Buserelin Promotes the Differentiation and Function of Macrophage-Colony-Stimulating Factor-Producing T Helper Cells.

BACKGROUND: Buserelin has been used to treat central precocious puberty (CPP). However, it could potentially result in immune dysregulation to undermine patients' health. Therefore, it is necessary to elucidate the effects of buserelin on immune cells. Here we explored buserelin-induced impacts on the differentiation and function of macrophage-colony-stimulating factor-producing T helper (ThGM) cells to uncover the immunoregulatory role of buserelin. METHODS: Rat CPP was induced by danazol injection followed by buserelin treatment. The frequencies of ThGM cells in the spleen and lymph nodes were evaluated by flow cytometry. ThGM cell generation and function were analyzed in cell culture assays. Cell signaling was measured by Immunoblotting. RESULTS: Buserelin increased the frequencies of splenic and lymph node ThGM cells. Buserelin promoted the in vitro differentiation and proliferation of ThGM cells. Buserelin-treated ThGM cells showed stronger supportive effects on other effector T helper cells. Buserelin induced the activation of the nuclear factor of activated T cells and extracellular signal-regulated kinase 1/2 in ThGM cells. CONCLUSION: Buserelin enhances the differentiation and function of pro-inflammatory ThGM cells, thus increasing the risk of autoimmune or inflammatory disorders. Therefore, it is necessary to monitor ThGM cells in buserelin-treated children to prevent latent immune dysregulation.

GnRH analogue triptorelin acetate induces ovulation in jennies during oestrus.

Most of the northern hemisphere donkey breeds are faced with the risk of extinction, thus donkey reproduction is considered an emerging branch of theriogenology, and the management of artificial insemination and induction of ovulation is a crucial point in setting up preservation protocols. For four consecutive cycles, six jennies' ovarian activity was routinely monitored; an ultrasound examination was performed daily from the evidence of a follicle diameter ≥27 mm until ovulation. Upon reaching a follicular diameter ≥32 ± 2 mm (Hour 0), oestrous jennies were treated alternatively with 0.1 mg triptorelin acetate, sc, (TRIP), 0.4 mg/sc of buserelin acetate (BUS) or saline, sc, (CTRL) and examined ultrasonographically at Hours 14, 24, 38, 42, 48, 62 and every 24 h until ovulation. Induction of ovulation was considered successful if ovulation occurred from 24 to 48 h after treatment. 11/12 cycles resulted in ovulation for TRIP and 12/12 for BUS and CTRL groups, respectively. Mean ± SD ovulation time after treatment was 37.3 ± 8.3, 47.1 ± 21.0 and 66.8 ± 25.9 h for BUS, TRIP and CTRL, respectively (p = .0032). Ovulation rates between h24 and h48 were 10/12 (83.3%) for both TRIP/BUS and 2/12 (16.7%) for CTRL, respectively (p = .003). Buserelin and triptorelin-treated jennies had a 25 times higher probability to ovulate between Hours 24 and 48 than controls (p = .003), while there were no jenny and cycle effects on the ovulatory rate. The results of this study show how triptorelin successfully induced ovulation in jennies, like other GnRH analogues previously evaluated.

Effect of the GnRH stimulation test on Canine Prostatic-Specific Esterase (CPSE).

The gonadotropin-releasing hormone (GnRH) stimulation test is used to investigate testicular production of testosterone (T) when performing a breeding soundness examination. In male dogs with fertility problems, the prostate should also be investigated as prostatic conditions may frequently lower semen quality. Serum concentrations of canine prostatic-specific esterase (CPSE) increase in dogs with benign prostatic hyperplasia (BPH). When performing a breeding soundness examination in a male dog, GnRH administration is frequently done at the beginning of the process and then both T and CPSE are assayed on the same serum sample collected 1 h following the GnRH injection. The aim of this study was to assess whether or not the administration of GnRH may alter CPSE concentrations in dogs with a healthy prostate. Twenty-eight client-owned intact adult male dogs were included in the study. Following a 7-day sexual rest all male dogs underwent a clinical examination and an ultrasonographic examination of the prostatic gland. Prostatic size and parenchyma of every tested dog were evaluated by ultrasonography to assess prostatic conditions. Two different GnRH stimulation protocols were used, A = gonadorelin 50µg/dog SC (n = 15) and B = buserelin 0.12 µg/kg IV (n = 13). T and CPSE concentrations were measured before and 1 h after GnRH administration by a laser-induced fluorescence analysis. Buserelin and gonadorelin were equally effective in causing a significant increase in serum T concentrations in the post GnRH sample. When considering the 28 dogs together, CPSE concentrations did not change following the stimulation test with either GnRH compound; however, in 4/28 cases, the post GnRH value was markedly increased to values compatible with a diagnosis of BPH. There was no difference in the action of buserelin or gonadorelin in causing an increase in serum T concentrations. CPSE secretion was increased in approximately 15% of dogs treated with either buserelin or gonadorelin. Therefore, whenever performing diagnostic testing in intact male dogs, CPSE should not be assayed on a post-GnRH serum sample.

The function of the pituitary-testicular axis in dogs prior to and following surgical or chemical castration with the GnRH-agonist deslorelin.

Chemical castration, that is the reduction of circulating testosterone concentrations to castrate levels by administration of a GnRH-agonist implant, is a popular alternative to surgical castration in male dogs. Detailed information concerning the pituitary-testicular axis following administration of a GnRH-agonist implant is still scarce. Therefore, GnRH-stimulation tests were performed in male dogs, prior to and after surgical and chemical castration. This approach also allowed us to determine plasma concentrations of testosterone and oestradiol in intact male dogs for future reference and to directly compare the effects of surgical and chemical castration on the pituitary-testicular axis. In intact male dogs (n = 42) of different breeds GnRH administration induced increased plasma LH, FSH, oestradiol and testosterone concentrations. After surgical castration basal and GnRH-induced plasma FSH and LH concentrations increased pronouncedly. Additionally, basal and GnRH-induced plasma oestradiol and testosterone concentrations decreased after surgical castration. After chemical castration, with a slow-release implant containing the GnRH-agonist deslorelin, plasma LH and FSH concentrations were lower than prior to castration and lower compared with the same interval after surgical castration. Consequently, plasma oestradiol and testosterone concentrations were lowered to values similar to those after surgical castration. GnRH administration to the chemically castrated male dogs induced a significant increase in the plasma concentrations of LH, but not of FSH. In conclusion, after administration of the deslorelin implant, the plasma concentrations of oestradiol and testosterone did not differ significantly from the surgically castrated animals. After GnRH-stimulation, none of the dogs went to pre-treatment testosterone levels. However, at the moment of assessment at 4,4 months (mean 133 days ± SEM 4 days), the pituitary gonadotrophs were responsive to GnRH in implanted dogs. The increase of LH, but not of FSH, following GnRH administration indicates a differential regulation of the release of these gonadotrophins, which needs to be considered when GnRH-stimulation tests are performed in implanted dogs.

Buserelin Inhibits the Immunosuppressive Activity of Regulatory T Cells through the Protein Kinase A Signaling in a Central Precocious Puberty Model.

BACKGROUND: Gonadotropin-releasing hormone analogs (GnRHas) are used for treating central precocious puberty (CPP). However, their roles in the regulation of immune cells especially regulatory T cells (Tregs) remains elusive. Therefore, we characterized buserelin-induced phenotypical and functional changes of Tregs. METHODS: A rat CPP model was established followed by administration of buserelin acetate. Flow cytometry was used to evaluate the expression of functional molecules in splenic Tregs. The suppressive activity of Tregs was determined by the suppression assay. GnRHR expression in Tregs was assessed by flow cytometry analysis and Immunoblotting. Normal Tregs were then stimulated and treated with buserelin acetate in vitro. After that, Foxp3 expression, Treg proliferation, and cytokine production were analyzed by flow cytometry. Intracellular signaling was evaluated by Immunoblotting, and Treg function was determined by the suppression assay. RESULTS: After in vivo buserelin treatment, the frequency of splenic Tregs was decreased, with the reduction in the expression of Foxp3, IL-10, and TGF-ß. The suppressive activity of Tregs was weakened. Buserelin down-regulated Foxp3 expression while promoting the expression of RORγt and IL-17 in Tregs through activating the protein kinase A (PKA) pathway in vitro. The PKA inhibitor H-89 abolished the effect of buserelin and enhanced Treg function. CONCLUSION: Buserelin impaired the immunosuppressive activity of Tregs through the PKA signal pathway. Buserelin-induced activation of PKA signaling down-regulated Foxp3 expression while promoting RORγt expression in Tregs, and subsequently weakened Treg function. Our study indicates the necessity of monitoring Treg activity in CPP patients to avoid potential autoimmunity or inflammation.

The pharmacokinetics of buserelin after intramuscular administration in pigs and cows.

BACKGROUND: Buserelin is a luteinizing hormone releasing hormone (LHRH) agonist used for the treatment of hormone-dependent diseases in males and females. However, the pharmacokinetics of buserelin in pigs and cows are not fully understood. This study was designed to develop a sensitive method to determine the concentration of buserelin in blood plasma and to investigate the pharmacokinetic parameters after intramuscular (i.m.) administration in pigs and cows. RESULTS: A sensitive and rapid stability method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed. The pharmacokinetic parameters of buserelin after i.m. administration were studied in five pigs and five cows at a single dose of 1 mg per pig and 3 mg per cow. The plasma kinetics were analyzed by WinNonlin 8.1.0 software using a non-compartmental model. The mean concentration area under the curve (AUC0-t) was 25.02 ± 6.93 h × ng/mL for pigs and 5.63 ± 1.86 h × ng/mL for cows. The maximum plasma concentration (Cmax) and time to reach the maximum concentration (tmax) were 10.99 ± 2.04 ng/mL and 0.57 ± 0.18 h for pigs and 2.68 ± 0.36 ng/mL and 1.05 ± 0.27 h for cows, respectively. The apparent volume of distribution (Vz) in pigs and cows was 80.49 ± 43.88 L and 839.88 ± 174.77 L, respectively. The elimination half-time (t1/2), and clearance (CL) were 1.29 ± 0.40 h and 41.15 ± 11.18 L/h for pigs and 1.13 ± 0.3 h and 545.04 ± 166.40 L/h for cows, respectively. No adverse effects were observed in any of the animals. CONCLUSION: This study extends previous studies describing the pharmacokinetics of buserelin following i.m. administration in pigs and cows. Further studies investigating other factors were needed to establish therapeutic protocol in pigs and cows and to extrapolate these parameters to others economic animals.

Effects of ranibizumab and zoledronic acid on endometriosis in a rat model.

PURPOSE: To investigate the histological efficacy of ranibizumab and zoledronic acid in an experimentally induced endometriosis model as compared with danazol, buserelin acetate and dienogest. METHODS: Endometrial implants were introduced in 52 female Wistar albino rats, which were then randomly divided into six groups. The animals were, respectively, given dienogest, danazol, buserelin acetate, zoledronic acid, ranibizumab and 0.9% NaCl. After 4 weeks, the volumes and histopathological properties of the implants were evaluated and the implants were excised completely at the third laparotomy. A histopathological scoring system was used to evaluate the preservation of epithelia. Endometrial explants were evaluated immunohistochemically. RESULTS: Among the groups, the histological score was significantly lower in the zoledronic acid and ranibizumab groups compared with the controls (p < 0.001). There were no significant differences regarding ellipsoidal volume levels between groups (p > 0.05). However, there was a statistically significant difference regarding cell numbers according to the degree of Bcl-2, NF-κB, and CD31 staining (p < 0.001). There was no statistically significant difference in Bcl-2, CD31, or NF-κB staining in the binary comparisons between the other groups (p > 0.05). For Bcl-2 staining, the staining rate of the group treated with zoledronic acid was significantly lower compared with the dienogest and danazol groups (p < 0.05). The staining rates of CD31 and NF-κB were significantly lower in the zoledronic acid and ranibizumab groups compared with the controls (p < 0.05). CONCLUSION: According to these results, zoledronic acid and ranibizumab may be putative candidates for the treatment of endometriosis.

Reproductive efficiency of sows inseminated at single dose fixed time with refrigerated, cryopreserved and encapsulated spermatozoa.

The aim was to assess the reproductive efficiency of different techniques used to preserve spermatozoa in artificial insemination semen doses (AI-doses) by evaluating refrigeration at 15°C, cryopreservation and encapsulation. Forty-two hyperprolific sows were treated with buserelin and inseminated once at a single fixed time. The fertility rate, embryonic vesicles viability and the early embryonic mortality (arrested conceptuses) evaluated post-mortem at 24th day of pregnancy, were analysed in order to assess the effectiveness of each proposed technique. Results show an overall reduction on fertility using the three proposal sperm preservation techniques (69.27%, 60.00% and 78.75% for refrigerated, frozen-thawed and encapsulated AI-doses, respectively). Total number of embryonic vesicles was very similar among the three treatments; yet, the number of viable vesicles was numerically different among groups, and thus, embryonic viability was 79.25%, 80.0% and 87.15% for refrigerated, frozen-thawed and encapsulated AI-doses, respectively.

Single injection of triptorelin or buserelin acetate in saline solution induces ovulation in mares the same as a single injection of hCG.

The aim of this study was to assess the efficacy of different doses of buserelin acetate and another GnRH agonist, triptorelin acetate, in saline solution in a single subcutaneous injection, to induce ovulation of growing pre-ovulatory follicle in mare and compare it with the classical treatment of a single injection of hCG. The study is split into 3 experiments over different breeding seasons in the same stud with a random distribution of treatment. The first one was to compare the injection of 6 mg of buserelin with 1,500 IU of hCG; the second one consisted of comparing different doses of buserelin (6 mg and 3 mg); and the third one compared three different doses of buserelin (3, 2 and 1 mg), 0.1 mg of triptorelin with 1,500 IU of hCG as a control group. The results of all experiments showed the same efficacy between all treatments with mares ovulating between 24 and 48 hr after injection: experiment 1: hCG (78% n = 41) and buserelin 6 mg (90% n = 50); experiment 2: buserelin 6 mg (78,1% n = 192) and buserelin 3 mg (78% n = 341); and experiment 3: hCG (87% n = 106), buserelin 3 mg (84,7% n = 137), buserelin 2 mg (82,7% n = 104), buserelin 1 mg (87% n = 54) and triptorelin 0.1 mg (84,7% n = 72). In conclusion, this study contributes to erasing the dogma that has been established since 1975 that a single injection in solution without any long-acting excipient of a GnRH agonist cannot induce ovulation in the mare. This study also shows that a injection of 0.1 mg of triptorelin in solution is a good alternative for ovulation induction and is comparable to small doses of buserelin acetate in solution (1 mg) and 1,500 IU of the gold standard trigger hCG, mainly in countries where human formulation of buserelin is not available.

The GnRH analogues affect novel neuropeptide SMIM20/phoenixin and GPR173 receptor expressions in the female rat hypothalamic-pituitary-gonadal (HPG) axis.

The recently discovered peptide phoenixin (PNX) and its receptor GPR173 are novel factors that exhibit a large spectrum of regulatory activity, especially when considered as a central modulator of GnRH-related hormonal control of reproductive processes. It has been already proven that GnRH agonists and antagonists can modulate peptidergic signalling in the HPG axis. Despite these findings, there is so far no information regarding the influence of treatment with GnRH analogues on SMIM20/phoenixin signalling in the hypothalamic-pituitary-gonadal axis. In the current study, SMIM20/phoenixin and GPR173 mRNA levels were measured in the hypothalamus, pituitary and ovaries of female rats in the dioestrus phase following treatment with GnRH-R agonist (buserelin) and antagonist (cetrorelix) using quantitative real-time PCR. The serum PNX concentrations were also estimated with ELISA technique. The hypothalamic, hypophyseal and especially ovarian levels of SMIM20 mRNA were increased after both buserelin and cetrorelix administration. The GPR173 expressions were in turn decreased in the hypothalamus and pituitary. Treatment with the GnRH analogues led to the modulation of SMIM20/phoenixin and GPR173 mRNA expression in the female rat hypothalamic-pituitary-gonadal axis. By identifying buserelin and cetrorelix as novel modulators of phoenixin signalling in the animal HPG axis, these results cast new light on the GnRH analogues mode of action and contribute to a better understanding of the mechanisms responsible for the hormonal control of reproduction.

Quantitative aspects of microchip isotachophoresis for high precision determination of main components in pharmaceuticals.

Although microchip electrophoresis (MCE) is intended to provide reliable quantitative data, so far there is only limited attention paid to these important aspects. This study gives a general overview of key aspects to be followed to reach high-precise determination using isotachophoresis (ITP) on the microchip with conductivity detection. From the application point of view, the procedure for the determination of acetate, a main component in the pharmaceutical preparation buserelin acetate, was developed. Our results document that run-to-run fluctuations in the sample injection volume limit the reproducibility of quantitation based on the external calibration. The use of a suitable internal standard (succinate in this study) improved the repeatability of the precision of acetate determination from six to eight times. The robustness of the procedure was studied in terms of impact of fluctuations in various experimental parameters (driving current, concentration of the leading ions, pH of the leading electrolyte and buffer impurities) on the precision of the ITP determination. The use of computer simulation programs provided means to assess the ITP experiments using well-defined theoretical models. A long-term validity of the calibration curves on two microchips and two MCE equipments was verified. This favors ITP over other microchip electrophoresis techniques, when chip-to-chip or equipment-to-equipment transfer of the analytical method is required. The recovery values in the range of 98-101 % indicate very accurate determination of acetate in buserelin acetate, which is used in the treatment of hormone-dependent tumors. This study showed that microchip ITP is suitable for reliable determination of main components in pharmaceutical preparations.

Enhanced permeability across Caco-2 cell monolayers by specific mannosylating ligand of buserelin acetate proliposomes.

CONTEXT: Oral delivery of peptide and protein drugs still remains the area of challenges due to their low stability and permeability across GI tract. Among numerous attempts, the receptor-mediated drug targeting is a promising approach to enhance GI permeability. OBJECTIVE: The aim of this study was to prepare mannosylated buserelin acetate (MANS-BA) proliposome powders grafted with N-octadecyl-d-mannopyranosylamine (SAMAN) as targeting moiety and evaluate their permeability across Caco-2 cell monolayers. MATERIALS AND METHODS: The MANS-BA proliposome powders were prepared by coprecipitation method. The targeting moiety SAMAN was synthesized in-house and confirmed by characterization using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC). RESULTS: The MANS-BA liposomes reconstituted from proliposome powders exhibited the oligolamellar vesicular structure of phospholipid bilayer. Their size, zeta potential and entrapment efficiency were in the ranges of 93.11-218.95 nm, -24.03 to -37.15 mV and 21.12-33.80%, respectively. The permeability of reconstituted MANS-BA liposomes across Caco-2 cell monolayers was significantly enhanced to about 1.2- and 2.2-fold over those of conventional BA liposomes and solution, respectively. DISCUSSION: Increase in dicetylphosphate, cholesterol and SAMAN contents resulted in significant increase in size and zeta potential of reconstituted MAN-BA liposomes. The entrapment efficiency was increased with increasing dicetylphosphate and mannitol contents in liposomes containing cholesterol. CONCLUSIONS: The significantly enhanced permeability across Caco-2 cell monolayers of MANS-BA liposomes might be due to the role of mannose receptor on intestinal enterocytes.

Effects of a single use of the GnRH analog buserelin on the induction of ovulation and endocrine profiles in heavy draft mares.

We observed structural changes in the follicles and uterus of heavy draft mares during estrus and examined the effect of a single injection of the gonadotropin-releasing hormone analog buserelin on ovulation and endocrine profiles. Twenty-two heavy draft mares were divided into a buserelin-treated group (n=8) and a control group (n=14). Mares were given an intramuscular injection of 40 µg buserelin when they presented signs of estrus to a teaser stallion, had ≥45 mm diameter follicles, and presented decreased uterine edema compared with the previous examination. The follicles and uterus were monitored using transrectal ultrasound imaging and measurement of blood levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and estradiol-17ß. The ovulation rates within 48 hr was significantly higher in the treated group (100%, 8/8) than in the control group (57.1%, 8/14; P=0.051). The mean ± SEM time before confirmation of ovulation was 29 ± 9 hr for the treated group and 59 ± 7 hr for the control group. There were no significant differences in mating frequency, double ovulation rate, or fertility rate between the two groups. One to two days after administering buserelin, LH and FSH temporarily increased, and in the control group, LH was high during ovulation, whereas FSH temporarily increased with the growth of the follicle. These results indicate that a single injection of 40 µg buserelin when follicles are at least 45 mm in diameter and uterine edema is decreased is effective for inducing ovulation.

GnRH analogs induce a LH peak and increase pregnancy per timed-AI in ewes.

To date, there have been no studies testing the capacity of GnRH analogs and respective doses to induce a LH peak in sheep. In this sense, the present study aimed to evaluate the capacity of different synthetic forms and doses of GnRH in inducing LH release in sheep, and the effect of GnRH administration at timed artificial insemination (TAI) on pregnancy per timed-AI. In experiment 1, ewes (n = 40) received an intravaginal device (IVD) of medroxyprogesterone acetate (MPA; 60 mg) for 7 d and prostaglandin F2α analog on Day 5. On Day 7, the ewes were allocated randomly into one of eight groups (n = 5/group), which received a GnRH analog at a specific dose, as follows: lecirelin (12.5 or 25 µg), gonadorelin (50 or 100 µg), buserelin acetate (4.2 or 8.4 µg), or deslorelin (375 or 750 µg). Blood samples for LH determination were obtained at 0, 2, 4, and 6 h after GnRH and the IVDs were removed after the last blood collection. The maximal LH concentration induced by gonadorelin at doses of 50 µg and 100 µg (12.0 ± 2.4 ng/mL and 28.6 ± 7.1 ng/mL, respectively) was lower (P < 0.05) than serum LH induced by 8.4 µg of buserelin (78.9 ± 12.9 ng/mL), 375 µg and 750 µg of deslorelin (75.6 ± 7.4 ng/mL and 72.1 ± 10.6 ng/mL, respectively) and 12.5 µg and 25 µg of lecirelin (73.3 ± 17.8 ng/mL and 61.6 ± 5.9 ng/mL, respectively). However, the maximal LH concentration induced by 4.2 µg of buserelin (49.4 ± 5.9 ng/mL) was similar (P > 0.05) to the 100 µg of gonadorelin. The total release of LH (area under the curve - AUC) after treatment with 50 µg of gonadorelin (31.7 ± 5.9 ng h/mL) was lower (P < 0.05) than after other agonists. In a second experiment, 330 ewes were treated with IVD containing MPA for 7 d. Simultaneously with IVD removal, 250 µg of cloprostenol and 200 IU of eCG were administered. Then, ewes were assigned randomly to either no further treatment (control); or to receive 4.2 µg of buserelin acetate (GnRH group) at cervical TAI, which was performed with fresh semen 54 h after IVD withdrawal in all the animals. Higher pregnancy per timed-AI was observed for GnRH (50.3 %) compared to control (40.7 %). We conclude that buserelin acetate (8.4 µg), lecirelin (12.5 and 25 µg) and deslorelin (375 and 750 µg) induced a greater stimulatory effect on LH secretion than gonadorelin treatment. Furthermore, buserelin acetate treatment at TAI increased pregnancy per timed-AI in ewes previously treated with MPA and eCG.

Effect of hormonal treatment on artificial propagation, spawning performance and embryonic development of striped snakehead Channa striata (Bloch, 1793).

Present study describes the spawning induction of striped Snakehead, Channa striata using carp pituitary extract (CPE) and LH-RH agonist i.e. Buserelin (Glp-His-Trp-Ser-Tyr-Ser-tBu-Leu-Arg-Pro-NHEt). Total four treatments were designed under both hormones trail and treated as control group, T1, T2, and T3 with three replications of each treatment. While breeders under all hormone treatments showed spawning performances, no spawning performance was observed in control group. Latency time after hormonal treatment was lowest (20-24 hrs) in case of CPE than Buserelin (25-30 hrs). Regarding to CPE, spawning, fertilization and hatching rate were higher with the increasing doses of CPE in different treatments. The highest mean ± Standard deviation spawning, fertilization and hatching rate were 85.60±8.58 %, 79.38±4.89 % and 64.33±6.60 % respectively in T3 where dose of CPE was 80 mg kg-1. Similarly, in case of Buserelin hormone highest spawning rate was found in T3 (80.61±5.59) where dose of Buserelin was 0.80 µg kg-1 body weight. Fertilization rate was on the level 48.57±5.99, 70.62±5.33 and 90.32±4.79 respectively for T1, T2, and T3.Whilst, hatching rate was found 20.81±4.91, 37.11±4.50 and 61.33±6.61 in T1, T2, and T3 treatments respectively. However, T3 exhibited best performance regarding spawning, fertilization and hatching rate which were significantly higher than other two treatments.The current study revealed that spawning induction using carp pituitary extract and Buserelin is effective and might be useful for artificial breeding of Channa striata. Regarding to dose application i.e. 80 mg kg-1 of CPE and 0.80 µg kg-1 of Buserelin may be successfully applied to ovulation stimulation of Channa striata.

Buserelin Acetate Added to Boar Semen Enhances Litter Size in Gilts in Tropical Environments.

The use of exogenous hormones has long been of interest for improving reproductive performance in swine production. Enhancing litter size directly impacts the economic efficiency of pig production. Various strategies, including nutritional, genetic, and hormonal approaches, have been explored with varying degrees of success. Administering a gonadotropin-releasing hormone (GnRH) agonist, such as buserelin, at the onset of estrus can induce ovulation and reduce the variation in ovulation timing among sows. This study assessed the impact of GnRH agonist supplementation in boar semen doses on the litter size of inseminated gilts. The research was conducted on a commercial swine herd in northern Thailand. A total of 231 Landrace × Yorkshire crossbred gilts, aged 224.5 ± 16.2 days at the onset of estrus synchronization, participated in the experiment. The gilts' estrus was synchronized with oral altrenogest supplementation at a dosage of 20 mg/day for 18 days. After exhibiting standing estrus, the gilts were randomly divided into three groups. Control group: gilts were inseminated at 0 and 12 h post standing estrus onset with a conventional semen dose (n = 94). Treatment 1: similar to the control group, but with an added 5 µg (1.25 mL) of buserelin acetate to the boar semen dose during the first insemination (n = 71). Treatment 2: similar to the control group, but with 10 µg (2.5 mL) of buserelin acetate added to the boar semen dose during the first insemination (n = 66). All gilts were inseminated twice during their standing estrus using the intrauterine artificial insemination method. Each semen dose contained 3.0 × 109 motile sperm in 80 mL. The farrowing rate averaged 78.8% and did not significantly differ between the groups (p = 0.141). The total number of piglets born per litter in the treatment 2 group was greater than in the control group (14.0 ± 0.3 vs. 13.2 ± 0.3, respectively, p = 0.049), but was not significantly different from the treatment 1 group (13.3 ± 0.3, p = 0.154). Similarly, the number of live-born piglets in the treatment 2 group was greater than in the control and treatment 1 groups (13.2 ± 0.4 vs. 12.3 ± 0.3 and 12.0 ± 0.4, respectively, p < 0.05). Moreover, the live-born piglets' litter birth weight in the treatment 2 group was greater than in the control group (17.0 ± 0.4 vs. 15.6 ± 0.3 kg, respectively, p = 0.008) and the treatment 1 group (15.7 ± 0.4 kg, p = 0.025). In conclusion, adding a GnRH agonist to boar semen appears to enhance the litter size of gilts. Further research should focus on understanding the underlying mechanisms and determining the optimal dose and timing for GnRH agonist supplementation.

Screening of Cyclodextrins in the Processing of Buserelin Dry Powders for Inhalation Prepared by Spray Freeze-Drying.

Purpose: In this study, we prepared inhalable buserelin microparticles using the spray freeze-drying (SFD) method for pulmonary drug delivery. Raffinose as a cryoprotectant carrier was combined with two levels of five different cyclodextrins (CDs) and then processed by SFD. Methods: Dry powder diameters were evaluated by laser light scattering and morphology was determined by scanning electron microscopy (SEM). Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis were utilized for the determination of crystalline structures. The aerodynamic properties of the spray freeze-dried powders were evaluated by twin stage impinger (TSI) and the stability of prepared samples was assessed under normal and accelerated conditions. Results: The prepared powders were mostly porous spheres and the size of microparticles ranged from 9.08 to 13.53 µm, which are suitable as spray-freeze dried particles. All formulations showed amorphous structure confirmed by DSC and XRD. The aerosolization performance of the formulation containing buserelin, raffinose and 5% beta-cyclodextrin (ß-CD), was the highest and its fine particle fraction (FPF) was 69.38%. The more circular and separated structures were observed in higher concentrations of CDs, which were compatible with FPFs. The highest stability was obtained in the formulation containing hydroxypropyl beta-cyclodextrin (HP-ß-16. CD) 5%. On the contrary, sulfobutylether beta-cyclodextrin (SBE-ß-CD) 5% bearing particles showed the least stability. Conclusion: By adjusting the type and ratio of CDs in the presence of raffinose, the prepared formulations could effectively enhance the aerosolization and stability of buserelin. Therefore, they can be proposed as a suitable career for lung drug delivery.

Gilt reproductive performance in a tropical environment after oestrus synchronization and fixed-time artificial insemination.

The present study was performed to evaluate the reproductive performance of gilts inseminated using fixed-time artificial insemination (FTAI) protocol. A total of 408 Landrace × Yorkshire crossbred gilts were included in the experiment. Gilts at 8 months of age were randomly allocated into three groups: control AI (n = 192), treatment 1-TAI (n = 117) and treatment 2-FTAI (n = 99). Gilts in the control AI group were inseminated 2-3 times during standing oestrus at 0, 12 and 24 h after the onset of oestrus. Gilts in the treatment 1-TAI group were orally administered 20 mg per day of altrenogest for 18 days and then inseminated 2-3 times during standing oestrus by conventional AI. Gilts in the treatment 2-FTAI group were synchronized like gilts in treatment 1-TAI group but then GnRH (10 µg of buserelin) was administered 120 h after the end of altrenogest treatment and fixed time artificial inseminated twice at 24 and 32 h after GnRH irrespective of the presence of oestrus or not. Conception rate of gilts in treatment 2-FTAI (87.9%) was similar to the treatment 1-TAI (94.9%) and control AI (83.3%) (P > 0.05). Conception rate in treatment 1-TAI (94.9%) was higher compared to control AI group (83.3%, P = 0.040). Farrowing rate of gilts in treatment 2-FTAI (83.8%) was similar to treatment 1-TAI (89.7%) and control AI (76.0%) (P > 0.05). Farrowing rate of treatment 1-TAI (89.7%) was higher than control AI gilts (76.0%, P = 0.033). In treatment 2-FTAI, the conception and farrowing rate of the nine gilts that were inseminated even if they were not detected in oestrus (all during warm season) was 44.4% and 44.4%, respectively. Regular return to oestrus was similar between groups (9.4%, 0.9% and 4.1% for control AI, treatment 1-TAI and treatment 2-FTAI, respectively, P > 0.05). The total number of piglets born per litter in treatment 1-TAI group was higher than control AI (13.1 ± 0.2 versus 11.6 ± 0.2, respectively, P < 0.001) and treatment 2-FTAI groups (12.2 ± 0.3, P = 0.019). The number of piglets born alive was higher in treatment 1-TAI (12.1 ± 0.3) compared to treatment 2-FTAI (11.3 ± 0.2) and control AI group (11.2 ± 0.3). The percentage of stillbirth and mummified foetus were not different between groups (P > 0.05). The present study indicated that fixed-time AI in gilts can be successfully performed by administration of altrenogest for 18 days, GnRH at 120 h after altrenogest withdrawal and then double fixed-time AI at 24 and 32 h after the administration of GnRH. Fertility metrics such as conception rate, farrowing rate and litter performances using this method were similar to gilts inseminated at oestrus with conventional AI.

Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment.

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 µg/day for 4 weeks followed by 8 µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function.

The Kisspeptin analogue C6 induces ovulation in jennies.

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (-24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies.