Estradiol Benzoate Research Articles

Comparison of Gonadotropin-Releasing Hormone versus Estrogen-Based Fixed-Time Artificial Insemination Protocols in Grazing Bos taurus Suckled Beef Cows.

Fixed-timed artificial insemination (FTAI) protocols for beef cattle in South America are primarily based on estradiol esters and intravaginal progesterone-releasing devices (IVPD). The objective of this study was to determine the optimal gonadotropin-releasing hormone (GnRH)-based protocol as an alternative to the use of estrogen-based protocols in grazing Bos taurus suckling beef cows. All cows received an IVPD on the day of protocol initiation and prostaglandin F2α (PG) plus equine chorionic gonadotropin (eCG) treatments at the time of IVPD removal. In Experiment 1, cows (n = 235) were randomly assigned to one of four treatments: (i) 7-day estradiol = 2 mg of estradiol benzoate (EB) at IVPD insertion on Day 9 and 1 mg of estradiol cypionate (ECP) at IVPD removal on Day 2; (ii) 7-day GnRH = 10 µg of GnRH at IVPD insertion on Day 10, IVPD removal on Day 3 and GnRH at FTAI; (iii) 7 & 7 estradiol = PG at IVPD insertion on Day 16, EB on Day 9 and ECP at IVPD removal on Day 2; (iv) 7 & 7 GnRH = PG at IVPD insertion on Day 17, GnRH on Day 10, IVPD removal on Day 3 and GnRH at FTAI. In Experiment 2, cows (n = 462) were randomly assigned to one of four treatments: (i) 6-day estradiol = EB at IVPD insertion on Day 9, IVPD removal on Day 3 and GnRH at FTAI; (ii) 7-day estradiol; (iii) 7-day GnRH; (iv) 7 & 7 GnRH. In Experiment 1, plasma progesterone concentrations and percentage of cows with a corpus luteum (CL) at IVPD removal, and pregnancy per AI (P/AI) were greater for cows subjected to GnRH-based protocols compared with cows subjected to estrogen-based protocols (p < 0.01). In Experiment 2, cows subjected to the 7 & 7 GnRH protocol had the greatest P/AI (p < 0.01). In summary, GnRH-based FTAI protocols resulted in similar or greater P/AI compared to estrogen-based FTAI protocols in grazing postpartum Bos taurus suckled beef cows. The greatest P/AI was attained with the 7 & 7 GnRH protocol.

Evaluation of minimally invasive estrus synchronization protocols in brown brocket deer (Subulo gouazoubira)

This study aimed to evaluate minimally invasive protocols for estrus synchronization in the brown brocket deer (Subulo gouazoubira). Females were submitted to Latin square design, in different treatments. All females received 0.25 mg of estradiol benzoate on the first day of treatment, concomitant with one of the following sources of progesterone: (1) DIP: an intravaginal progesterone releasing device for eight days, (2) MGA1x: once a day (in the morning) oral dose of 1 mg melengestrol acetate for eight days, (3) MGA2x: twice a day (morning and afternoon) oral doses of 0.5 mg of MGA for eight days, (4) P4LA: a single i.m. administration of 75 mg of long-acting progesterone (P4LA). Eight days after the beginning of each treatment, females received an i.m. administration of 265 µg of prostaglandin (PGF2α; cloprostenol). Treatment efficacy was evaluated by manifestation of behavioral estrus after treatment and concentration of fecal progesterone metabolites (FPM). The time to onset of estrus in treatment P4LA was significantly longer (180 ± 38.9 h) compared to DIP (63 ± 6.6 h), MGA1x (53 ± 14.4 h) and MGA2x (41 ± 10.1 h) (P = 0.008). According to individual baseline FPM and FPM concentration during the days after estrus, the corpus luteum formation was suggested in all females which responded to the treatments (93.75 %). Low synchrony, longer interval between PGF2α administration and onset of estrus suggest that the P4LA dose (75 mg) is too high and not effective for S. gouazoubira. DIP, MGA 1x and MGA 2x, were effective in estrus synchronization.

Cheminformatics Strategies Unlock Marburg Virus VP35 Inhibitors from Natural Compound Library.

The Ebola virus and its close relative, the Marburg virus, both belong to the family Filoviridae and are highly hazardous and contagious viruses. With a mortality rate ranging from 23% to 90%, depending on the specific outbreak, the development of effective antiviral interventions is crucial for reducing fatalities and mitigating the impact of Marburg virus outbreaks. In this investigation, a virtual screening approach was employed to evaluate 2042 natural compounds for their potential interactions with the VP35 protein of the Marburg virus. Average and worst binding energies were calculated for all 20 poses, and compounds that exhibited binding energies <-6 kcal/mol in both criteria were selected for further analysis. Based on binding energies, only six compounds (Estradiol benzoate, INVEGA (paliperidone), Isosilybin, Protopanaxadiol, Permethrin, and Bufalin) were selected for subsequent investigations, focusing on interaction analysis. Among these selected compounds, Estradiol benzoate, INVEGA (paliperidone), and Isosilybin showed strong hydrogen bonds, while the others did not. In this study, the compounds Myricetin, Isosilybin, and Estradiol benzoate were subjected to a molecular dynamics (MD) simulation and free binding energy calculation using MM/GBSA analysis. The reference component Myricetin served as a control. Estradiol benzoate exhibited the most stable and consistent root-mean-square deviation (RMSD) values, whereas Isosilybin showed significant fluctuations in RMSD. The compound Estradiol benzoate exhibited the lowest ΔG binding free energy (-22.89 kcal/mol), surpassing the control compound's binding energy (-9.29 kcal/mol). Overall, this investigation suggested that Estradiol benzoate possesses favorable binding free energies, indicating a potential inhibitory mechanism against the VP35 protein of the Marburg virus. The study proposes that these natural compounds could serve as a therapeutic option for preventing Marburg virus infection. However, experimental validation is required to further corroborate these findings.

High estradiol reduces adult neurogenesis but strengthens functional connectivity within the hippocampus during spatial pattern separation in adult female rats

Adult neurogenesis in the dentate gyrus plays an important role for pattern separation, the process of separating similar inputs and forming distinct neural representations. Estradiol modulates neurogenesis and hippocampus function, but to date no examination of estradiol's effects on pattern separation have been conducted. Here, we examined estrogenic regulation of adult neurogenesis and functional connectivity in the hippocampus after the spatial pattern separation task in female rats. Ovariectomized Sprague-Dawley rats received daily injections of vehicle, 0.32 μg (Low) or 5 μg (High) of estradiol benzoate until the end of experiment. A single bromodeoxyuridine (BrdU) was injected one day after initiation of hormone or vehicle treatment and rats were tested in the delayed nonmatching to position spatial pattern separation task in the 8-arm radial maze for 12 days beginning two weeks after BrdU injection. Rats were perfused 90 min after the final trial and brain sections were immunohistochemically stained for BrdU/neuronal nuclei (NeuN) (new neurons), Ki67 (cell proliferation), and the immediate early gene, zif268 (activation). Results showed that high, but not low, estradiol reduced the density of BrdU/NeuN-ir cells and had significant inter-regional correlations of zif268-ir cell density in the hippocampus following pattern separation. Estradiol treatment did not influence pattern separation performance or strategy use. These results show that higher doses of estradiol can reduce neurogenesis but at the same time increases correlations of activity of neurons within the hippocampus during spatial pattern separation.

Effects of Quince Gel and Hesperidin Mixture on Experimental Endometriosis.

Endometriosis (EM) is the presence of endometrial tissue outside the uterus. This study aimed to examine the effects of quince gel and hesperidin treatment on uterine tissue in an experimental endometriosis model. Thirty-two rats were categorized into four groups as sham, EM, EM+quince gel (QG), and EM+QG+Hesperidin (HES). The endometriosis (EM) model was induced with surgical intervention. Estradiol benzoate (EB) was used to induce endometrial hyperplasia. In the EM group, EB was given to rats for 7 days. The EM+QG group received 2 cc QG for 21 days. HES treatment was given for 21 days after EM induction. At the end of the experiment, blood was taken from the animals and the serum total antioxidant status (TAS) and total oxidant status (TOS) values were studied. Uterine tissues were dissected and processed for histological paraffin embedding. Tissues were fixed in 4% glutaraldehyde solution and processed for ultrastructural analysis. After EM, QG and HES treatment significantly increased the TAS and decreased the TOS value. EM caused epithelial and glandular degeneration, thinning of the basal membranes, and vascular dilatation with increased fibrosis and edema. QG+HES restored the pathology and showed protective effects in uterine tissues. Caspase-3 expression was increased in the epithelium, glands, and muscle layers of the EM group. In EM+QG+HES, hesperidin protected cell survival and decreased Caspase-3 expression in uterine tissues. TNF-α expression was intense in inflammatory cells and the muscle layer in the EM group. HES reduced inflammation by decreasing the TNF-α expression. MAPK expression was increased after EM induction in epithelial, glandular, and inflammatory cells in the EM group. After HES treatment, MAPK expression was mainly negative in cells of uterine tissue in the EM+QG+HES group. Ultrastructurally, in the EM group, organelles were disrupted and dilated and degenerated after EM induction. QG and HES treatment improved cellular organelles. Local vaginal applications can be an alternative treatment method in the endometriosis model via QG+HES treatment promoting cell proliferation and angiogenesis and preventing cell death.

Impact of a constant current electrical stimulation (CCES) system and hormonal growth-promoting (HGP) implants on meat quality and palatability of finished steers

This study evaluated the effects of a constant current electrical stimulation (CCES) system and hormonal growth-promoting (HGP) implants on the quality and palatability of the longissimus thoracis et lumborum (LTL) from yearling-finished steers. The experiment used a total of 46 Angus cross steers, which were either non-implanted (n = 20) or implanted with trenbolone acetate and estradiol benzoate (n = 26). The CCES was applied to one side of each carcass during the slaughter process, whereas the other side remained unstimulated. Regardless of the application of HGP implants, the CCES reduced pH at 3 and 72 h post-mortem and shear force at all ageing times (P < 0.05), improved colour at 72 h post-mortem and during the retail display (P < 0.05), increased initial and overall tenderness (P < 0.01), and decreased the amount of perceived connective tissue and the proportion of trained panelists detecting spongy texture (P < 0.05) compared to meat from unstimulated carcass sides. Although CCES increased meat purge losses and reduced moisture content (P < 0.05), this did not affect meat juiciness (P > 0.10). CCES interacted with HGP to prevent increase in drip loss (P > 0.10), increase frequency of panelists detecting bloody/serumy flavour and typical texture, and reduce the proportion of panelists detecting rubbery texture in meat (P < 0.05). Regardless of stimulation treatment, meat from implanted animals had a more pronounced pH decline at 72 h post-mortem (P < 0.05) and a higher proportion of panelists finding no off-flavours (P < 0.05) or bloody/serumy flavour (P < 0.01) than non-implanted cattle. The CCES system tested in this study improved LTL quality and palatability of heavier beef carcasses.

Plasma estradiol profile after administration of different types of estradiol esters in acyclic mares.

Although the use of different estradiol esters has been extensively studied in hormonal protocols in cows, such information is lacking in mares. The present study aimed to assess the effects of treatment with the same doses and administration frequency of estradiol cypionate, estradiol benzoate and 17β estradiol on plasma estradiol (E2) concentrations of acyclic mares and correlate the E2 profile to the endometrial edema score. Sixteen treatments were performed in 14 mares randomly divided into three groups: EB (n=5), EC (n=5) and 17β (n=6), receiving 10mg on day 0 (D0), 6mg on D1 and 4mg on D2 of estradiol benzoate, estradiol cypionate, and estradiol 17β, respectively. Blood samples, rectal palpations and ultrasound evaluations were performed once daily, starting before the first estradiol treatment (D0) until edema disappearance or D8. Moderate to high edema was observed in all groups 24h after the first estradiol administration. Edema persisted above score 2 until D7, D5 and D4 in groups EC, EB and 17β, respectively. Higher edema was found on D2 in EB group compared to 17β, on D6 and D7 in EC compared to 17β, and on D8 in EC compared to EB (p≤0.05). Maximum E2 concentrations were detected on D1 in groups: EB, showing a sharp decrease from D2 to D3 (p<0.0001); and 17β, where no differences were observed between treatment days (p≥0.05). In the EC group, maximum concentrations were observed on D2, which remained high from D2 to D4 compared to the other days (p<0.0001). Plasma concentration of E2 was higher in EB than the other groups on D0 and D1, and EB>EC>17β on D2. Plasma E2 concentrations and edema score were positively correlated, being moderate in groups EB and EC and weak in the 17β group. In conclusion, the most pronounced plasma E2 concentration was observed in EB group 24h after the administration of the first dose. Estradiol concentrations peaked 48h after EC administration, while a distinct peak was not observed in a 24h interval evaluation in the 17β group. Moreover, high edema does not necessarily reflect on high E2 concentrations in acyclic mares.

Effects of estradiol cypionate dosage and body condition on reproductive performance of Nellore cattle synchronized for timed-artificial insemination

This experiment was designed to evaluate the effects of increasing doses of estradiol cypionate (ECP) and different body condition score (BCS) on reproductive performance of Bos indicus beef females assigned to a timed-artificial insemination (TAI) management. In this experiment, 1683 Bos indicus Nellore cows were blocked by parity and assigned to receive 1) an intravaginal P4 device (1.9 g of P4) and 2.0 mg of estradiol benzoate on day −11, 12.5 mg (i.m.) of dinoprost tromethamine, 300 IU (i.m.) of equine chorionic gonadotrophin, 0.6 mg (i.m.) of estradiol cypionate and CIDR withdrawal on day −2, followed by TAI on day 0 (n = 849; 0.6ECP) or 2) the same synchronization protocol with 1.0 mg of ECP on day −2 (n = 834; 1.0ECP). In both treatments, estrus expression was measured between days −2 and 0. Body condition score (BCS) was evaluated on days −11, 31, and 71 of the experiment and the BCS variation (Δ) was also determined between these timepoints. Transrectal ultrasonography was performed on days 31, 71, and 111 for pregnancy rate determination. All binary data were analyzed using cow as the experimental unit with GLIMMIX, whereas continuous variables were analyzed with the MIXED procedure of SAS. No treatment effects were observed on estrus expression rate. Treatment × BCS interactions were observed for pregnancy rates in all time points (days 31, 71, and 111), as 1.0ECP cows with a LOW BCS also had a greater P/AI than cows assigned to 0.6ECP. In summary, increasing the dose of ECP benefited the reproductive performance of Nellore beef cows with a reduced BCS (≤2.75), whereas no benefits were seen when the BCS was considered adequate (>2.75).

Administration of Estradiol Benzoate Enhances Ovarian and Uterine Hemodynamics in Postpartum Dairy Buffaloes.

The postpartum (PP) period is a crucial stage for the resumption of reproductive performance and ovarian cyclicity in dairy buffaloes. The present study aimed, for the first time, to assess the effect of the administration of estradiol benzoate (EB) on ovarian and uterine hemodynamics in PP dairy buffaloes. Eight pluriparous acyclic domestic buffaloes were enrolled in the present experiment and received a dose of 10 mg of estradiol benzoate (EB) intramuscularly 4 weeks after parturition. All animals were examined two times before EB administration (days -3, and -1) and on the day of EB administration (day 0), followed by examinations on days 1, 3, 5, 7, and 9 post-EB administration. The middle uterine artery (MUA) and ovarian artery (OA) blood flow patterns were assessed using a color Doppler ultrasound device. The reproductive parameters were (1) the cross-sectional diameters (cm) of the OA and MUA, (2) cranial uterine horn thickness (UHT; cm), and (3) hemodynamic changes within the MUA on both the ipsi- and contra-lateral sides of the previous pregnant horn and within the OA corresponding to the ovarian tissues. The examined blood flow parameters were the pulsatility index (PI), resistance index (RI), peak systolic/end-diastolic ratio (S/D), time-averaged maximum velocity (TAV; cm/s), uterine blood flow rate (BFR; bpm), and uterine blood flow volume (BFV; mL/min). Concomitantly, blood samples were collected from the coccygeal vein, and the sera were stored at -18 °C for use in estradiol (E2-17β) and nitric oxide (NO) assays. The results revealed increases in both OA and MUA cross-sectional diameter (cm) on the ipsi-lateral and contra-lateral (p < 0.05) sides within 24 h until day 9 post-treatment. The values of the RI and PI of blood flow within the OA and MUA on the ipsi-lateral and contra-lateral sides of the previous pregnancy were obviously lower (p < 0.05) at 24 h after the administration of EB, and then, started to gradually elevate, reaching the pre-treatment values on day 9 after EB administration. Both the BFR and BFV in the OA and MUA significantly increased from 24 h to 72 h after EB administration on both the ipsi-lateral and contra-lateral sides (p < 0.05); then, their values started to decrease to reach the pretreatment value on day 9 after EB administration. Both E2 and NO concentrations significantly increased (p < 0.05) from 24 h until day 3 after EB injection and then started to decline after that, reaching the pre-treatment value on day 9. In conclusion, the administration of EB enhances the ovarian and uterine blood flow concomitantly with increased levels of NO in PP dairy buffaloes.

Morphologic, Steroidogenic, and Transcriptomic Assessment of the Corpus Luteum in Holstein Cows after Spontaneous or Hormone-Induced Ovulation.

There is evidence that replacing the gonadotropin-releasing hormone (GnRH) with porcine luteinizing hormone (pLH) to synchronize ovulation prior to artificial insemination (AI) increased pregnancy per AI in dairy cows without affecting blood progesterone (P4) concentrations. Whether morphologic, steroidogenic, and transcriptomic differences exist among corpora lutea (CL) formed after ovulation induced by GnRH and pLH is unclear. Our main objective, therefore, was to compare CL characteristics between GnRH- and pLH-induced CL. In 24 non-lactating Holstein cows, ovulations were spontaneous (Spont-Ov) or induced with 100 µg GnRH, 25 mg pLH, or 1 mg estradiol benzoate (EB), with CL excised 12 d after ovulation. In pLH- versus GnRH-treated cows, the duration of elevated LH (above baseline) was prolonged (10 versus 6 h, respectively, p < 0.01), but CL dimensions, pixel intensity of CL images, proportions of steroidogenic and non-steroidogenic luteal cells, and mean plasma LH did not significantly differ. Post-ovulation mean plasma P4 (ng/mL) did not differ among Spont-Ov (3.0) pLH (3.1) or GnRH (3.0) cows but were lower in EB cows (2.0). In vitro P4 concentration was greater in luteal explants of pLH-treated cows than in all other groups (combined means, 16.0 vs. 12.3 µg/mL, p < 0.02). Relative abundance of mRNA for oxytocin receptor (OXTR) was 2-fold higher (p < 0.01) in CL of pLH vs. GnRH cows and highest in Spont-Ov CL. In summary, pLH-treated cows had a longer LH peak, and greatest luteal tissue concentrations and in vitro production of P4. We inferred that increased P4 concentrations at the ovarian-uterine level in pLH-treated cows could have promoted embryo development and increased pregnancy per AI.

Chloroform Fraction of Drymaria cordata Linn (CFDC) Suppresses Estradiol Benzoate- Induced Endometrial Hyperplasia

The diagnosis of uterine dysfunction (endometrial hyperplasia) is on the rise. The available treatment is quite expensive and associated with some side effects. The therapeutic potential of natural products is now being explored, as they are easily available with little or no side effects. Drymaraia cordata is folklorically utilized in the treatment of diverse ailments including uterine fibroids. This study aims to investigate the potential therapeutic effect of chloroform fraction of methanol extract of Drymaria cordata (CFDC) in estradiol benzoate (EB)-induced endometrial hyperplasia. Thirty-six rats were randomly divided equally into six groups. These included control group, CFDC: (100 mg/kg), CFDC: (200 mg/kg), EB: (2 mg/kg), EB + CFDC (100 mg/kg), and EB + CFDC (200 mg/kg). Endometrial hyperplasia (EH) was induced by intraperitoneal injection of EB. The levels of estrogen (E2), progesterone (PG), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Malondialdehyde (MDA), Superoxide dismutase (SOD), and Glutathione peroxidase (GSH-Px) activities were determined using ELISA technique. The uterine histological assessment and immunohistochemical expression levels of estrogen receptor, Ki-67, cytochrome c, and caspase 3 were carried out. EH was severely expressed in the uterine section of EB-treated rats. However, CFDC administration improved the pathological features of the animal model. The sex hormones levels were increased in the EB-treated group, which were significantly reduced by CFDC. The antioxidant indices were also restored by CFDC. Immunoexpression levels of ERα and Ki-67 were downregulated while cytochrome c and caspase 3 were upregulated by CFDC. This study suggests that CFDC contains phytochemicals that can protect against EB-induced EH via modulation of hormonal signaling, apoptotic machinery, and oxidative indices.

Wenjing Zhitong recipe exhibits potential anti-primary dysmenorrhea properties by inhibiting COX2 and PKC signaling pathway in rats induced by estradiol benzoate and oxytocin

Wenjing Zhitong recipe exhibits potential anti-primary dysmenorrhea properties by inhibiting COX2 and PKC signaling pathway in rats induced by estradiol benzoate and oxytocin

GnRH34 with or without estradiol cypionate in timed AI in Bos indicus beef cows

Two experiments were performed to evaluate the effects of GnRH treatment on the fertility of suckled Nelore beef cows treated with an estradiol/progesterone (E2/P4)-based protocol for timed artificial insemination (TAI). Experiment 1 focused on determining the effects of estradiol cypionate (EC) on ovulation in TAI cows treated with GnRH 34 h after removal of the intravaginal P4 device (IPD). Suckled cows (n = 26) were treated with 2 mg estradiol benzoate (EB) and IPD containing 1 g P4. After 8 days, IPDs were removed, and all cows were treated with 150 μg of d-cloprostenol (prostaglandin F2 alpha analog) and 300 IU of equine chorionic gonadotropin (eCG), then separated into two treatment groups consisting of cows who received 1) saline 0.9% i.m. (GnRH34 group) or 2) 0.6 mg i.m. of EC (EC-GnRH34 group). On day 9 (05:00 p.m.), all cows were given GnRH (10.5 μg of buserelin acetate) i.m. No differences were observed between the groups (P > 0.05) in the time of ovulation after IPD removal or in the proportion of cows ovulating. Experiment 2 focused on determining the effects of GnRH34 along with or in the absence of EC on day 8 on pregnancy per AI (P/AI) in postpartum beef cows. Cows (n = 981) were treated similarly to those in Experiment 1, but an additional group, the EC-GnRH48 group, was included, in which cows received EC on day 8 whereas those that did not show estrus received GnRH at TAI. Thus, in this experiment, groups consisted of GnRH34 (n = 322), EC-GnRH34 (n = 335), and EC-GnRH48 (n = 324). A higher rate of estrus expression was observed in cows treated with EC following IPD removal (EC-GnRH34: 69%, EC-GnRH48: 64.8%) than in cows in the GnRH34 group (45.6%). No difference in P/AI was observed between the treatment groups (P = 0.45), but P/AI in cows in the EC-GnRH34 group (64.2%) tended to be greater (P = 0.1) than in cows in the GnRH34 group (58%). In summary, although ovulation synchrony did not differ among the groups, P/AI in cows treated with EC and GnRH 34 h after IPD removal tended to be greater than in cows treated solely with GnRH; this was most likely due to a shorter proestrus/estrus period, considering the lower proportion of cows that displayed estrus in the GnRH34 group. Finally, given that P/AI did not differ between the EC-GnRH34 and EC-GnRH48 groups, our results suggest that, for cows not displaying estrus, administration of EC at the time of IPD removal followed by treatment with GnRH 48 h afterward represents the most cost-efficient TAI strategy for South American Zebu-based beef operations.

EFFICACY OF OVULATION SYNCHRONIZATION REGIMENS ON PREGNANCY RATE TO TIMED ARTIFICIAL INSEMINATION IN ACYCLIC POST-PARTUM NILI-RAVI BUFFALOES

The present study compared the two ovulation synchronization treatments combined with a progesterone release device in buffaloes. Ninety acyclic buffaloes were divided into three experimental The blood samples were collected before the start of the trial to determine the reproductive status (anestrous and sub-estrous). In G I and G II, CIDR was inserted for seven days and treated with on the 6th day of protocol. In the protocol, the first estradiol benzoate (EB) and doses were given on day 0 and 2nd EB and doses on day 8 to G I and G II groups, respectively. Whereas, G III served as a control group. The G I and G II groups were observed for heat signs, but fixed time twice AI was done at 48 or 60 hours of CIDR removal, whereas G III was inseminated at detected estrus. The blood samples were also collected prior to the execution of the trial from experimental buffaloes for observing the cyclicity status through serum progesterone level. The results of cyclicity showed 78% anestrous and 22% sub-estrous status in included buffaloes. The effects of synchronization protocols revealed that estrus response was significantly greater in G I and G II groups than in the G III group. The ovulation occurrence, ovulatory follicle szie and pregnancy outcomes were also significantly higher in G I and G II compared to G III. Conclusively, including EB or at the CIDR insertion and removal times effectively enhances estrus, ovulation and pregnancy in sub-estrous and anestrous buffaloes.&#x0D;

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2–3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Plasma estradiol profile after administration of different types of estradiol esters in acyclic mares.

Although the use of different estradiol esters has been studied extensively in hormonal protocols in cows, such information is lacking in mares. The present study aimed to assess the effects of treatment with the same doses and administration frequency of estradiol cypionate (EC), estradiol benzoate (EB) and 17β estradiol (17β) on plasma estradiol (E2) concentrations of acyclic mares and correlate the E2 profile to the endometrial edema score. Sixteen treatments were performed in 14 mares randomly divided into three groups: EB (n=5), EC (n=5) and 17β (n=6), receiving 10mg on day 0 (D0), 6mg on D1 and 4mg on D2 of estradiol benzoate (Sincrodiol®, Ouro Fino, Brazil), estradiol cypionate (E.C.P®, Zoetis, Brazil), and estradiol 17β (17βeta®, Botupharma, Brazil), respectively. Blood samples, rectal palpations and ultrasound evaluations were performed once daily, starting before the first estradiol treatment (D0) until edema disappearance or D8. Plasma E2 was quantified by radioimmunoassay. Endometrial edema was scored as: 0= lack of edema, 1=mild, 2=moderate, 3=high and 4=exaggerated. Mean estradiol plasma concentrations were analyzed by general linear model of variance with repeated measures. Median edema scores were compared by Kruskal Wallis followed by Mann Whitney tests while Spearman's correlation test was used to correlate E2 concentrations and edema score. Moderate to high edema was observed in all groups 24h after the first estradioladministration. Edema persisted above score 2 until D7, D5 and D4 in groups EC, EB and 17β, respectively. Higher edema was found on D2 in group EB compared to 17β, on D6 and D7 in group EC compared to 17β, and on D8 in group EC compared to EB (p≤0.05). Maximum E2 concentrations were detected on D1 in groups: EB (190±83.3 pg/mL), showing a sharp decrease from D2 to D3 (p<0.0001); and 17β (44.99±15.0 pg/mL), where no differences were observed between treatment days (p≥0.05). In the EC group, maximum concentrations were observed on D2 (81±43.8 pg/mL), which remained high from D2 to D4 compared to the other days (p<0.0001). Plasma concentration of E2 was higher in group EB than the other groups on D0 and D1, and EB>EC>17β on D2. Plasma E2 concentrations and edema scores were positively correlated, being moderate in groups EB and EC (EB: r=0.47; EC: r=0.45, p<0.01) and weak in the 17β group (r=0.39, p<0.0001). In conclusion, the most pronounced plasma E2 concentration was observed in group EB 24h after the administration of the first dose. Estradiol concentrations peaked 48h after EC administration, while a distinct peak was not observed in a 24h interval evaluation in the 17β group. Moreover, high edema does not necessarily reflect on high E2 concentrations in acyclic mares.

Estradiol treatment prior to progesterone favors embryo survival in anestrous recipient mares

Previous studies have shown a positive correlation between the length of estrus prior to ovulation and fertility in cyclic donors and embryo recipient mares. Furthermore, higher endometrial expression of Uterocalin (P19) was found in anestrous mares treated with a 5 day estradiol protocol, compared to recipient mares with a short or no estradiol priming. However, it is not known how this extended estradiol exposure influences embryo survival. The objective of this study was to determine the effect of different lengths of estradiol treatment on embryo survival, growth and proportion of mitotic cells. Three experimental groups of anestrous recipient mares were used: (1) mares treated with long-acting progesterone (LA P4) only (NE); (2) mares treated with a single administration of 2.5mg of estradiol benzoate (SE) 2 days before LA P4; and (3) mares treated with 8mg of estradiol split into increasing doses for 5 consecutive days (LE) ending 3 days before LA P4 treatment. Embryos were recovered from donor mares 8 days after ovulation, and embryo diameter and grade were recorded. Only grade I and II expanded blastocysts were used for this study; they were transferred into recipient mares using a modified Wilsher's technique 4 days after LA P4. Embryo recovery was performed 2 days after transfer by uterine lavage to examine embryo survival and growth rate inthe three experimental groups. After a positive recovery, embryos were evaluated for grade, size and fixed in 1% paraformaldehyde for subsequent staining with Hoechst 33342 for nuclear evaluation by confocal microscopy. The proportion of mitotic cells was assessed for 4 areas (10,000µm2) per embryo. The likelihood of recovering an embryo 2 days after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (p =.09). The embryos recovered from the three different groups of recipients 48h after transfer did not, however, show any differences in size, morphology or in the proportion of nuclei undergoing mitosis (p>0.05). In conclusion, the duration of estradiol treatment before progesterone administration tended to influence embryo survival in anestrous recipients within 2 days of transfer. However, the grade and growth rate of surviving embryos did not differ among groups. This may indicate that if the endometrial environment is not optimal to support embryo development following transfer, the embryo dies and disappears relatively quickly, i.e. within 48 h, whereas surviving embryos continue to develop normally.

Effects of electroacupuncture on N-methyl-D-aspartate receptor and mitogen activated protein kinase in spinal cord of rats with primary dysmenorrhea

To observe the effects of electroacupuncture (EA) on the expression levels of N-methyl-D-aspartate receptor (NMDAR), extracellular signal-regulated kinase (ERK)1/2, p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) in the spinal cord of rats with primary dysmenoramia (PDM), so as to explore the underlying mechanism of EA treating PDM. Thirty female SD rats were randomly divided into normal group, model group and EA group, with 10 rats in each group. The PDM rat model was established by subcutaneous injection of estradiol benzoate and oxytocin into the thigh. At the same time of modeling, rats in the EA group were treated with EA (50 Hz) at "Sanyinjiao" (SP36) and "Guanyuan" (CV4) once daily, 20 min each time, for 10 consecutive days. The writhing times, writhing score and writhing latency were observed within 30 min after oxytocin injection. The uterine pathological morphology was observed by HE staining, and pathological score was calculated. Serum prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) were determined by ELISA. The protein expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were detected by Western blot. Compared with the normal group, the writhing times and writhing score were significantly increased (P<0.05); the endometrial epithelial cells showed vacuolar degeneration, death and hyperemia, the uterine pathological score was increased (P<0.05); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly increased (P<0.01), while the content of serum PGE2 was significantly decreased (P<0.01); the expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were significantly increased (P<0.05, P<0.01) in the model group. Compared with the model group, the writhing times and writhing score were significantly decreased (P<0.05), the writhing latency was prolonged (P<0.05); the endometrial epithelial cells still showed vacuolar degeneration, death and hyperemia, and the uterine pathological score was decreased (P<0.01); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly decreased (P<0.01), while the content of serum PGE2 was significantly increased (P<0.01); the protein expression levels of ERK1/2 and JNK in spinal cord were significantly decreased (P<0.01) in the EA group. EA intervention at SP36 and GV4 has obvious analgesic effect on PDM rats, and its mechanisms may be related to reducing serum prostaglandin, alleviating uterine inflammation, and inhibiting the protein expressions of NMDAR, ERK1/2, p38 MAPK and JNK in spinal cord.

(103) Olfactory Investigation of the Male is Stimulated by Administration of Dihydrotestosterone and Blunted by Co-administration of Flutamide in Ovariectomized Rats Primed with Estradiol

Abstract Introduction Although Hypoactive Sexual Desire Disorder (HSDD) is a common condition with a relevant impact on women’s quality of life, approved androgenic formulations for its treatment are lacking. In previous studies, we demonstrated that the administration of Dihydrotestosterone (DHT), a non-aromatizable androgen, in ovariectomized (OVX) Long-Evans rats primed with Estradiol Benzoate (EB), enhanced measures of sexual behavior. Objective To further clarify the mechanisms underlying the facilitating action of testosterone on female sexual function by investigating the effect of AR (androgen receptor) activation in a validated animal model. Methods After sexual training and hormonal wash-out, OVX Long-Evans rats were treated with different hormonal regimens: oil (O) + O (negative control group), 10 mcg EB+500 mcg Progesterone (P) (positive control group), 10 mcg EB+500 mcg DHT, and 10 mcg EB+500 mcg DHT+10 mg flutamide (FLU), a nonsteroidal AR antagonist (n=4 per group). EB was administered s.c. 48 h, P and DHT s.c. 4 h, and flutamide i.m. 48 and 24h prior to video-recorded sexual behavioral testing sessions. These took place in bisected unilevel pacing chambers, with sexually experienced males. Five testing sessions were conducted every 8 days. The main outcome measures were appetitive behaviors: the number of hops/darts (partial solicitation), full solicitations and olfactory investigations. Results A significant difference emerged among the 4 groups for all the outcome measures. Regarding solicitations, animals treated with EB+DHT displayed a higher number of hops/darts and full solicitations than the negative control group (O+O), although significantly lower than the positive control group (EB+P) for hops/darts (p=0.025). As a novel finding, we observed that the stimulating effect of EB+DHT was blunted in rats treated with EB+DHT+FLU, which showed a lower number of behaviors than EB+DHT rats (p=0.014 for hops/darts, and p=0.083, approaching statistical significance, for full solicitations). Interestingly, a significant difference was detected in the frequency of olfactory investigations, with the EB+DHT group displaying a higher frequency not only when compared with O+O rats (p=0.047), but also with EB+DHT rats (p=0.047 for both comparisons). Once again, the co-administration of FLU significantly reduced the stimulating effect (p=0.005 for olfactory investigations for EB+DHT+FLU vs. EB+DHT). Conclusions These data, to be confirmed in larger samples, indicate that the positive modulation exerted by androgens on appetitive behaviors, homologous to sexual interest in women, are independent of aromatization. In addition, a specific stimulating effect was detected for olfactory investigations. Disclosure No

Gut Microbiota and Acylcarnitine Metabolites Connect the Beneficial Association between Estrogen and Lipid Metabolism Disorders in Ovariectomized Mice.

Decreased estrogen level is one of the main causes of lipid metabolism disorders and coronary heart disease in women after menopause. Exogenous estradiol benzoate is effective to some extent in alleviating lipid metabolism disorders caused by estrogen deficiency. However, the role of gut microbes in the regulation process is not yet appreciated. The objective of this study was to investigate the effects of estradiol benzoate supplementation on lipid metabolism, gut microbiota, and metabolites in ovariectomized (OVX) mice and to reveal the importance of gut microbes and metabolites in the regulation of lipid metabolism disorders. This study found that high doses of estradiol benzoate supplementation effectively attenuated fat accumulation in OVX mice. There was a significant increase in the expression of genes enriched in hepatic cholesterol metabolism and a concomitant decrease in the expression of genes enriched in unsaturated fatty acid metabolism pathways. Further screening of the gut for characteristic metabolites associated with improved lipid metabolism revealed that estradiol benzoate supplementation influenced major subsets of acylcarnitine metabolites. Ovariectomy significantly increased the abundance of characteristic microbes that are significantly negatively associated with acylcarnitine synthesis, such as Lactobacillus and Eubacterium ruminantium group bacteria, while estradiol benzoate supplementation significantly increased the abundance of characteristic microbes that are significantly positively associated with acylcarnitine synthesis, such as Ileibacterium and Bifidobacterium spp. The use of pseudosterile mice with gut microbial deficiency greatly facilitated the synthesis of acylcarnitine due to estradiol benzoate supplementation and also alleviated lipid metabolism disorders to a greater extent in OVX mice. IMPORTANCE Our findings establish a role for gut microbes in the progression of estrogen deficiency-induced lipid metabolism disorders and reveal key target bacteria that may have the potential to regulate acylcarnitine synthesis. These findings suggest a possible route for the use of microbes or acylcarnitine to regulate disorders of lipid metabolism induced by estrogen deficiency.