Optogenetic Activation of Arcuate Kisspeptin Neurons Generates a Luteinizing Hormone Surge-Like Secretion in an Estradiol-Dependent Manner

Traditionally, the anteroventral periventricular (AVPV) nucleus has been the brain area associated with luteinizing hormone (LH) surge secretion in rodents. However, the role of the other population of hypothalamic kisspeptin neurons, in the arcuate nucleus (ARC), has been less well characterized with respect to surge generation. Previous experiments have demonstrated ARC kisspeptin knockdown reduced the amplitude of LH surges, indicating that they have a role in surge amplification. The present study used an optogenetic approach to selectively stimulate ARC kisspeptin neurons and examine the effect on LH surges in mice with different hormonal administrations. LH level was monitored from 13:00 to 21:00 h, at 30-minute intervals. Intact Kiss-Cre female mice showed increased LH secretion during the stimulation period in addition to displaying a spontaneous LH surge around the time of lights off. In ovariectomized Kiss-Cre mice, optogenetic stimulation was followed by a surge-like secretion of LH immediately after the stimulation period. Ovariectomized Kiss-Cre mice with a low dose of 17β-estradiol (OVX+E) replacement displayed a surge-like increase in LH release during period of optic stimulation. No LH response to the optic stimulation was observed in OVX+E mice on the day of estradiol benzoate (EB) treatment (day 1). However, after administration of progesterone (day 2), all OVX+E+EB+P mice exhibited an LH surge during optic stimulation. A spontaneous LH surge also occurred in these mice at the expected time. Taken together, these results help to affirm the fact that ARC kisspeptin may have a novel amplificatory role in LH surge production, which is dependent on the gonadal steroid milieu.

Metabolomics Study of Guizhi Fuling Capsules in Rats With Cold Coagulation Dysmenorrhea

Dysmenorrhea refers to a kind of uterine cramping pain that occurs in women during the period of menstrual. Guizhi Fuling Capsules are mainly used for the treatment of various pain syndromes and especially effective in treating primary dysmenorrhea. However, the research on its modern pharmacology and mechanism of action have not been thoroughly carried out. It is not clear about the main active ingredients, potential targets and metabolic pathways involved in its efficacy. Therefore, this research project employed estradiol benzoate sensitization combined with oxytocin pain to construct the cold coagulation syndrome dysmenorrhea model, observed the anti-dysmenorrhea effect of Guizhi Fuling Capsules, and used the metabolomics to explore its mechanism. The results showed that Guizhi Fuling Capsules could considerably reduce the number of writhing times in dysmenorrhea rats, increasing the level of PGE2 and β-EP and reducing the contents of PGF2α in rat serum. Pathological sections of uterus and ovaries also showed that Guizhi Fuling Capsules could significantly relieve endometrial hyperplasia and improve ovarian function. The LC/MS-based metabolomics of rat uterine indicated that the model group has a great deviation from the control group. Compared with the model group, the Guizhi Fuling Capsules group had a tendency to shift to the control group, and the main metabolic changes was mainly concentrated on saturated and unsaturated fatty acids. Among them, arachidonic acid is in a pivotal position, and the expression of its rate-limiting enzyme (COX-2) involved in its cyclooxygenase metabolic pathway was significantly up-regulated in the model group, but significantly decreased after the intervention of Guizhi Fuling Capsules. In conclusion, Guizhi Fuling Capsules can effectively relieve primary dysmenorrhea, and this effect may be attributed to the regulation effects of Guizhi Fuling Capsules on endogenous metabolism, such as inhibiting arachidonic acid converted to prostaglandins through downregulate the expression of COX-2, which plays an anti-inflammatory effect.

PSX-B-5 Effect of replacement of dry-rolled corn with un-processed rye on growth performance, efficiency of dietary net energy utilization, and carcass traits of finishing heifers

Continental × British beef heifers were used in a randomized complete block design experiment to evaluate the effects of replacing dry-rolled corn with unprocessed rye on growth performance, efficiency of dietary net energy (NE) utilization, and carcass trait responses in finishing heifers. Heifers (n = 56; 433 ± 34.0 kg) were transported 241 km from a regional sale barn to the Ruminant Nutrition Center in Brookings, SD. Heifers were blocked by weight grouping and then allotted to pens (n = 7 heifers/pen and 4 pens/treatment). Treatments included a finishing diet that contained 60% grain (DM basis) as dry-rolled corn (DRC) or unprocessed rye grain (RYE). On d 14, heifers were consuming the final diet and were implanted with 200 mg of trenbolone acetate and 28 mg of estradiol benzoate (Synovex-Plus, Zoetis, Parsippany, NJ). RYE heifers had decreased (P ≤ 0.01) final body weight, average daily gain, and gain efficiency; but tended (P = 0.08) to have a greater dry matter intake compared to DRC. RYE had decreased (P ≤ 0.01) observed dietary NE and decreased (P ≤ 0.01) observed-to-expected dietary NE ratio for maintenance and gain compared to DRC. Dressing percentage, 12th rib fat thickness, ribeye area, and the distribution of USDA yield and quality grades were not altered (P ≥ 0.12) by diet. Hot carcass weight, yield grade, estimated empty body fat (EBF), and body weight at 28% EBF decreased (P ≤ 0.02) and retail yield increased (P= 0.01) in RYE compared to DRC. These data indicate that unprocessed rye is a palatable feed ingredient for inclusion in finishing diets for beef cattle and that rye inclusion only minimally influences carcass quality. The feeding value of unprocessed rye is considerably less (21.4%) than that of dry-rolled corn using current standards and approximately 91% of the NE value of processed rye.

PHYTOCHEMICAL SCREENING AND APHRODISIAC EFFECT OF ETHANOLIC EXTRACT OF MASSULARIA ACUMINATA(G. DON) BULLOCK EX HOYL. (RUBIACEAE)IN MALE WISTAR RATS

Objective: The objective of the present study is to determine, through phytochemical screening, the major chemical compounds and to evaluate the aphrodisiac effect of the ethanolic extract of M.acuminata stems in adult male rats. Methods :To achieve our objectives, a phytochemical study was conducted to determine the major chemical compounds present in the ethanolic extract of M. acuminatastems. The phytochemical screening was carried out following standard analytical procedures using thin layer chromatography.The aphrodisiac effect was evaluated in sexually naïve male rats. In sexually naïve male rats, a single administration of the ethanolic extract was performed at doses of 10, 20 and 40 mg/kg versus 0.5 mL/100g distilled water and 715 µg/kg Sildenafil Pfizer 50 mg in the negative and positive control rats, respectively. Female rats were induced to oestrus by sequential administration of estradiol benzoate (Sigma - Aldrich) (25 μg/rat) to make them receptive to males. Results :The results of the phytochemical screening revealed that the ethanolic extract of M. acuminata stems contains coumarins, flavonoids, tannins, triterpenes of triterpenesaponins. The biological study showed that at doses of 20 and 40 mg/kg, ethanolic extract of M. acuminatastems resulted in a significant (p < 0.05) and highly significant (p < 0.001) increase in the number of sexual mounts, the number of erections, the number of ejaculations and a significant (p < 0.01) decrease in the latency time between sexual mounts in male rats. Conclusions:The sexual stimulating effects of the ethanolic extract of M. acuminata observed in this study could be attributed to the presence of the identified chemical compounds, hence the interest in using this plant in traditional aphrodisiac medicine.

Gestational losses after innovating with two embryos in a fixed-time embryo transfer program

The aim of this study was to quantify the pregnancy rate after implantation of two embryos after FTET protocols, as well as to monitor pregnancy losses until parturition, evaluating, mainly, if this strategy results in more number of animals born. Therefore, 423 multiparous recipients were selected, standardized in terms of body score, who had high-quality corpora lutea. Animals were randomly divided into two groups according to one or two embryos transferred (1 embryo = Control, n = 237; 2 embryos = Group 1, n = 186). All recipients received the same hormonal treatment, which consisted of administering, on Day 0, 2 mL of estradiol benzoate (Gonadiol, ZOETIS) + 1.9 g multidose 1st use progesterone implant (CIDR, ZOETIS); on Day 8 the implants were removed + injected 0.4 mL of estradiol cypionate (E.C.P, ZOETIS) + 1.5 mL of eCG (Novormon, ZOETIS) + 1 mL of dinoprost tromethamine (Lutalyse, ZOETIS). The animals were evaluated by ultrasonography at 30 and 60 days after embryo transfer, to diagnose the success rate and embryo losses during this period. Furthermore, information was collected on births, length of gestation, number of twin births, number of childbirth assistance and the weight of the calves. The results showed that Group 1 had better success than the Control, with higher conception rates at 30 days (68.3% vs. 53.2%, P<0.001) and at 60 days (62.9% vs. 52.3%; P<0 .05). The number of animals born was also higher for Group 1 (53.3% vs. 43.3%, P<0.01). The percentage of twins born in Group 1 was 17.9%, and the animals had lower weight compared to the Control (34.29 + 7.36 vs 37.63 + 5.73, P<0.05). The length of pregnancy and the number of assistances were similar between groups. In conclusion, the strategy adopted in this experiment suggests a considerable increase in the calf birth rate, but losses during pregnancy and their mechanisms need to be elucidated.

GnRH or estradiol benzoate combination with CIDR improves in-vivo embryo production in bovines (Bos indicus and Bos taurus) under subtropics

Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species’ genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology’s widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60 ± 4.08 vs 04.31 ± 0.98 and 09.33 ± 1.78 vs 05.22 ± 1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60 ± 4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions.

GPGMH, a New Fixed Timed-AI Synchronization Regimen for Swamp and River Crossbred Buffaloes (Bubalus bubalis)

The crossbreeding of Swamp and River type buffalo breeds is practiced for the improvement of milk yield and reproductive performance in swamp buffalo herds. This study aimed to modify the Ovsynch synchronization protocol (GPG) and improve the fixed-timed artificial insemination (FTAI) for better reproductive performance of crossbred buffaloes. Comparison of four conventional synchronization protocols [pregnant mare gonadotropin-prostaglandin F2α-gonadotropin-releasing hormone (PmPG), gonadotropin-releasing hormone-prostaglandin F2α-gonadotropin-releasing hormone (GPG), prostaglandin F2α-gonadotropin-releasing hormone-prostaglandin F2α-estradiol benzoate (PGPE), and progesterone-pregnant mare gonadotropin-prostaglandin F2α-gonadotropin-releasing hormone (P4PmPG)] in crossbred buffaloes showed that the GPG protocol treated buffaloes displayed higher (P &lt; 0.05) estrus response with an increasing tendency in ovulation (84.6%) and pregnancy rates (30.8%) than PmPG, PGPE, and P4PmPG treated buffaloes. Buffaloes treated with a dose of 0.4 (mg/kg) mifepristone combined with GPG, exhibited higher (P &lt; 0.05) estrous response (82.4%), ovulation (94.1%), and pregnancy (47.1%) rates compared with other doses (0, 0.3, or 0.5 mg/kg) groups. Injection of mifepristone along second GnRH injection in buffaloes improved (P &lt; 0.05) pregnancy rate (35.3%) when compared to before or after the second GnRH of GPG protocol. Single AI after 24 h of mifepristone or second GnRH injection seems the best time to enhance the pregnancy rates in buffaloes compared to double or other single AI times in the modified GPGMH protocol. In comparison, GPGMH reduced the follicular cyst incidence (P &lt; 0.05) with increasing ovulation (P &gt; 0.05) and pregnancy rates (P &gt; 0.05) than the P4GPG and GPG protocols in crossbred buffaloes. The current study supported that new synchronization protocol (modified of GPG protocol; GPGMH) by the inclusion of mifepristone (with a dose of 0.4 mg/kg along second GnRH), AI after 24 h of mifepristone or second GnRH, and human chorionic gonadotropin (hCG at day 5 of AI) enhance the ovulation and pregnancy rates in crossbred buffaloes.

RNA-sequencing of AVPV and ARH reveals vastly different temporal and transcriptomic responses to estradiol in the female rat hypothalamus

In females, estrogens have two main modes of action relating to gonadotropin secretion: positive feedback and negative feedback. Estrogen positive and negative feedback are controlled by different regions of the hypothalamus: the preoptic area/anterior portion (mainly the anteroventral periventricular nucleus, AVPV) of the hypothalamus is associated with estrogen positive feedback while the mediobasal hypothalamus (mainly the arcuate nucleus of the hypothalamus, ARH), is associated with estrogen negative feedback. In this study, we examined the temporal pattern of gene transcription in these two regions following estrogen treatment. Adult, ovariectomized, Long Evans rats received doses of estradiol benzoate (EB) or oil every 4 days for 3 cycles. On the last EB priming cycle, hypothalamic tissues were dissected into the AVPV+ and ARH+ at 0 hrs (baseline/oil control), 6 hrs, or 24 hrs after EB treatment. RNA was extracted and sequenced using bulk RNA sequencing. Differential gene analysis, gene ontology, and weighted correlation network analysis (WGCNA) was performed. Overall, we found that the AVPV+ and ARH+ respond differently to estradiol stimulation. In both regions, estradiol treatment resulted in more gene up-regulation than down-regulation. S100g was very strongly up-regulated by estradiol in both regions at 6 and 24 hrs after EB treatment. In the AVPV+ the highest number of differentially expressed genes occurred 24 hrs after EB. In the ARH+, the highest number of genes differentially expressed by EB occurred between 6 and 24 hrs after EB, while in the AVPV+, the fewest genes changed their expression between these time points, demonstrating a temporal difference in the way that EB regulates transcription these two areas. Several genes strongly implicated in gonadotropin release were differentially affected by estradiol including Esr1, encoding estrogen receptor-α and Kiss1, encoding kisspeptin. As an internal validation, Kiss1 was up-regulated in the AVPV+ and down-regulated in the ARH+. Gene network analysis revealed the vastly different clustering of genes modulated by estradiol in the AVPV+ compared with the ARH+. These results indicate that gene expression in these two hypothalamic regions have specific responses to estradiol in timing and direction.