Estradiol Benzoate Research Articles

ATR-FTIR spectroscopy to evaluate serum protein expression in a murine cerebral ischemia model

Stroke is a prevalent vascular disease that causes disability and death worldwide. Molecular techniques have been developed to assess serum concentrations of biomarkers associated with this disease, such as some proteins. ATR-FTIR was proposed as an alternative technique to determine protein expression during the early stages of stroke. Serum samples from sham, ischemic, and ischemic treated with estradiol benzoate (EB; as a neuroprotective agent) male rats were evaluated at 0, 2-, 4-, 6-, 12-, and 24-hours post-ischemia. The analysis was developed in the mid-infrared region but mainly focused on the protein region (1500–1700 cm−1), where it was possible to observe the modulation in the absorbance intensity. The peaks at 1545, 1645, 1635, and 1650 cm−1 associated with amide II, amide I, β-sheets, and α-helixes, respectively, were prominent peaks where protein modulation was observed. The results demonstrate that infrared spectroscopy could be a good alternative technique to determine the modulation of protein expression during stroke events.

Role of Membrane Estrogen Receptor Alpha on the Positive Feedback of Estrogens on Kisspeptin and GnRH Neurons.

Estrogens act through nuclear and membrane-initiated signaling. Estrogen receptor alpha (ERα) is critical for reproduction, but the relative contribution of its nuclear and membrane signaling to the central regulation of reproduction is unclear. To address this question, two complementary approaches were used: estetrol (E4) a natural estrogen acting as an agonist of nuclear ERs, but as an antagonist of their membrane fraction, and the C451A-ERα mouse lacking mERα. E4 dose- dependently blocks ovulation in female rats, but the central mechanism underlying this effect is unknown. To determine whether E4 acts centrally to control ovulation, its effect was tested on the positive feedback of estradiol (E2) on neural circuits underlying luteinizing hormone (LH) secretion. In ovariectomized females chronically exposed to a low dose of E2, estradiol benzoate (EB) alone or combined with progesterone (P) induced an increase in the number of kisspeptin (Kp) and gonadotropin-releasing hormone (GnRH) neurons coexpressing Fos, a marker of neuronal activation. E4 blocked these effects of EB, but not when combined to P. These results indicate that E4 blocked the central induction of the positive feedback in the absence of P, suggesting an antagonistic effect of E4 on mERα in the brain as shown in peripheral tissues. In parallel, as opposed to wild-type females, C451A-ERα females did not show the activation of Kp and GnRH neurons in response to EB unless they are treated with P. Together these effects support a role for membrane-initiated estrogen signaling in the activation of the circuit mediating the LH surge.

7288 A Preclinical Mouse Model of Gender-Affirming Hormone Therapy: Metabolic and Behavioral Outcomes

Abstract Disclosure: A. Yasrebi: None. K. Otersen: None. O. Groh: None. E. Guthman: None. I. Shmarakov: None. S. Campbell: None. T.A. Roepke: None. The long-term metabolic effects of gender-affirming hormone therapy (GAHT) is largely unknown, in part, due to the lack of properly designed preclinical animal models. Here we propose a rodent study design, beyond the classical endocrine model of gonadectomy and steroid treatment, to accurately reflect the reality of the trans experience. Specifically, trans women do not all seek or undergo orchidectomy (ORX) instead take estrogen supplementation along with an androgen blocker such as finasteride. In this study, 40 WT C57 male mice were divided into 4 groups of 10 mice - 1) intact with oil vehicle, 2) intact with estradiol benzoate (EB, 150 μg/kg/d) and finasteride (F, 0.25 mg/kg/d), 3) ORX with oil; and ORX with EB (150 μg/kg/d). Each mouse was dosed daily for 8 weeks while on a standard chow diet. Mice were pair housed and food intake and body weight were collected weekly prior to behavioral (open field, elevated plus, Y-maze) and metabolic (body composition, metabolic rates, activity, tolerance testing) phenotyping. Intact mice treated with EB+F exhibited less avoidance behaviors, potentially due to differences in movement, with no differences between treatments in the Y-maze. Cumulatively, Intact:EB+F gained less weight than Intact:oil (3.4 ± 0.4 g vs 5.3 ± 0.4 g), while ORX:oil mice gained less than ORX:EB (2.2 ± 0.5 g vs 5.5 ± 0.7 g). These interactive effects were due to differences in the gain of fat or lean mass between ORX and intact and lower cumulative food intake in ORX:oil mice (136 ± 3.8 g vs Intact:oil: 159 ± 4.9 g; Intact:EB+F: 167 ± 6.1 g; ORX:EB: 171 ± 3.4 g). Metabolic assessments found that Intact:EB+F exhibited elevated V.O2, V.CO2, and heat production in the light and dark cycle compared to Intact:oil, while ORX:EB only exhibited elevated heat production compared to ORX:oil. Similarly, Intact:EB+F and ORX:EB moved more than their counterparts during the dark cycle; however, only Intact:EB+F exhibited elevated wheel running. Tolerance tests (i.p.) found that glucose clearance was augmented in ORX:EB mice while insulin-induced glucose clearance was lower in Intact:EB+F compared to Intact:oil but modestly elevated by EB in ORX mice. These data, along with suppressed fasting blood glucose in ORX:EB mice, suggest that glucose homeostasis is differentially modulated by GAHT paradigms in mice. Analysis of hypothalamic, hepatic, adipose, and intestinal gene expression is currently underway. Collectively, these data, generated by using a translationally-relevant preclinical animal model, are the first to establish that metabolism is differentially affected by both steroid treatments and ORX and suggesting that the long-term metabolic effects of GAHT are dependent on the type of treatment. The underlying mechanisms and sites of action remain to be investigated. Presentation: 6/3/2024

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

Combined developmental exposure to estrogenic endocrine disruptor and nutritional imbalance induces long term adult prostate inflammation through inflammasome activation

Increasing number of studies suggested that environmental deleterious impacts (such as estrogen-like endocrine disruptors, EDCs, unhealthy diet) during early human development affect the risk of developing non-communicable diseases including prostate cancer (PCa) later in life. To test if the combination of EDCs and unhealthy induces adult prostate lesions, we developed an experimental model of adult male Sprague Dawley rats exposed during gestation (from day 7) to weaning to high fat diet (HFD 60 % fat), or to a xenoestrogen (estradiol benzoate, EB, 2.5 µg/d) from post-natal days 1–5, or to a combination of both. EB and EB+HFD exposures induced decreased prostate weight in adult rats along with inflammatory status. A white blood cell infiltrate was observed after EB exposure and more dramatic lesions were observed with the combined exposure, along with a gland destruction. The lesions, following EB or EB+HFD exposure, are associated with elevated mRNA levels for TNFa, IL6 and CCL2/MCP1 pro-inflammatory cytokines while the levels of the anti-inflammatory IL10 cytokine remained unchanged. This activation of NLRP3 and elevated levels of CASP1 were observed following EB or EB+HFD exposures associated with elevated mRNA levels for IL1b, substrates for the NLRP3 complex. HFD exposure alone has mild if not pro-inflammatory effects in adult prostate. In conclusion, we showed that developmental combined exposure to EB and HFD programmed prostate inflammatory lesions in adult prostate. Since proliferative inflammatory atrophy and chronic inflammation of prostate may drive cell to become cancer cells, our model might be useful for study onset of PCa.

A new hormonal protocol supports early development of invitro-produced embryos after transfer to anoestrus mares.

The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 67% showed evident intrauterine development. Hence, ICSI-derived embryos can be successfully transferred to primed anoestrus mares, but more studies are required to ensure further embryo development and foaling.

Effect of the Application of PGF2α Associated With Ovulation Induction in a Fixed-Time Superovulation Programme for Precocious Nellore Heifers.

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

(-)-Carvone Inhibits Oxytocin-induced Writhing Via Uterine Relaxation in Rodents.

(-)-Carvone, a ketone monoterpene, is the main component of essential oils from several medicinal plants and has been reported to have anti-arthriric, anticonvulsive, antidiabetic, anti-inflammatory, anticancer, and immunomodulatory effects. Therefore, this study aimed to investigate the spasmolytic activity of (-)-carvone in rodent models. The isolated virgin rat uterus was mounted in an organ bath apparatus, and the relaxing effect of ( -)-carvone and its mechanism of action were evaluated in tonic contractions induced by carbachol, KCl, PGF2α, or oxytocin. The animal model of primary dysmenorrhea was replicated with the injection of estradiol benzoate in female mice for three consecutive days, followed by intraperitoneal administration of oxytocin. Non-clinical acute toxicity evaluation was also performed. (-)-Carvone potency and effectiveness were larger in carbachol (pEC50 = 5.41 ± 0.14 and Emax = 92.63 ± 1.90% at 10-3M) or oxytocin (pEC50 = 4.29 ± 0.17 and Emax = 86.69 ± 1.56% at 10-3M) contractions. The effect of ( -)-carvone was altered in the presence of 4-aminopyridine, glibenclamide, L-NAME, or methylene blue. Mice pre-treated with (-)-carvone at a dose of 100mg/kg showed a significant reduction in the number of writhing after oxytocin administration. No toxicity was observed after oral administration of 1g/kg ( -)-carvone. Taken together, we showed that (-)-carvone reduced writhing by a spasmolytic effect, probably through the participation of KV and KATP channels and the nitric oxide pathway.

Therapeutic Efficacy of Wet Cupping on Uterine Inflammation in Primary Dysmenorrhea: An In Vivo Investigation Targeting Bilateral L4/L5 Vertebrae

Primary dysmenorrhea (PD) is a prevalent problem in gynecology affecting the quality of life for young women. This medical condition is often responsible for the absence of young women from school or work, increasing the risk of developing hyperemesis gravidarum in the future. Furthermore, PD causes pain due to the activation of the local PGF2α pathway in the endometrium, which triggers oxidative stress processes, uterine ischemia and spasmodic uterine contractions. The first-line therapies for PD, Nonsteroid anti-inflammatory drugs (NSAIDs) and oral contraceptives, can cause side effects such as headache, nausea, intestinal ulcers and platelet abnormalities. To overcome the impact of PD, cupping has been proven effective as an alternative pain therapy with minimal side effects. Therefore, this study aimed to evaluate the effects of cupping on writing latency threshold time, PGF2α levels and FP receptor expression in PD model rats. The experiment was conducted using female Sprague Dawley rats to analyze the effects of wet cupping therapy (WCT) on inflammatory repair in the uterus. The method used was a pre- and post-test control group design, with 35 female rats randomly divided into 5 groups. These consisted of normal control (NC), negative control (C−), positive control (C+) + ibuprofen 7.2 mg, as well as treatment groups, comprising dry cupping (DC) and WCT. The results showed that writhing time threshold of WCT was the same as C+ (p = 0.368), while PGF2α decreased in DC (43.43 pg/mL, p = 0.008) and WCT (189.25 pg/mL, p = 0.0154). Similar to C+, WCT significantly decreased serum PGF2α levels compared to C−. WCT also influenced the expression of PTGFR subunit similarly to NC, showing the capacity to control the inflammatory pathway. The study suggested that WCT was a promising therapeutic strategy for PD by decreasing PGF2α and PTGFR expression. HIGHLIGHTS Previous investigations conducted on experimental animals using wet cupping therapy (WCT) have shown histopathological results associated with primary dysmenorrhea (PD) caused by estradiol benzoate and oxytocin. Moreover, this study shows the potential of WCT in reducing inflammation-induced PD pain by inhibiting the expression of PGF2α and PTGFR in the cyclooxygenase pathway, potentially increasing the pain threshold. The results show that WCT offers a potential alternative therapy for PD caused by estradiol benzoate and oxytocin. GRAPHICAL ABSTRACT

The expressions of nr0b1/2 genes in the Chinese giant salamander and their response to estradiol benzoate/17α-methyltestosterone treatment

Previously, the nr0b1 and nr0b2 genes have been identified in various animals and found to be closely associated with sex determination. In order to investigate the regulatory roles of nr0b1 and nr0b2 genes in the sex determination of Chinese giant salamander (CGS, Andrias davidianus), we obtained the full-length cDNA sequences of nr0b1 and nr0b2 through cloning, determined the characteristics of their tissue expressions and the responses of nr0b1 and nr0b2 to estradiol benzoate (EB) and 17α-methyltestosterone (MT) by using quantitative real-time PCR (qRT-PCR), and evaluated gonad development through histological sections. The full-lengths of cDNA of nr0b1/2 in C GS are separately 1278bp and 789bp. They have complete open reading frames (ORF) of 864bp and 774bp and encode 287 and 257 amino acids respectively. The 3' untranslated regions (UTR) are 276bp and 15bp, and the 5' UTR of nr0b1 is 138bp. In the 3' UTR of nr0b1, a putative polyadenylation signal (AATAAA) is identified. Tissue distribution analysis indicates that nr0b1 is mainly expressed in the pituitary, kidney, and testes, with significant differences between females (ZW-F) and males (ZZ-M) expression in the testes. On the other hand, nr0b2 is mainly expressed in the liver, followed by gonadal tissues. After immersion and feeding with EB and MT, EB's promotion on nr0b1 expression in CGS ovaries and testes is shown, by contrast MT only has a positive effect on nr0b2 in CGS testes. Histological sections show that after EB treatment, both genetic females (ZW-F) and sex-reversed females (ZZ-F) of CGS contain oogonia, indicating that EB can cause sex reversal in CGS; however, after MT treatment, no cellular morphological changes are observed either in genetic females (ZW-F) or in genetic males (ZW-M), indicating that this concentration cannot induce a shift in the sex ratio of CGS. These findings lay the foundation for further investigating the molecular mechanisms by which nr0b1/2 influence the differentiation and maintenance of CGS gonads.

Effects of Two Types of Estrogen on the Follicular Wave for in Vivo Oocyte Collection in Brown Swiss Cows

The manipulation of follicular waves through hormonal treatments, such as estrogen administration, plays a crucial role in optimizing in-vivo oocyte collection for assisted reproductive technologies. The present study aimed to evaluate the effect of two specific types of estrogen on follicular wave dynamics and their impact on in-vivo oocyte collection in Brown Swiss cows. Fourteen cows, in their first lactation, weighing approximately 340 kg were randomly assigned to one of two treatments including T1 (estradiol cypionate) and T2 (estradiol benzoate). Both treatments were administered at 1.2 mg of estrogen, at day 0 of the experiment. All Brown Swiss cows were provided with a diet entirely consisting of alfalfa grazing. On day 7, follicular wave dynamics were assessed using a DP-50 vet ultrasound device equipped with a 7.5 MHz transducer for transvaginal follicular aspiration guidance. Follicle counts were categorized into three size ranges including 2-4 mm (small), 4-8 mm (medium), and greater than 8 mm (large). Additionally, the quantity and quality (viable oocytes) of the collected oocytes were evaluated by the Ovum Pick Up (OPU) team for oocyte viability on day 7. The study assessed the follicular dynamics (number of follicles) and efficiency of oocyte collection (viable oocytes) in cows treated with Estradiol Cypionate (T1) and Estradiol Benzoate (T2). The average number of small, medium-sized, and large follicles size were 6.048 ± 6.037, 3.16 ± 2.01, and 0.53 ± 0.67 respectively. The total number of follicles was 9.59 ± 3.56. The mean number of viable oocytes recovered was 3.024 ± 1.66, while the mean number of non-viable oocytes was 1.47 ± 1.01. The results indicated no significant differences between treatments in the size of small, medium, and large follicles, nor in the total number of follicles and viable oocytes recovered. However, a significant difference was observed in the number of non-viable oocytes recovered, with a higher mean in T2 (1.86) compared to T1 (1.09). The results indicated an adequate follicular response and viable oocyte recovery in both treatment groups (estradiol cypionate and estradiol benzoate). However, variations in oocyte viability were observed, with estradiol cypionate showing a slight advantage.

PSVIII-9 Effects of growth implants and timing of vaccination on calf immunity and growth performance

Abstract This study assessed calf growth performance and blood parameters of beef calves under different growth implants and timing of vaccination protocols. Crossbreed beef calves (n = 59) were stratified by birth body weight (BW) and age (41 ± 5.2 kg of BW and 70 ± 8 d of age) and randomly assigned in a split-plot design with implant strategy as the main plot and timing of vaccination as the subplot. The main plot (treatment) consisted of 3 implant strategies: 1) Synovex C (SC; n = 20; 100 mg progesterone/10 mg estradiol benzoate, or 2) Synovex One Grower (SG; n = 19; 150 mg trenbolone acetate and 21 mg estradiol benzoate; or 3) no implant (CON; n = 20). Subplot treatments were the timing of vaccination booster against bovine respiratory disease pathogens on 1) weaning (WV; n = 29) or 2) 7-d post-weaning (PWV; n = 30). Cow-calf pairs were treated alike in a single pasture during the pre-weaning phase, receiving the same vaccine (Bovi-Shield Gold One Shot and Ultrabac 7) on d 0 (implantation day) and booster time (WV or PWV). After weaning (approximately 187 d of age), calves were allocated in a single pen receiving ad libitum hay until d 60 post-weaning. Body weight and blood were collected on d 0, weaning, d 0 and 28 relative to vaccination booster, and d 60 post-weaning. Blood was collected to determine insulin-like growth factor (IGF-1), cortisol, and leptin concentrations and to perform a complete blood cell count. WV calves had decreased cortisol concentrations on booster day and 28 d post-vaccine (P = 0.01). Regardless of implant treatment, calves tended to have decreased IGF-1 concentrations 28-d post-vaccine booster (P = 0.09). An effect of treatment (P &amp;lt; 0.01) was detected for lymphocyte counts, with CON having greater concentrations vs. SG and SC calves. A treatment × timing of vaccination (P &amp;lt; 0.01) interaction was detected for monocyte counts, where SG-PWV calves had the greatest concentrations. A treatment effect (P &amp;lt; 0.02) was observed for eosinophil percentage, where SG had reduced percentages than CON, while SC was intermediate. An effect of treatment was detected for leptin (P &amp;lt; 0.01), with SG calves having the least, CON being intermediate, and SC the greatest concentrations. From d 0 to 60 d post-weaning, SG tended to have greater ADG (P ≤ 0.07) compared with CON, while SC was intermediate. On d 28 and d 35 post-weaning, calves implanted with SG were heavier (P ≤ 0.04) than the CON but not than SC. In summary, this study demonstrates that growth implants and timing of vaccination significantly affect both physiological parameters and growth performance in beef calves. Implanting calves with Synovex One Grower resulted in improved growth performance.

PSIV-6 Blood biomarkers of oxidative stress in offspring from dams infected with bovine viral diarrhea virus during late-term gestation

Abstract The objective of this experiment was to examine the impact of dams infected with bovine viral diarrhea virus (BVDV) during late gestation on inflammatory blood biomarkers in their offspring. Fetal infection with BVDV before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). It was hypothesized that offspring of dams transiently infected with BVDV on d 175 of gestation would have greater inflammatory blood biomarkers than control offspring. Unvaccinated, yearling Hereford heifers (n = 24), seronegative for antibodies to BVDV1 and BVDV2 were bred by artificial insemination with X chromosome-bearing sperm from an Angus sire. On d 175 of pregnancy, heifers were intranasally inoculated with either Dulbecco’s Modified Eagle Medium + 2% horse serum (sham control) or 4.0 log TCID50 noncytopathic type 2 BVDV to generate control and transient infected calves, respectively. All BVDV-inoculated dams seroconverted by d 14 post-inoculation and all sham-inoculated control dams remained seronegative. Twelve, seronegative control heifer calves were born to the control dams, and 11 seropositive (to type 2 BVDV) TI heifer calves were born to BVDV-inoculated dams. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate body weight (BW) of 600 kg. Upon arrival, all animals received a standard heifer growth implant containing 200 mg of testosterone propionate and 20 mg of estradiol benzoate, a modified live viral vaccine containing IBR-BRSV-PI3, and were dewormed. Jugular blood samples were obtained every 28 d until harvest (280 d on feed) and analyzed for biomarkers of inflammation. Data were analyzed as a randomized block design with the MIXED procedures of SAS. There was a treatment x time interaction (P &amp;lt; 0.05) for plasma ceruloplasmin and the oxidized form of glutathione. On d 0 and 28 of the feeding period, all biomarkers were similar across treatments. By d 56 of the feeding period, ceruloplasmin (P &amp;lt; 0.03) and the oxidized form of glutathione (P &amp;lt; 0.01), indicators of chronic inflammation, were increased in plasma samples from TI heifers compared with controls. These parameters remained greater in TI heifers compared with controls throughout the feeding period. There were no treatment or treatment x time interactions for plasma interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and haptoglobin. These data suggest that TI heifers may be experiencing a greater level of oxidative stress later in the finishing period. This research was supported by USDA-NIFA Grant #2019-67015-29866.

Integrated metabolomics and lipidomics investigation of the mechanism of Danggui Sini Decoction on improving lipid homeostasis in primary dysmenorrhea

BackgroundDanggui Sini Decoction (DGSND) is a classic prescription for treating primary dysmenorrhea (PD), while, the ameliorating effects of DGSND on PD and its mechanisms are not yet fully understood. PurposeThe present study is devoted to investigate the protective effect of DGSND against PD and the possible mechanism from the perspective of metabolomics as well as lipidomics. MethodsDGSND was characterized by UPLC-Q-TOF/MS. The PD rat model was induced by estradiol benzoate and oxytocin, and traditional pharmacology, including writhing times, latency time, biochemical index, organ index, and histopathology were performed to evaluated the efficacy of DGSND on PD. Urine metabolomics strategy combined with functional analysis was adopted to delineate the therapeutic effect of DGSND on PD rats and anchor the crucial pathway, and lipidomics analysis was further performed with the uterine tissue as the research object to elucidate the protective mechanism of DGSND from the perspective of lipid homeostasis. Finally, western blot analysis was used to validate the expression of key metabolic enzymes in lipid metabolism. ResultsDGSND was effective in ameliorating writhing times, latency time, the value of prostaglandin F2α (PGF2α)/PGE2, uterus index, and morphological changes of PD rats. Metabolic signature of PD rats was primarily characterized by the disturbance of steroid hormone metabolism, amino acid metabolism, and lipid metabolism. Functional analysis revealed the urine biomarkers of PD were most related with lipid abnormality. Further lipidomics analysis indicated DGSND exerted anti-PD effects by remodeling lipid homeostasis, which might be due to the significant correlations between different kinds of lipids, especially the extremely high correlation of phosphatidylethanolamine, phosphatidylcholine, and fatty acids. Moreover, the key metabolic enzymes expression of CK, PLA2, LPCAT3, COX-2, and 5-LOX can be greatly downregulated by DGSND. ConclusionOur findings demonstrated a novel protective mechanism of DGSND against PD by regulating lipid homeostasis.

Does the Uterine Ozone Therapy Alter the Transcript Profile of Anti- and Proinflammatory Genes in Mares With Endometritis?

This study aimed to evaluate the localised effects of intrauterine ozone therapy on endometrial recovery in mares with endometritis. Our investigation assessed changes in gene expression profiles of anti-inflammatory (IL-1RA and IL-10), proinflammatory (IL-R1B3i and TNFα) and pleiotropic (IL-6) cytokines, along with detailed histological measurements of epithelial and endometrial thickness and the glandular area ratio. Twenty mares were assigned to a 2 × 2 factorial design based on endometritis diagnosis and treatment (control or 42 μg/mL ozone insufflation), resulting in four groups: NC (negative for endometritis/control), NO (negative/ozone), PC (positive/control) and PO (positive/ozone). Oestrus was induced with 2 mg of oestradiol benzoate on Days -1, 1 and 3, plus 1 mg on Day 5. Day 0 marked the initial uterine treatment, followed by insufflations on Days 1 and 2 with O3 (ozone) or O2 (control). Uterine biopsies were taken before treatment on Day 0 and Day 6 for histological analysis and gene expression assessment. Data were analysed using a statistical model that included endometritis status, treatment type, biopsy times (D0 and D6) and their interactions, analysed with Proc Glimmix. Regardless of treatment or endometritis status, significant biopsy effects (p < 0.01) indicated increased epithelial height and endometrial thickness in Day 6 samples. Analysis of IL-1 and TNFα revealed a significant interaction (p < 0.05) among endometritis, treatment and biopsy, with higher IL-1B3i expression on Day 6 in the PC group. The treatment effect (p < 0.04) showed a higher frequency (p < 0.01) of animals with positive modulation in the PC group (66.7%) versus the PO group (0.0%). An interaction effect (p = 0.08) between endometritis and treatment resulted from higher IL-1RA expression on Day 6 in the PC group compared to the PO group. Biopsy effect was significant for IL-10 (p < 0.01), indicating higher values in the second sample associated with tissue repair. In the short-term evaluation, ozone therapy did not influence endometrial morphology and may modulate cytokine expression, specifically the reduction in IL-1 and TNFα levels. Therefore, this therapy appears to be a safe and potentially effective treatment for modulating the inflammatory response in mares with endometritis.

The protective effect of vinpocetine against Estradiol-benzoate induced cervical hyperkeratosis in female rats via modulation of SIRT1/Nrf2, and NLRP3 inflammasome

The current study was assigned to determine the putative preventive role of vinpocetine (VIN) in cervical hyperkeratosis (CHK) in female rats. Estradiol Benzoate (EB) was utilized in a dose f (60 μg/100 g, i.m) three times/week for 4 weeks to induce cervical hyperkeratosis. VIN was administered alone in a dose of (10 mg/kg/day, orally) for 4 weeks and in the presence of EB. Levels of malondialdehyde (MDA), total nitrites (NOx), reduced glutathione (GSH), interleukin-18 (IL-18), IL-1β, tumor necrosis factor-alpha (TNF-α) were measured in cervical tissue. The expression of NLRP3/GSDMD/Caspase-1, and SIRT1/Nrf2 was determined using ELISA. Cervical histopathological examination was also done. EB significantly raised MDA, NOx, TNF-α, IL-18, IL-1β, and GSDMD and up-regulated NLRP3/Caspase-1 proteins. However, GSH, SIRT1, and Nrf2 levels were reduced in cervical tissue. VIN significantly alleviates all biochemical and histopathological abnormalities. VIN considerably mitigates EB-induced cervical hyperkeratosis via NLRP3-induced pyroptosis and SIRT1/Nrf2 signaling pathway.

Improving Milk Yield, Milk Quality, and Follicular Functionality Behavior in Dairy Cows from the Implementation of Microencapsulated Chili Pepper Supplements in Their Diets.

The present study evaluates the effect of including microencapsulated hot chili pepper (MHCP) in the diet of crossbred dairy cows on the volume and quality of milk and on ovarian morphofunctionality. Twenty-four crossbred females in their lactating period were used. The cows were divided into two experimental groups, a control (CT) and an MHCP -supplemented group (CP) given 1 g a day per animal of microencapsulated hot chili in concentrate for 42 days. Over seven weeks of daily milk production was measured, and sample milk was collected weekly for composition analysis. Animals were subject to an ovulation synchronization protocol on day 0 (D0), and an intravaginal progesterone (P4) implant, estradiol benzoate, and prostaglandin (PGF2α) were administered. On D8, the P4 implant was removed and PGF2α, equine chorionic gonadotropin, and estradiol cypionate were administered to the animals. The ovarian dynamics were evaluated in B mode and color Doppler. There were significant differences (p < 0.05) in the group X time interaction, the volume of milk produced, and the amount in kg/day of milk components. There was a higher percentage of vascularization in the preovulatory follicle in the CP group (p ≥ 0.10). The findings show that the inclusion of MHCP in the diet of dairy cows does influence their milk production and reproduction.

Reproductive outcomes in Holstein heifers synchronized with timed-AI protocols that provide for a lengthened proestrus.

This study compared reproductive outcomes among two protocols for synchronization of ovulation that provide for a lengthened proestrus with the conventional oestradiol-based protocol currently used for timed-AI (TAI). Holstein heifers (13-15 months) at one location were assigned randomly to one of three TAI protocols. Heifers (n = 150) in the 7-day oestradiol benzoate (EB) group received a progesterone device (Cue-Mate) and 2 mg EB on Day 0; 500 μg of cloprostenol (PGF) and Cue-Mate removal on Day 7; 1 mg of EB on Day 8 and TAI on Day 9 (54 h after Cue-Mate removal). Heifers (n = 150) in the 5-day CO-Synch (CO) group received a Cue-Mate and 100 μg of gonadotropin-releasing hormone (GnRH) on Day 2; Cue-Mate removal and PGF (twice, 12 h apart) on Day 7; and GnRH along with TAI on Day 10 (72 h after Cue-Mate removal). Heifers (n = 150) in the J-Synch (JS) group received a Cue-Mate and 2 mg of EB on Day 1; PGF and Cue-Mate removal on Day 7; GnRH and TAI on Day 10 (72 h after Cue-Mate removal). Heifers were inseminated by one technician with frozen-thawed conventional semen from one of four commercially available sires. Plasma progesterone (P4) concentrations (ng/mL) were determined at Cue-Mate removal and TAI. Ovarian ultrasonography was done in a subset of 217 heifers at the initiation of protocols, at Cue-Mate removal; TAI; and 7 days after TAI. Approximately, 28 and 50 days after TAI pregnancy status was determined by ultrasonography. Mean (±SEM) plasma P4 concentration at Cue-Mate removal was greater (p < .01) in CO (6.02 ± 0.2) and JS (6.51 ± 0.2) compared to EB heifers (4.53 ± 0.2). Mean (±SEM) plasma P4 concentration at TAI was lowest in the JS (0.28 ± 0.05), intermediate in CO (0.46 ± 0.02), and greatest in EB heifers (0.66 ± 0.05, p < .01). The diameter of the ovulatory follicle (mean ± SEM) was the smallest in the JS group compared to that in the CO and EB groups (15.8 ± 0.5; 13.9 ± 0.5; and 12.7 ± 0.5 mm for EB, CO and JS, respectively). More (p < .01) heifers in the JS group had their oestrous cycle synchronized (50.0, 78.8 and 82.4% for EB, CO and JS groups), and were pregnant at 28 (40.3, 51.3 and 63.3% for EB, CO and JS groups) and 50 days after TAI (32.6, 46.0 and 60.0% for EB, CO and JS groups). In summary, heifers subjected to the J-Synch TAI protocol had lower P4 at TAI, and better overall response to hormonal treatments, which resulted in increased P/AI at 28 and 50 days after TAI compared to those heifers subjected to either a 7-day EB protocol or a 5-day CO-synch protocol.

The use of a recombinant equine chorionic gonadotropin (reCG) in fixed-time AI programs in beef cattle

The aim of the current study was to evaluate the effect of a novel recombinant eCG (reCG) on pregnancy rates to AI (P/AI) in suckled beef cows of different breeds that were synchronized with an estradiol/progesterone (P4)-based protocol for fixed-time AI (TAI). In experiment 1, 1244 Bos taurus suckled cows were used. On Day 0 all cows received an intravaginal P4 device (600 mg P4) and 2 mg of estradiol benzoate. On Day 7, devices were removed, and all cows received 0.150 mg of D-cloprostenol plus 1 mg of estradiol cypionate and were randomly divided to receive 140 IU or 105 IU of reCG or no reCG treatment (controls) at that time. Cows were tail painted for estrus detection and those in estrus by 48 h after P4 device removal were inseminated; whereas those not showing estrus were also inseminated and received GnRH at the same time. In experiment 2, 818 Bos taurus x Bos indicus crossbred suckled cows received the same FTAI protocol used in Experiment 1. Cows were randomly divided at the time of P4 device removal into 4 groups to receive 140 IU, 105 IU or 84 IU of reCG or no reCG treatment. In experiment 3, 345 Bos indicus suckled cows were submitted to the same FTAI protocol as those in previous experiments and were randomly divided into three groups to receive 140 IU or 105 IU of reCG, or 300 IU of serum derived eCG (PMSG). In Experiment 1, estrus rate and P/AI was greater (P < 0.05) in cows treated with reCG (79.9 and 53.5 %, 76.9 and 52.3 % for the 105 UI and 140 UI reCG groups, respectively) than those in the control group (69.9 and 44.4 %, respectively). In Experiment 2, cows treated with reCG tended (P < 0.1) to achieve a greater P/AI than control cows (38.6 %, 37.1 %, 36.2 % and 28.2 % for those receiving 84 IU, 105 IU,140 IU of reCG, and those in the control group); but when P/AI of all cows treated with reCG was contrasted to that of control cows, the difference was significant (P < 0.01). In Experiment 3, P/AI in cows treated with 84 IU of reCG (54 %) did not differ from that of cows treated with serum derived eCG (59 %) but both were greater (P < 0.05) than cows treated with 105 UI of reCG (41 %). In conclusion, treatment with reCG improved fertility in suckled Bos taurus and Bos taurus x Bos indicus beef cows. In suckled Bos indicus cows, although treatment with reCG and serum derived eCG were comparable, the higher dosage of reCG was detrimental to their P/AI.

Therapeutic effects of curculigoside on cyclophosphamide-induced premature ovarian failure in mice

Objective The main purpose of this study was to elucidate the anti-apoptotic effects of curculigoside (CUR) on ovarian granulosa cells (GCs) in a mouse model of cyclophosphamide (CTX)-induced premature ovarian failure (POF). Method Intraperitoneal injection of CTX (100 mg/kg body weight) induced POF in mice. Thirty-six female mice were divided into six groups: blank group; POF model group; low-dose CUR group; medium-dose CUR group; high-dose CUR group; and estradiol benzoate group. Mice were orally administered for 28 consecutive days. Twenty-four hours after the completion of treatment, mice were weighed and euthanized, and blood was collected from the eyeball under anesthesia. The ovaries were surgically separated and weighed, and the ovarian index was calculated. Hematoxylin–eosin (HE) staining was used to observe follicular development and corpus luteum morphology in the ovaries. Serum levels of follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH) and estradiol (E2) were measured. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) content and malondialdehyde (MDA) levels in ovarian tissue were determined. The GC apoptosis level was measured. Western blotting was used to detect protein expression levels of Beclin-1, LC3, P62, AKT, p-AKT, mTOR and p-mTOR in the ovaries. Results The results showed that CUR can improve body weight and ovarian index; promote follicular development and reduce follicular atresia; improve FSH, AMH and E2 levels; downregulate MDA levels and restore antioxidant enzyme activity; inhibit the autophagy level; activate the AKT/mTOR signaling pathway; and alleviate GC apoptosis. Conclusion CUR improves POF by activating the AKT/mTOR signaling pathway, inhibiting autophagy and alleviating GC apoptosis.