermy in an Ovine Model of Polycystic Ovary Syndrome

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30–100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.     

Using vaginal discharge score (VDS) grading system to evaluate the effect of clinical endometritis on reproductive performance of dairy cows in China

Clinical endometritis (CE) is a major cause in affecting the reproductive performance of dairy cows. The objectives of this study were to ascertain the prevalence of CE and to evaluate the effect of CE on reproductive performance in dairy cows using vaginal discharge score (VDS) grading system. 803 dairy cows were examined by vaginoscope with 4-point VDS at 26 ± 3 days in milk (DIM) and classified into six groups: non-endometritis with VDS 0 (control; CON), endometritis with VDS 1 (MEM), non-treated endometritis with VDS 2 (NTME), treated endometritis with VDS 2 (TME), non-treated endometritis with VDS 3 (NTPE), and treated endometritis with VDS 3 (TPE). Cows in TME and TPE groups were treated with 200 mL of 50% dextrose solution by intrauterine infusion. The prevalence of CE was 33% at 26 ± 3 DIM. Binary logistic regression analysis revealed cows in MEM, NTME and NTPE groups had a less likelihood of first artificial insemination (AI) pregnancy than those in CON group (P < 0.05). Kaplan-Meier survival curves for days open were statistically different (P = 0.004). In Cox regression model, cows in NTME and NTPE groups had a reduced pregnancy rate than those in CON group (P < 0.05). The hazard of pregnancy in NTME group was lower than that in TME group (P = 0.044). Similarly, it was lower for the hazard of pregnancy in NTPE group than in TPE group (P = 0.048). Cows in MEM, NTME, and NTPE groups required more services per pregnancy than those in CON group (P < 0.05). In conclusion, CE examined by the VDS grading system impaired reproductive performance, and mild endometritis with VDS 1 should be treated in the early postpartum period to ameliorate fertility in dairy herds.     

TNFα Altered Inflammatory Responses,Impaired Health and Productivity,but Did Not Affect Glucose or Lipid Metabolism in Early-Lactation Dairy Cows

Inflammation may be a major contributing factor to peripartum metabolic disorders in dairy cattle. We tested whether administering an inflammatory cytokine, recombinant bovine tumor necrosis factor-α (rbTNFα), affects milk production, metabolism, and health during this period. Thirty-three Holstein cows (9 primiparous and 24 multiparous) were randomly assigned to 1 of 3 treatments at parturition. Treatments were 0 (Control), 1.5, or 3.0 µg/kg body weight rbTNFα, which were administered once daily by subcutaneous injection for the first 7 days of lactation. Statistical contrasts were used to evaluate the treatment and dose effects of rbTNFα administration. Plasma TNFα concentrations at 16 h post-administration tended to be increased (P<0.10) by rbTNFα administration, but no dose effect (P>0.10) was detected; rbTNFα treatments increased (P<0.01) concentrations of plasma haptoglobin. Most plasma eicosanoids were not affected (P>0.10) by rbTNFα administration, but 6 out of 16 measured eicosanoids changed (P<0.05) over the first week of lactation, reflecting elevated inflammatory mediators in the days immediately following parturition. Dry matter and water intake, milk yield, and milk fat and protein yields were all decreased (P<0.05) by rbTNFα treatments by 15 to 18%. Concentrations of plasma glucose, insulin, β-hydroxybutyrate, non-esterified fatty acids, triglyceride, 3-methylhistidine, and liver triglyceride were unaffected (P>0.10) by rbTNFα treatment. Glucose turnover rate was unaffected (P = 0.18) by rbTNFα administration. The higher dose of rbTNFα tended to increase the risk of cows developing one or more health disorders (P = 0.08). Taken together, these results indicate that administration of rbTNFα daily for the first 7 days of lactation altered inflammatory responses, impaired milk production and health, but did not significantly affect liver triglyceride accumulation or nutrient metabolism in dairy cows.     

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×10⁷ PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3⁺ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.     

Altered Theca and Cumulus Oocyte Complex Gene Expression,Follicular Arrest and Reduced Fertility in Cows with Dominant Follicle Follicular Fluid Androgen Excess

Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.     

Synchronization of follicular wave emergence does not improve embryonic yield in superovulated ewes

The present study aimed to investigate the effects of a combination of progesterone with different doses of E-17β on following end points: (1) ovarian follicular dynamics and emergence of a new follicular wave, and (2) superovulatory response and embryo yield. In Experiment 1, 28 ewes were randomly divided into four groups (n = 7) to receive either 2.0 mg, 1.0 mg, 0.5 mg or none E-17β one day after insertion of a progesterone device. The different doses of estradiol similarly delayed the moment of follicular emergence (overall mean = 3.1 ± 1.0 days vs. control group = 0.86 ± 1.0 days; P < 0.01), but the emergence of the new wave showed greater synchronization with the 0.5 mg dosage of E-17β. In Experiment 2, sixty-two donor ewes received an internal progesterone release device (day -1) for 7 d and 1 d after the insertion of this device (day 0) were allocated randomly to receive 0.5 mg of E-17β or only the vehicle (control group). Superstimulation was initiated on day 3 with the administration of 133 mg of pFSH in eight decreasing doses. Contrary to expectations, the protocol with the administration of 0.5 mg E-17β did not improve the percentage of donors with > 2 CL, the number of CL and the production of embryos (P > 0.05). It was concluded that the combination of progesterone and 0.5 mg E-17β was more efficient in synchronizing the emergence of the new follicular wave, however this approach seems to be unnecessary in ewe’s superovulation programs.     

Exogenous progesterone enhances ova and embryo quality following superstimulation of the first follicular wave in Nelore (Bos indicus) donors

Three experiments were conducted to evaluate the effects of exogenous progesterone on superovulatory response and ova/embryo quality in Bos indicus donors superstimulated during the first follicular wave (FFW). We hypothesized that exogenous progesterone during gonadotropin treatments would improve ova and embryo quality. In Experiment 1, 18 Nelore cows were randomly allocated to three groups: (1) FFW; (2) FFW plus a progesterone-releasing device (FFW+P4); and (3) control (E2+P4). Cows in the FFW groups were superstimulated beginning at synchronized ovulation, whereas cows in the control group were superstimulated after synchronization of follicular wave emergence with estradiol plus progesterone (E2+P4). There were no differences in mean (± SD) numbers of transferable embryos between FFW+P4 (8.0 ± 4.5) and control (6.7 ± 4.8) groups, but both were higher (P = 0.006) than the FFW group (0.2 ± 0.4). In Experiment 2, FFW and FFW+P4 were compared in 20 Nelore donors; exogenous progesterone increased the number of transferable embryos (3.9 ± 3.4 vs. 1.3 ± 4.1, P = 0.003). In Experiment 3, FFW and FFW+P4 were compared in 10 Nelore donors except that cows were slaughtered 12 h after pLH (Lutropin-V^®, Bioniche Animal Health, Belleville, ON, Canada) treatment. More mature cumulus oocyte complex (COC) (expanded cumulus cell layers) were collected in the FFW+P4 group than in the FFW group (21.8 ± 13.1 vs. 10.8±14.7; P = 0.003). In summary, superovulatory response was satisfactory when FSH (Folltropin-V^®, Bioniche Animal Health) treatment was initiated at emergence of the first follicular wave in Nelore (Bos indicus) donors, and the hypothesis that administration of exogenous progesterone during the treatment will improve oocyte and embryo quality was supported.     

Protective effect of mirtazapine and hesperidin on cyclophosphamide-induced oxidative damage and infertility in rat ovaries

Cyclophosphamide (CP) causes infertility due to ovarian toxicity. The toxicity mechanism suggests oxidative stress. We assessed whether mirtazapine (MTZ) and hesperidin (HSP) could promote ovarian protection against damage due to CP chemotherapy. Female Wistar rats aged 14 weeks were used. Animals were divided into four groups: control vehicle group (n = 8); CP group (n = 8, rats received 150 mg/kg of CP, single intraperitoneal [i.p.] injection); CP + MTZ group (n = 8, rats received same dose of CP + 30 mg/kg of MTZ, orally, daily); and HSP + CP group (n = 8, rats received same dose of CP + 100 mg/kg of HSP, orally, daily). After eight days of medication, ovaries were removed and ovarian toxicity was assessed by counting follicles and corpora lutea. Nitric oxide (NO) and malondialdehyde (MDA) levels, myeloperoxidase (MPO), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities were estimated in ovarian tissue. NO level, MDA level, and MPO activity were increased (P < 0.001), while, GPx and SOD activities were lowered significantly (P < 0.001) in CP-treated group compared with control vehicle. In addition, ovulation, number of follicles, and ovarian weight were reduced by CP treatment. On the contrary, rats pretreated with MTZ and HSP showed significant decrease in NO, MDA levels, and MPO activity, while, activities of SOD and GPx were increased (P < 0.001). Oxidative stress induced by CP in the rat ovary causes infertility in the female rats. HSP and MTZ could reverse this effect and provide protection of fertility against CP-induced toxicity.     

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in Granulosa Cells Using pDmrt-1-Cre or Amhr2-Cre Reduces Fertility by Arresting Follicular Development and by Reducing Litter Size in Female Mice

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa^-/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa^-/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P < 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa^-/- females (P < 0.007). The time between the first and second parturitions was also increased for heterozygous females (P < 0.04) and tended to be increased for pDmrt1-Cre;Vegfa^-/- females (P < 0.07). pDmrt1-Cre;Vegfa^-/- females had smaller ovaries (P < 0.04), reduced plasma estradiol (P < 0.007), fewer developing follicles (P < 0.008) and tended to have fewer corpora lutea (P < 0.08). Expression of Igf1r was reduced (P < 0.05); expression of Foxo3a tended to be increased (P < 0.06); and both Fshr (P < 0.1) and Sirt6 tended to be reduced (P < 0.06) in pDmrt1-Cre;Vegfa^-/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa^-/- mice that required more time from mating to first parturition (P < 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter.     

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.     

Mechanism of programmed cell death factor 4/nuclear factor-κB signaling pathway in porcine coronary micro-embolization-induced cardiac dysfunction

The aim of this study was to investigate the role of the programmed cell death factor 4 (PDCD4)/nuclear factor-κB (NF-κB) signaling pathway in coronary micro-embolism (CME)-induced inflammatory responses and cardiac dysfunction in a porcine model. Bama miniature pigs were randomly divided into four groups (n = 5 per group). Micro-embolization balls or saline were infused through a microcatheter in the left anterior descending (LAD) artery in the CME and Sham groups, respectively. PDCD4 siRNA or control siRNA mixed with transfection reagent was infused via the LAD artery 72 h before CME induction in the CME + siRNA-PDCD4 and siRNA-control groups, respectively. Cardiac function was evaluated with ultrasound. Tissue biopsy was stained with hematoxylin–eosin (HE) and hematoxylin basic fuchsin picric acid (HBFP) to measure infarction area. Myocardial PDCD4 and tumor necrosis factor-α (TNF-α) mRNA and protein expression were analyzed by quantitative PCR and Western blotting. NF-κB activity was evaluated in gel electrophoretic mobility shift assay. Echocardiographic parameters showed that compared with the sham group, the CME group had impaired heart function, manifested as systolic dysfunction and left ventricular dilatation (reduced left ventricular ejection fraction [LVEF], left ventricular fractional shortening [FS], and cardiac output [CO] [P < 0.05] and increased left ventricular end-diastolic diameter [LVEDd] [P < 0.05]). Compared with the CME group, the CME + siRNA-PDCD4 group had attenuated CME-induced cardiac function damage (increased LVEF, FS and CO [P < 0.05] and reduced LVEDd [P < 0.05]). Compared with the sham group, the CME group had significantly increased PDCD4 and TNF-α mRNA and protein expression and increased NF-κB activity (P < 0.05). These effects were significantly inhibited in the CME + siRNA-PDCD4 group (P < 0.05). In conclusion, PDCD4/NF-κB signaling pathway activation is an important mechanism for CME-induced cardiac dysfunction, suggesting that inhibition of PDCD4/NF-κB signaling pathway may be a potential target for the prevention and treatment of CME.     

Superovulation and recovery of zygotes suitable for microinjection in different breeds of sheep

This study investigated the appropriateness of different superovulatory protocols in various breeds of sheep for obtaining a maximum of zygotes suitable for microinjection. Animals were mated either once or two to three times to fertile rams. In Experiment 1, a 24 h interval between a two to three times mating and egg recovery resulted in 42.2% suitable zygotes whereas with single mating only 10.4% fertilized eggs were obtained. The extension of the interval to 40 h associated with a two to three times mating resulted in a recovery of 42.9% fertilized eggs but most (70%) of these were already at the two-cell stage. In Experiment 2, eCG resulted in similar superovulatory responses in Merino ewes as the more labour requiring FSH treatment (8.1 ± 4.5 versus 7.5 ± 4.1 corpora lutea (CL); 6.3 ± 3.0 versus 6.8 ± 4.0 oocytes/ zygotes; 39.4% versus 40.6% fertilization rate). In Experiment 3, following superovulation with pFSH (Folltropin®) the number of CL was not different among Merino, Finn, Crossbreds (Blackface X Finn) and Texel sheep (8.6 ± 5.2; 10.3 ± 4.5; 8.5 ± 3.8; 8.2 ± 2.8, respectively) as was the number of recovered oocytes/ zygotes (7.4 ± 5.6; 9.8 ± 4.3; 7.3 ± 3.8; 6.4 ± 2.9, respectively). However, the number of unfertilized ova was higher (P < 0.05) in Finn sheep as compared with Crossbreds and Texel sheep (5.0 ± 3.3 versus 2.2 ± 2.3 and 1.9 ± 2.6). Similarly, the fertilization rate was higher (P < 0.05) in Crossbreds and Texel sheep (64.4% and 65.5%) as compared with Finn and Merino sheep (38.3% and 42.5%). In Experiment 4, it was shown that in Merino sheep purified FSH supplemented with 68.6% LH resulted in lower (P < 0.05) superovulatory responses as compared with purified FSH supplemented with 133.1% LH or Folltropin (LH contamination 0.1%) (4.7 ± 3.3 versus 8.8 ± 3.8 and 8.6 ± 5.2 CL; 3.8 ± 2.5 versus 7.4 ± 3.6 and 7.4 ± 5.6 oocytes/zygotes, respectively). A three times repeated superovulatory treatment and oviductal flush per animal at monthly intervals did reduce (P < 0.05) the number of CL, but had no deleterious effect on zygote yields and the percentage of microinjectable zygotes. We conclude that (1) at least a two to three times mating is required to obtain acceptable fertilization rates; (2) the interval between mating and recovery should be 24–26 h in order to obtain zygotes; (3) eCG results in similar superovulatory responses as FSH; (4) Folltropin® is a suitable drug to induce superovulation in sheep; (5) the LH content of the FSH preparation plays a significant role in the superovulatory response of sheep; (6) superovulation and embryo recoveries can be repeated at least three times per animal without decrease in efficiency.     

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.     

Elevated Progesterone Levels on the Day of Oocyte Maturation May Affect Top Quality Embryo IVF Cycles

In contrast to the impact of elevated progesterone on endometrial receptivity, the data on whether increased progesterone levels affects the quality of embryos is still limited. This study retrospectively enrolled 4,236 fresh in vitro fertilization (IVF) cycles and sought to determine whether increased progesterone is associated with adverse outcomes with regard to top quality embryos (TQE). The results showed that the TQE rate significantly correlated with progesterone levels on the day of human chorionic gonadotropin (hCG) trigger (P = 0.009). Multivariate linear regression analysis of factors related to the TQE rate, in conventional IVF cycles, showed that the TQE rate was negatively associated with progesterone concentration on the day of hCG (OR was -1.658, 95% CI: -2.806 to -0.510, P = 0.005). When the serum progesterone level was within the interval 2.0–2.5 ng/ml, the TQE rate was significantly lower (P <0.05) than when the progesterone level was < 1.0 ng/ml; similar results were obtained for serum progesterone levels >2.5 ng/ml. Then, we choose a progesterone level at 1.5ng/ml, 2.0 ng/ml and 2.5 ng/ml as cut-off points to verify this result. We found that the TQE rate was significantly different (P <0.05) between serum progesterone levels < 2.0 ng/ml and >2.0 ng/ml. In conclusion, the results of this study clearly demonstrated a negative effect of elevated progesterone levels on the day of hCG trigger, on TQE rate, regardless of the basal FSH, the total gonadotropin, the age of the woman, or the time of ovarian stimulation. These data demonstrate that elevated progesterone levels (>2.0 ng/ml) before oocyte maturation were consistently detrimental to the oocyte.     

Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery

Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50. mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF(2)alpha (prostaglandin F(2)alpha) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P>0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; P<0.01) and with the number of ova and/or embryos recovered (r=0.88; P<0.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%.